DIAGNOSIS OF MAJOR DEPRESSIVE DISORDER, MILD

COGNITIVE IMPAIRMENT, AND ALZHEIMER'S DISEASE AND OTHER NEUROLOGIC AND PSYCHIATRIC DISORDERS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/126,347, filed February 27, 2015, the disclosure is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Major Depressive Disorder (MDD) is a neuropsychiatric condition that afflicts anywhere from 10 to 20% of the population. The most common age of onset is between 30 and 40 years, with a later peak between 50 and 60 years of age. In the United States, MDD is a contributing cause to the majority of the approximately 30,000 annual deaths by suicide. Diagnosis and severity of MDD are based on a clinician assessment of a defined set of specific signs and symptoms experienced by the subject during one or more major depressive episodes (See, e.g., American Psychiatric Association (2013), Diagnostic and statistical manual of mental disorders: DSM-5, Washington, D.C. American Psychiatric Association). This subjective diagnostic method relies on the subject's self-reported experiences and observed behavior. In addition, many MDD patients do not adequately respond to antidepressant treatment, with 70% of patients failing to achieve remission on their initial antidepressant and 2 out of 3 patients requiring more than one antidepressant (see, Trivedi MH, et al. AM J Psychiatry, 2006; 163(1): 28-40. Warden D, et al. Curr Psychiatry Rep. 2007; 9(6): 449-59). Since major depressive disorder is a heterogeneous illness, there is a need in the art for more effective methods of objectively diagnosing and/or assessing the severity and prognosis of MDD.

[0003] Individuals diagnosed with mild cognitive impairment (MCI) have cognitive impairments beyond that expected for their age and education, but that typically do not interfere significantly with their daily activities (see, e.g., Petersen et al., Arch Neuro,

56(3):303-308, 1999; Gauthier et al., Lancet, 367: 1262-1270, 2006; Petersen et al., Arch Neurol, 66: 1447-1455, 2009). MCI is considered to be a clinical stage distinct from normal aging and dementia. Diagnosis of MCI can involve a comprehensive clinical assessment including clinical observation, neuroimaging, blood tests and neuropsychological testing, similar to that used for diagnosing Alzheimer's disease. MCI can be diagnosed if there is evidence of: a cognitive or memory complaint by the individual and/or a family member; cognitive or memory impairment for age; a change in functional abilities; generally preserved activities of daily life, possibly with increased difficulty; and an absence of dementia.

Current methods for diagnosing the disorder are based on cognitive or memory assessments performed by a clinician. For example, subjects scoring higher than 24 of a Folstein Mini- Mental State Examination (MMSE) can be diagnosed with MCI. [0004] Alzheimer's disease and related disorders (ADRD) are debilitating conditions that impair memory, thought processes, and functioning, primarily among older adults. The effects of these diseases can be devastating, both for individuals afflicted with ADRD and for their families. People with ADRD require significant amounts of health care and intensive long-term services and support such as management of chronic conditions, medications, supervision and care, or assistance with personal care activities, such as eating, bathing, and dressing. In the United States, ADRD affects as many as 5 million people and nearly 40% of the population aged 85 and older. Roughly 13.2 million older Americans are projected to have ADRD by 2050.

[0005] Alzheimer's disease (AD) is an age-related, non-reversible brain disorder that develops over a period of years. The symptoms of AD gradually lead to behavior and personality changes, a decline in cognitive abilities such as decision-making and language skills, and problems recognizing family and friends. AD ultimately leads to a severe loss of mental function. These losses are related to the worsening breakdown of the connections between certain neurons in the brain and their eventual death. AD is one of a group of disorders called dementias that are characterized by cognitive and behavioral problems. It is the most common cause of dementia among people age 65 and older.

[0006] Cerebral folate deficiency (CFD) is defined as any neurological syndrome associated with low cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF) levels, the active folate metabolite, in the presence of normal folate metabolism outside the nervous system. CFD can result from either disturbed folate transport or from increased folate turnover within the central nervous system (CNS). [0007] There remains a need in the art for molecular marker-based methods for diagnosing the risk or aiding in the diagnosing of the risk for major depressive disorder (MDD), mild cognitive impairment (MCI), AD-related disorders and CFD in an individual. The present invention fulfills this and other needs as well.

BRIEF SUMMARY OF THE INVENTION

[0008] In one embodiment, the present invention provides a method for aiding in the diagnosis of a risk for major depressive disorder (MDD) and/or the risk of inadequate response to antidepressant treatments in a subject. The method comprises detecting or quantitating the level or amount of autoantibodies to folate receptor alpha (FRa) in a sample from the subject; and diagnosing the subject as having a risk for MDD by the presence of autoantibodies to folate receptor alpha (FRa). In some aspects, detecting or quantitating comprises performing an immunoassay with a folate receptor as an antigen. In other aspects, detecting or quantitating comprises using a homogeneous mobility shift assay (HMSA). [0009] In yet another embodiment, the present invention provides a method for treating a subject having MDD. The method comprises detecting or quantitating the level of autoantibodies to folate receptor alpha (FRa) in a sample from the subject; and administering a treatment comprising a therapeutically effective amount of a folate containing formulation and a therapeutically effective amount of an antidepressant drug to the subject to ameliorate at least one symptom of MDD if the presence of autoantibodies to FRa is detected or determined.

[0010] In some aspects, the sample is selected from the group of whole blood, serum, plasma, urine, saliva and cerebral spinal fluid (CSF). In some embodiments, the sample is a serum sample. In other embodiments, the sample is a cerebral spinal fluid (CSF) sample. [0011] In some aspects, the subject has lower than normal levels of MTHF in CSF. The subject may have normal levels of MTHF in whole blood.

[0012] In some aspects, the subject is resistant to at least one antidepressant drug, which can be, for example, a selective serotonin reuptake inhibitor (SSRI) or selective

norepinephrine reuptake inhibitor (S RI). [0013] In some aspects, the autoantibodies to folate receptor alpha (FRa) comprise blocking autoantibodies and/or binding autoantibodies. The binding autoantibodies can be IgG antibodies and/or IgM antibodies against FRa. [0014] The level or amount of autoantibodies can be in a range of from about 0 to 74 pg/ml in the sample. In some instances, the level or amount of autoantibodies can be in a range of from about 75 to 160 pg/ml in the sample. In other instances, the level or amount of autoantibodies is in a range of from about 161 to 260 pg/ml in the sample. In yet other instances, the level or amount of autoantibodies is in a range of from about 261 to 742 pg/ml in the sample.

[0015] In some aspects, the method further comprises treating the subject with a folate containing formulation and an antidepressant drug. The antidepressant drug can be a selective serotonin reuptake inhibitor (SSRI) or selective norepinephrine reuptake inhibitor (S RI).

[0016] In some aspects, the presence or level of autoantibodies is correlated to the

HAMD28 scale. The presence of blocking and/or binding autoantibodies can correlate to a HAMD28 score that indicates depression, e.g., severe to very severe depression, or major depressive disorder. [0017] In yet another embodiment, the present invention provides a method for aiding in the diagnosis of a risk for mild cognitive impairment (MCI) or Alzheimer' s disease and related dementias (ADRD) in a subject. The method comprises detecting or quantitating autoantibodies to folate receptor alpha (FRa) in a sample from the subject; and diagnosing the subject as having a risk for MCI or ADRD by the presence of autoantibodies to folate receptor alpha (FRa). In some aspects, detecting or quantitating comprises performing an immunoassay with a folate receptor as an antigen. In other aspects, detecting or quantitating comprises using a homogeneous mobility shift assay (HMSA).

[0018] In still yet another embodiment, the present invention provides a method for treating a subject having MCI. The method comprises detecting or quantitating autoantibodies to folate receptor alpha (FRa) in a sample from the subject; and administering a treatment comprising a therapeutically effective amount of a folate containing formulation to the subject to ameliorate at least one symptom of MCI if the presence of autoantibodies to FRa is determined.

[0019] In some aspects, the sample is a member selected from the group of whole blood, serum, plasma, urine, saliva and cerebral spinal fluid (CSF). In some embodiments, the sample is a serum sample. In other embodiments, the sample is a cerebral spinal fluid (CSF) sample. [0020] In some aspects, the subject has lower than normal levels of MTHF in CSF. In some cases, the subject can have normal levels of MTHF in whole blood. The level of MTHF can be determined according to any method recognized by those of ordinary skill in the art. [0021] In some aspects, the autoantibodies to folate receptor alpha (FRa) comprise blocking autoantibodies and/or binding autoantibodies. The binding autoantibodies can be IgG autoantibodies and/or IgM autoantibodies.

[0022] The level or amount of autoantibodies can be in a range of from about 0 to 74 pg/ml in the sample. In some instances, the level or amount of autoantibodies can be in a range of from about 75 to 160 pg/ml in the sample. In other instances, the level or amount of autoantibodies is in a range of from about 161 to 260 pg/ml in the sample. In yet other instances, the level or amount of autoantibodies is in a range of from about 261 to 742 pg/ml in the sample.

[0023] In some aspects, the method further comprises treating the subject with a folate containing formulation. In other aspects, the method further comprises treating the subject with a folate containing formulation that includes vitamin B _i2 and other B-vitamins, and an intracellular antioxidant such as N-acetyl-cysteine and a drug such as an acetylcholinesterase inhibitor and/or memantine (e.g., an NMD A antagonist drug).

[0024] In some aspects, the presence or level of autoantibodies is correlated to a cognitive or memory test scale.

[0025] In some aspects, the treatment further comprises a therapeutic effective amount of an acetylcholinesterase inhibitor or memantine drug. In other aspects, the treatment further comprises a therapeutic effective amount of an acetylcholinesterase inhibitor and a memantine drug (e.g., an NMDA antagonist drug). [0026] In some aspects, the sample is a member selected from the group of whole blood, serum, plasma, urine, saliva and cerebral spinal fluid (CSF). In some embodiments, the sample is a serum sample. In other embodiments, the sample is a cerebral spinal fluid (CSF) sample.

[0027] In some aspects, the autoantibodies to folate receptor alpha (FRa) comprise blocking autoantibodies and/or binding autoantibodies. The binding autoantibodies can be IgG antibodies and/or IgM antibodies. In some aspects, detecting or quantitating comprises performing an immunoassay with a folate receptor as an antigen. In other aspects, detecting or quantitating comprises using a homogeneous mobility shift assay (HMSA).

[0028] In still yet another embodiment, the present invention provides a method for treating a subject having ADRD, the method comprising: detecting or quantitating autoantibodies to folate receptor alpha (FRa) in a sample from the subject; and administering a treatment comprising a therapeutically effective amount of a folate containing compound to the subject to ameliorate at least one symptom of ADRD if the presence of autoantibodies to FRa is determined.

[0029] In some aspects, the treatment further comprises a therapeutic effective amount of an acetylcholinesterase inhibitor or memantine drug (e.g., an MDA antagonist drug). In other aspects, the treatment further comprises a therapeutic effective amount of an acetylcholinesterase inhibitor and a memantine drug.

[0030] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows the distribution of propensity scores in age and gender matched TRD cases with controls.

[0032] FIGS. 2A-2C provide analysis results for the TRD study cases. FIG. 2A shows matching cases and controls in six sub-classes based on the propensity scores. FIG. 2B shows the full data of the Chi-square test for deviance to detect effect-modification. FIG. 2C shows the matched data of the Chi-square test for deviance to detect effect-modification.

[0033] FIG. 3 shows the distribution of propensity scores in age and gender matched NAC cases with controls.

[0034] FIGS. 4A-4C provide analysis results for the NAC study cases. FIG. 4A shows matching cases and controls in six sub-classes based on the propensity scores. FIG. 4B shows the full data of the Chi-square test for deviance to detect effect-modification. FIG. 4C shows the matched data of the Chi-square test for deviance to detect effect-modification. DETAILED DESCRIPTION OF THE INVENTION I. INTRODUCTION

[0035] The present invention is based, in-part, on the surprising discovery of molecular markers of major depressive disorder, mild cognitive impairment and Alzheimer' s disease and other disorders. Provided herein are methods for diagnosing MDD or MCI or ADRD or CFD based on the presence or level of circulating autoantibodies against folate receptor a (FRa) in a sample, such as a serum sample from a subject. Without being bound to a particular theory, it is believed that individuals with MDD or MCI or ADRD or CFD can develop or have blocking and/or binding autoantibodies to FRa. These autoantibodies are polyclonal and may be blocking autoantibodies, binding autoantibodies, or both. The autoantibodies can interfere with folate transport across the blood-brain barrier. These biomarkers are indicative of MDD or MCI or ADRD or CFD. In certain instances, the autoantibodies are correlated to man-made disease scoring scales or indexes such as the HAMD scale, and cognitive or memory test scales. In certain aspects, the methods described herein include features that are significantly more than correlating a nature-based principle and a disease state.

[0036] In certain other instances, the L-5-methyltetrahydrofolate dose is tailored for patients suffering from depression, schizophrenia, MCI, MDD, ADRD or CFD who need higher L-5-methyltetrahydrofolate intake caused by genetic polymorphisms (like for instance MTHFR C677T) or due to blocked L-5-methyltetrahydrofolate passage at the blood-brain barrier. Dose escalation of L-5-methyltetrahydrofolate is warranted if there are confounding effects such as the severity of the indication, polymorphisms or the amount and type of autoantibodies. In certain aspects, dose escalation is warranted if blocked folate transfer across the blood-brain barrier is due to FRa-autoantibodies, or a patient history of bad food habits or diseases leading to nutritional deficiencies amongst others systemic folate deficiency (like fast food overconsumption, alcoholism, hidden celiac disease, and the like).

[0037] In some instances, a subject carrying one or more polymorphisms associated with brain folate deficiency (e.g., MTHFR C677T) may require a dose of L-methylfolate that is about 2 to 4 times higher than a subject without the polymorphism(s). In other instances, a subject carrying one or more polymorphisms associated with brain folate deficiency (e.g., MTHFR C677T) also has autoantibodies against FRa, then an even higher dose of L- methylfolate should be administered to the subject to impact brain folate levels. In some cases, subjects with the autoantibodies may have unmetabolized folic acid.

[0038] It is believed that at physiological L-5-methyltetrahydrofolate concentrations in the nanomolar range, L-5-methyltetrahydrofolate will bind to the FRa attached to choroid epithelial cells. Whereas at high serum concentrations within the micromolar range, induced by pharmacologic supplement administration, the low-affinity high capacity transporter RFCl will also be enabled to transport methylfolate to the CNS across the blood-brain barrier. In CFD syndromes due to circulating FRa autoantibodies, membrane attached FRa antigen can complex with these antibodies and the natural route of FRa-mediated transport is blocked. Thus, transport of methylfolate across the blood-brain barrier via RFCl occurs at plasma concentrations significantly above the physiological nanomolar range. As such, administration of high doses of 5-methyltetrahydrofolate can achieve the principle of therapy for CFD syndromes due to FRa-autoantibodies or due to FRa gene mutations wherein higher doses are needed. [0039] In certain instances, the presence and or titer of the autoantibodies assist or aid in the diagnostic of a variety of diseases. Further, the methods of treating is influenced by the presence of the autoantibodies and other factors or conditions that affect folate brain levels, such as for example, one-carbon polymorphisms (e.g., MTHFR 677C→T or 1298A→C or others). In certain instances, unmetabolized folic acid can also be an indicator. [0040] In some instances, a patient needs more folate to reach the brain (and effectively be part of the normal treatment regimen) when autoantibodies are present, when unmetabolized folic acid is present then when just dealing with a genetic polymorphism alone. In effect, detection, presence or levels of autoantibodies, unmetabolized folic acid and polymorphisms help increase diagnosis and prognosis of a variety of folate-associated diseases. [0041] The methods are useful in a variety of indications including, but not limited to, neuropsychiatric and neurological diseases and disorders (e.g. autism, schizophrenia) hearing loss (noise induced or age-related) and tinnitus, macular degeneration, visual disturbances, anosmia, migraine, headache, inflammation linked diseases, myelination related diseases (e.g., multiple sclerosis), epilepsy and seizures in general, sleep disturbances, insomnia, irritability associated with neuropsychiatric disorders, metabolic diseases, including diabetic peripheral neuropathy and neuropathy in general, renal diseases, lung diseases, and or thyroid diseases. II. DEFINITIONS

[0042] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0043] The terms "a," "an," or "the" as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth. [0044] The term "depression" includes a mental state of depressed mood characterized by feelings of sadness, despair and discouragement. In some instances, depression is a clinical symptom, and can include, but not limited to, major depressive disorder (including single episode and recurrent), unipolar depression, treatment-refractory depression, resistant depression, anxious depression and dysthymia (also referred to as dysthymic disorder).

Further, the term "depression" can encompass any major depressive disorder, dysthymic disorder, mood disorders due to medical conditions with depressive features, mood disorders due to medical conditions with major depressive-like episodes, substance-induced mood disorders with depressive features and depressive disorder not otherwise specific as defined by their diagnostic criteria, as listed in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) or any later edition thereof, or the World Health Organization's International Statistical Classification of Diseases and Related Health Problems (ICD-10).

[0045] The term "major depressive disorder" or "MDD" includes a mental/mood condition characterized by two or more major depressive episodes. Symptoms of MDD may include fatigue, feelings of worthlessness or guilt, impaired concentration or indecisiveness, insomnia or hypersomnia, markedly diminished interest or pleasure in almost all activities, restlessness, recurring thoughts of death or suicide, and significant weight loss or gain (5% weight change). Diagnostic criteria for MDD and depressive episodes are list in the DSM-IV and are useful for assessing a subject. [0046] Generally, depression is evaluated by a clinician using, e.g., the criteria listed in the DSM-IV or efficacy measures (neuropsychological assessments) such as the Hamilton Depression Rating Scale (HAMD-28, HAMD-21 or HAMD-7), the Clinical Global Impression (CGI) Scale, the Montgomery -Asberg Depression Rating Scale (MADRS), the Beck Depression Inventory (BDI), the Zung Self-Rating Depression Scale, the Wechsler Depression Rating Scale, the Raskin Depression Rating Scale, the Inventory of Depressive Symptomatology (IDS), and the Quick Inventory of Depressive Symptomatology (QIDS). For example, measurable lessening of depression (e.g., clinical improvement) includes any clinically significant decline in a measurable marker or symptom, such as measuring markers for depression in the blood or CSF e.g., red blood cell folate, serum folate, serum MTHF, or assessing the degree of depression, e.g., using a neuropsychological assessment.

[0047] For example, a score of 0-7 on HAMD-7 is typically considered to be normal. Scores of 20 or higher indicate moderate, severe, or very severe depression. Questions 18-21 may be recorded to give further information about the depression (such as whether diurnal 1 variation or paranoid symptoms are present), but are not necessary part of the scale. Thus, a reduction of symptoms can be considered clinically relevant if, e.g., the HAMD score is decreased to under, e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6. [0048] The term "mild cognitive impairment" or "MCI" includes a brain function syndrome or condition that is characterized by symptoms including problems with memory, language, thinking and judgment that are greater than normal age-related changes. In most cases, mild cognitive impairment does not significantly impact daily functioning of the individual. Tests such as the mental examinations, e.g., Mini-Mental State Examination (MMSE), neurological examinations, neuropsychological evaluations, and evaluations of medical history can be performed by a clinician to assess a subject and aid in the evaluation of possible MCI. There may be a higher likelihood of MCI to progress to an Alzheimer' s disease-related disorder.

[0049] The term "Alzheimer' s disease and related disorders," "Alzheimer' s disease and related dementia" or " ADRD" includes debilitating conditions that impair memory, thought processes, and functioning, primarily among older adults. Alzheimer's disease (AD) is an age-related, non-reversible brain disorder that develops over a period of years. The symptoms of AD gradually lead to behavior and personality changes, a decline in cognitive abilities such as decision-making and language skills, and problems recognizing family and friends. AD ultimately leads to a severe loss of mental function or dementia. These losses are related to the worsening breakdown of the connections between certain neurons in the brain and their eventual death. Cognitive, functional, global and caregiver based assessments of Alzheimer' s disease and related disorders can performed to evaluate an individual' s overall condition. For example, useful cognitive assessments include the Alzheimer's Disease Assessment Scale, cognitive subsection (ADAS-cog), Blessed information-memory- concentration test (BIMC), clinical dementia rating scale (CDR), and mini-mental state examination (MMSE). Useful function assessment tools include, but are not limited to, the function assessment questionnaire (FAQ), instrumental activity of daily living (IADL), physical self-maintenance scale (PSMS), and progressive deterioration scale (PDS). Non- limiting global assessment tools include the clinical global impression of change (CGIC), clinical interview-based impression (CIBI) and global deterioration scale (GDS). Useful caregiver-based assessments include the behavioral pathology in Alzheimer' s disease rating scale (BEHAVE-AD) and neuropsychiatry inventory (NPI). Additional methods for assessing cognitive impairment in patients suspected of having Alzheimer' s disease are described, for example, in Cordell et al., Alzheimer' s & Dementia, 2013, 9: 141-150.

[0050] The term "acetylcholinesterase inhibitor" or "AChEI" is used herein to refer to a drug that enhances function of cholinergic neurons by inhibiting the catabolic enzyme acetylcholinesterase (AChE). The term encompasses reversible, pseudo-reversible and irreversible AChEIs as well as AChEIs that selectively inhibit AChE, and AChEIs, that are less selective (e.g., also target butyrylcholinesterase, BuChE). Preferably, AChEIs useful in the methods of the present invention are reversible or pseudo-reversible. Specific examples of AChEIs useful in the methods and compositions of the present invention include, but are not limited to, tacrine (THA; l,2,3,4-tetrahydro-9-aminoacridine hydrochloride), donepezil, galantamine, rivastigmine, huperzine A, zanapezil, ganstigmine, phenserine,

phenethylnorcymserine (PENC), cymserine, thiacymserine, SPH 1371 (galantamine plus), ER 127528, RS 1259, and F3796. [0051] The term "NMDA antagonist drugs" is used to refer to drugs, that can suppress the normal triggering of NMDA receptor-mediated neuronal firings. Preferred NMDA antagonist drugs of the invention are 1-aminocyclohexane derivatives such as memantine and neramexane. These compounds also have 5HT ₃ antagonist activity and/or neuronal nicotinic receptor antagonist activity. [0052] The term "sample" includes any biological specimen obtained from an individual. Suitable samples for use in the present invention include, without limitation, whole blood, plasma, serum, cerebrospinal fluid, saliva, urine, stool, tears, any other bodily fluid, tissue samples (e.g., biopsy), and cellular extracts thereof (e.g., red blood cellular extract). In a preferred embodiment, the sample is a blood, plasma, serum, urine, saliva or cerebrospinal fluid (CSF) sample. The use of samples such as plasma, serum, saliva, urine and CSF is well known in the art (see, e.g., Hashida et al., J. Clin. Lab. Anal., 1 1 :267-86 (1997)). One skilled in the art will appreciate that samples such as whole blood, plasma, serum, saliva, urine or CSF samples can be diluted prior to the analysis of marker levels.

[0053] The term "marker" or "biomarker" includes any biochemical marker, serological marker, protein markers, genetic marker, metabolic markers, or other clinical characteristic that can be used in the selection of therapy for depression, in the prediction of the probable course and outcome of depression, and/or in the prediction of the likelihood of recovery from the disorder. In some aspects, the markers are utilized in combination with one or more (e.g., a plurality of) statistical analyses to aid or provide a prognosis of depression in an individual. In other aspects, the markers are used to aid or provide a prediction of therapeutic response to a treatment (e.g., antidepressant drug monotherapy) in an individual diagnosed with depression (e.g., major depressive disorder).

[0054] The present invention relies, in part, on determining the presence (or absence) or level (e.g., concentration) of an autoantibody against folate receptor a in a sample obtained from an individual. As used herein, the term "detecting the presence of an autoantibody" includes determining the presence of each marker of interest by using any quantitative or qualitative assay known to one of skill in the art. In certain instances, qualitative assays that determine the presence or absence of a particular trait, variable, genotype, and/or biochemical or serological substance (e.g., protein or antibody) are suitable for detecting each marker of interest. In certain other instances, quantitative assays that determine the presence or absence of protein, antibody, or activity are suitable for detecting each marker of interest. As used herein, the term "detecting the level of an autoantibody" includes determining the level of each marker of interest by using any direct or indirect quantitative assay known to one of skill in the art. In certain instances, quantitative assays that determine, for example, the relative or absolute amount of protein, antibody, or activity are suitable for detecting the level of each marker of interest. One skilled in the art will appreciate that any assay useful for detecting the level of a marker is also useful for detecting the presence or absence of the marker. [0055] The term "individual," "subject," or "patient" are used interchangeably and typically includes humans, but also includes other animals such as, e.g., other primates, rodents, canines, felines, equines, ovines, porcines, and the like.

[0056] The term "antidepressant" or "antidepressant drug" includes any pharmaceutical agent which treats depression. In some aspects, the antidepressant drug administered to the subject in accordance with the methods described herein can be any conventional

pharmaceutical agent which is commonly indicated for treating depression. Examples of antidepressant drugs include, but are not limited to, selective serotonin reuptake inhibitors (SSRIs), serotonin and dopamine reuptake inhibitors (SDRIs), serotonin-norepinephrine reuptake inhibitors (S RIs), serotonin-noradrenaline-dopamine reuptake inhibitors

(S DRIs), norepinephrine-dopamine reuptake inhibitors ( DRIs), norepinephrine

(noradrenaline) reuptake inhibitors (NRIs), monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake enhancers (SSREs), melatonergic agonists, tryptamines, tricyclic antidepressants, and atypical anti-depressants. [0057] The term "drug monotherapy" includes a treatment regimen that includes the use of one therapeutic agent (e.g., drug) during the course of therapy. The agent can be

administered once or repeatedly during the course of therapy.

[0058] The term "adjunctive therapy" includes a treatment regimen that includes another therapeutic agent (e.g., drug) used together with another therapeutic agent (e.g., drug) during the course of therapy. The adjunctive agent can be administered once or repeatedly during the course of therapy and given simultaneously or subsequently to the primary agent.

[0059] The terms "treatment" and "treating" as used herein, with respect to treatment of a disease, refers to preventing the progression of the disease, altering the course of the disorder (for example, but are not limited to, slowing the progression of the disorder), partially reversing a symptom of the disorder or reducing one or more symptoms and/or one or more biochemical markers in a subject, preventing one or more symptoms from worsening or progressing, promoting recovery, and/or improving prognosis.

[0060] The terms "effective amount," "amount effective" and "therapeutically effective amount" include an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, these terms include an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a "prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition.

[0061] The term "memory test scale" or "cognitive or memory test scale" includes a neuropsychological test designed to measure different cognitive or memory functions in a subject. Non-limiting examples of cognitive or memory tests useful for diagnosing MCI include the Williams Scale for the Measurement of Memory, Wechsler Memory Scale, Rivermead Behavior Memory Test, Wechsler Adult Intelligence Scale, Mattice Dementia Rating Scale (DRS), the Everyday Memory Questionnaire, Free and Cued and Selective Reminding Test, Rey Auditory Verbal Learning Test, California Verbal Learning Test, Hopkins Verbal Learning Test (HVLT), Trail Making test, Boston Naming Test, Stroop Color Word Test, Cambridge Neuropsychological Test Automated Battery (CANTAB), Motor Control Task (MT) test, Pattern Recognition Memory (PRM) test, Paired Associates Learning (PAL) test, Spatial Working Memory (SWM) test, and other tests to evaluate episodic memory (i.e., the ability to learn and retain new information), executive functions (e.g., set-shifting, reasoning, problem-solving, and planning), language (e.g., naming, fluency, expressive speech and comprehension), visuospatial skills, and/or attention control.

[0062] Cerebral folate deficiency (CFD) syndrome is a neurodevelopmental disorder typically caused by folate receptor autoantibodies that interfere with folate transport across the blood-brain barrier. In cerebral folate deficiency the serum or plasma blood levels of folate are typically normal, but a sample of cerebral spinal fluid indicates decreased levels of folate.

III. DETAILED DESCRIPTIONS OF EMBODIMENTS

A. Autoantibodies Against Folate Receptor a (FRA)

[0063] In some embodiments, the present invention provides methods comprising measuring the presence (or absence) or level of autoantibodies to FRa in a sample, e.g., blood, serum, plasma, urine, saliva or CSF sample, from a subject. The subject can be a human subject. In some embodiments, the subject is suspected of or has MDD. In some instances, the subject has one or more MTHFR S Ps (e.g., one or more MTHFR C677T mutations). Some cases, the subject has impaired or blocked folate transport at the blood- brain barrier. In other embodiments, the subject is suspected of or has MCI. In yet other embodiments, the subject is suspected of or has ADRD. The subject can have or is suspected of having cerebral folate deficiency syndrome, or the subject can have or is suspected of having sub-normal cerebral folate level e.g., a neurological syndrome or condition characterized by having lower levels of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid and normal levels of folate in the blood and plasma. In some

embodiments, the subject has normal folate levels in serum, plasma and/or whole blood compared to a healthy subject, yet has a lower than normal level of 5-methyltetrahydrofolate (5MTHF) in cerebrospinal fluid. [0064] In certain instances, the serum autoantibody titer can be used to predict the CSF methylfolate level. The level of CSF methylfolate can then be used to administer the necessary dose of methylfolate. In certain instances, the CSF methylfolate levels are stratified depending on age, sex, indication and combinations thereof.

[0065] The method provided herein includes measuring the presence or level of blocking autoantibody, the presence or level of binding IgG autoantibody, the presence or level of binding IgM autoantibody, and any combination thereof. Levels of autoantibodies to FRa can be detected by any known methods in the art. Autoantibodies to FRa can be detected using immunoassays as described in, e.g., Ramaekers et al, N Eng J Med, 352: 1985-91, 2005, Molloy et al., N Engl J Med, 361(2): 152-160, 2009, U.S. Pat. No. 7,846,672 and U.S. Pat. App. No. 2010/0179073, which are incorporated herein by reference in their entirety for all purposes. In an ELISA based assay, recombinant or purified folate receptor a or apo-FRa (i.e., FRa without bound folate) can be coated onto a plate and the test sample (serum or plasma) can be added and incubated such that autoantibodies to FRa present in the sample complex with the bound FRa. Subsequently, the complexed autoantibodies are detected using a detectable, labeled secondary antibody against human IgG or IgM and, in some instances, a colorimetric reaction. The test sample can be pre-treated to remove endogenous folate, such as by treating with acid/charcoal.

[0066] Alternatively, autoantibodies against FRa can be detected using a homogenous mobility shift assay (HMSA). Autoantibodies against FRa can be detected by using fluorescently labeled folate receptor and FIPLC on a size exclusion column. The presence of complexes comprising labeled FRa and FRaAuAb can be monitored by fluorescence and compared to free labeled FRa.

[0067] Autoantibodies against FRa can be detected using a homogenous mobility shift assay (FEVISA) as disclosed in U.S. Patent No. 8,574,855, U. S. Patent Pub. No.

2013/0344621 and U.S. Patent Appl. Pub. No. 2014/0045276. In one instance, the present invention provides a method for determining the presence or level of an autoantibody to FRa in a sample, the method comprising: (a) contacting a fluorescently labeled folate receptor with the sample to form a labeled complex with the autoantibody; (b) subjecting the labeled complex to size exclusion chromatography to separate the labeled complex from free labeled folate receptor and to detect an amount of the labeled complex and an amount of free labeled folate receptor; and (c) comparing the amount of the labeled complex and the amount of the free labeled folate receptor detected in step (b) to a standard curve of known amounts of the autoantibody, thereby determining the presence or level of the autoantibody.

[0068] In some instances, mass spectrometry (MS) can be used, e.g., the biological sample is infused directly into the mass spectrometer which provides both separation and detection of autoantibodies to FRa. In some instances, the autoantibodies to FRa can be optionally separated (e.g., prior to detection) from a biological sample by gas chromatography (GC), e.g., when interfaced with mass spectrometry (GC-MS), and/or high performance liquid chromatography (HPLC), and/or capillary electrophoresis (CE).

[0069] Other useful methods for detecting an autoantibody include nanostructure-initiator mass spectrometry, laser-desorption/ionization mass spectrometry, e.g., matrix assisted laser desorption/ionization (MALDI) mass spectrometry, surface-enhanced laser

desorption/ionization (SELDI) mass spectrometry, secondary ion mass spectrometry (SIMS), desorption electrospray ionization (DESI) mass spectrometry, nuclear magnetic resonance ( MR) spectroscopy, on-mobility spectrometry, electrochemical detection (coupled to HPLC) and radiolabel (when combined with thin-layer chromatography), mass spectrometry such as MALDI/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass

spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.).

[0070] Blocking autoantibodies to FRa refer to autoantibodies that block (e.g., sterically hinder or prevent) the binding of folic acid or folate to folate receptor a. Methods for detecting blocking autoantibodies include an assay that uses radiolabeled folic acid. The assay can include the steps of incubating the individual's sample, e.g., serum, that is free of folate with solubilized, purified folate receptors and adding radiolabeled folic acid, e.g., [ ³ H]folic acid. If the blocking autoantibodies are present in the sample, [ ³ H]folic acid will be prevented from binding the folate receptor. In some cases, the method comprises incubating the sample with solubilized apo-folate receptor and [ ³ H]folic acid such that the folic acid can bind to the folate receptor, removing free [ ³ H]folic acid by adsorption to dextran-coated charcoal, and measuring receptor-bound radioactivity in the supernatant fraction. The amount of blocking autoantibody can be determined because it is inversely proportional to the amount of radioactivity bound to the receptor. An exemplary embodiment of a method of measuring the level of blocking autoantibodies to FRa is found in, e.g., Ramaekers et al, N Eng J Med, 352: 1985-91, 2005.

[0071] Binding autoantibodies to FRa refers to autoantibodies that specifically bind to folate receptor a. In other words, a FRa epitope is recognized by a binding autoantibody against FRa. An exemplary embodiment of a method for measuring the level of binding autoantibodies is an ELISA assay using purified or recombinant folate receptors as the immobilized antigen. Secondary antibodies known to those of ordinary skill in the art can be used to determine whether the autoantibody is an IgG or IgM antibody type.

[0072] In some aspects, the level or amount of autoantibodies detected in an individual' s sample is from about 0 to about 1000 pg/ml, e.g., about 0 pg/ml-about 900 pg/ml, about 50 pg/ml-about 1000 pg/ml, about 0 pg/ml-about 100 pg/ml, about 0 pg/ml-about 74 pg/ml, about 75 pg/ml-about 160 pg/ml, about 161 pg/ml-about 260 pg/ml, about 261 pg/ml-about 1000 pg/ml, about 0 pg/ml-about 250 pg/ml, about 251 pg/ml-about 500 pg/ml, or about 501 pg/ml-about 1000 pg/ml. B. Predicting Diagnosis of MDD or MCI or ADRD or CFD

[0073] Provided herein are methods for diagnosing MDD or MCI or ADRD or CFD in a subject by measuring the level of blocking and/or binding autoantibodies to FRa in a sample taken from the subject. The subject may be suspected of having MDD or MCI or ADRD or CFD. If the presence of blocking and/or binding (e.g., IgG binding autoantibodies and/or IgM binding autoantibodies) is determined, the subject is determined to have MDD or MCI or ADRD or CFD.

[0074] In some cases, the presence or level of autoantibodies is correlated to a depression criteria or scale. As an example, the level of autoantibodies to FRa can correspond to a value or value range on a depression scale such as the HAMD-28 scale and the like. In some cases, the level of autoantibodies represents a score indicating major depressive disorder.

[0075] In some cases, the presence or level of autoantibodies is correlated to a cognitive or memory test scale. For example, the level of autoantibodies determined using the method provided herein can represent a value or value range corresponding to MCI on a cognitive or memory test scale. In other cases, the level of autoantibodies can represent a value or value range corresponding to ADRD on a cognitive or memory test scale.

[0076] The present invention is based, in part, on the surprising discovery that subjects with MDD or MCI or ADRD have a statistically higher probability or incidence of having blocking and/or binding autoantibodies to FRa compared to normal subjects who do not have MDD or MCI or ADRD. For instance, the odds of a subject with MDD having blocking autoantibodies is significantly higher than for a normal subject (odds ratio (OR)=2.16; 95% confidence interval (CI): 1.02-4.66; p-value=0.004). The odds of a MDD subject having IgM autoantibodies is significantly higher than for a normal subject (OR=4.21 ; 95% CI: 1.98-9.46; p-value=0.001). In contrast, the odds of a MDD subject having IgG autoantibodies is not significantly different than for a normal subject (OR=1.28; 95% CI: 0.6-2.73; p-value=0.518). The odds of a MDD subject having either blocking autoantibodies or binding autoantibodies (e.g., IgG or IgM autoantibodies) is significantly higher than for a normal subject (OR=3.24; 95% CI: 1.70-6.33; p-value<0.001). The test subject and normal (control) subject can be age- matched and gender-matched. [0077] In some cases, the odds of a subject with cognitive impairments such as MCI or ADRD having binding IgG autoantibodies is significantly higher than for a normal subject (OR=3.16; 95% CI: 1.54-6.68; p-value=0.002). Also, the odds of a subject with cognitive impairments having binding IgM autoantibodies is significantly higher than for a normal subject (OR=6.73; 95% CI: 2.94-16.34; p-value<0.001). In contrast, the odds of a subject with cognitive impairments having blocking autoantibodies is not different than for a normal subject (OR=0.88; 95% CI: 0.37-2.09; p-value=0.772). The odds of a cognitively impaired subject having either blocking autoantibodies or binding autoantibodies (e.g., IgG or IgM autoantibodies) is significantly higher than for a normal subject (OR=2.58; 95% CI: 1.32- 5.12; p-value=0.006). The test subject and normal (control) subject can be age-matched and gender-matched.

[0078] In some embodiments, the method for aiding the diagnosis of MDD or MCI or ADRD includes using a statistical algorithm to process the information obtained from detecting or quantitating the presence (or absence) or level of the autoantibodies. In some instances, the statistical algorithms independently comprise one or more learning statistical classifier systems. In particular embodiments, statistical algorithms advantageously provide improved sensitivity, specificity, negative predictive value, positive predictive value, and/or overall accuracy for diagnosing MDD or MCI or ADRD.

[0079] The term "statistical algorithm" or "statistical process" includes any of a variety of statistical analyses used to determine relationships between variables. The variables can be the presence or level of at least one marker of interest and/or the assessment of at least one psychological measure. Any number of markers and/or psychological measures can be analyzed using a statistical algorithm described herein. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more biomarkers and/or psychological measures can be included in a statistical algorithm. In one embodiment, logistic regression is used. In another embodiment, linear regression is used. In certain instances, the statistical algorithms of the present invention can use a quantile measurement of a particular marker within a given population as a variable. Quantiles are a set of "cut points" that divide a sample of data into groups containing (as far as possible) equal numbers of observations. For example, quartiles are values that divide a sample of data into four groups containing (as far as possible) equal numbers of observations. The lower quartile is the data value a quarter way up through the ordered data set; the upper quartile is the data value a quarter way down through the ordered data set. Quintiles are values that divide a sample of data into five groups containing (as far as possible) equal numbers of observations. The present invention can also include the use of percentile ranges of marker levels (e.g., tertiles, quartile, quintiles, etc.), or their cumulative indices (e.g., quartile sums of marker levels, etc.) as variables in the algorithms (just as with continuous variables).

[0080] In certain embodiments, the present invention involves detecting or determining the presence, absence or level of autoantibodies using quartile analysis. In this type of statistical analysis, the level of autoantibodies is defined as being in the first quartile (<25%), second quartile (25-50%), third quartile (51%-<75%), or fourth quartile (75-100%) in relation to a reference database of samples. These quartiles may be assigned a quartile score of 1, 2, 3, and 4, respectively. In certain instances, autoantibodies that are not detected in a sample are assigned a quartile score of 0 or 1, while autoantibodies that are detected (e.g., present) in a sample (e.g., sample is positive for the molecular marker) is assigned a quartile score of 4. In some embodiments, quartile 1 represents samples with the lowest autoantibody levels, while quartile 4 represent samples with the highest autoantibody levels. The reference database of samples can include a large spectrum of patients with MDD, MCI or ADRD. From such a database, quartile cut-offs can be established. [0081] Statistical algorithms (e.g., learning statistical classifier systems) can be trained and tested using a cohort of samples (e.g., serological samples) from healthy individuals, MDD patients, non-MDD patients, MCI patients, non-MCI patients, AD patients, non-AD patients, ADRD patients and/or non-ADRD patients. Samples from patients diagnosed with MDD can be stratified according to HAMD-28 score. Samples from patients diagnosed with MDD using a published criteria such as the HAMD-28 diagnostic criteria are suitable for use in training and testing the statistical algorithms described herein. Samples from healthy individuals can include those that were not identified as MDD, MCI, ADRD or AD samples. One skilled in the art will know of additional techniques and diagnostic criteria for obtaining a cohort of patient samples that can be used in training and testing the statistical algorithms described herein.

[0082] The term "sensitivity" refers to the probability that a method, system, or code of the invention gives a positive result when the sample is positive, e.g., having MDD, MCI or ADRD. Sensitivity is calculated as the number of true positive results divided by the sum of the true positives and false negatives. Sensitivity essentially is a measure of how well a method, system, or code of the invention correctly identifies those with MDD, MCI or

ADRD from those without the disease. The statistical algorithms can be selected such that the sensitivity is at least about 60%, and can be, for example, at least about 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0083] The term "specificity" refers to the probability that a method, system, or code of the invention gives a negative result when the sample is not positive, e.g., not having MDD, MCI or ADRD. Specificity is calculated as the number of true negative results divided by the sum of the true negatives and false positives. Specificity essentially is a measure of how well a method, system, or code of the invention excludes those who do not have MDD, MCI or ADRD from those who have the disease. The statistical algorithms can be selected such that the specificity is at least about 70%, for example, at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0084] The term "negative predictive value" or "NPV" refers to the probability that an individual identified as not having MDD, MCI or ADRD actually does not have the disease. Negative predictive value can be calculated as the number of true negatives divided by the sum of the true negatives and false negatives. Negative predictive value is determined by the characteristics of the method, system, or code as well as the prevalence of the disease in the population analyzed. The statistical algorithms can be selected such that the negative predictive value in a population having a disease prevalence is in the range of about 70% to about 99% and can be, for example, at least about 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0085] The term "positive predictive value" or "PPV" refers to the probability that an individual identified as having MDD, MCI or ADRD actually has the disease. Positive predictive value can be calculated as the number of true positives divided by the sum of the true positives and false positives. Positive predictive value is determined by the

characteristics of the method, system, or code as well as the prevalence of the disease in the population analyzed. The statistical algorithms can be selected such that the positive predictive value in a population having a disease prevalence is in the range of about 80% to about 99% and can be, for example, at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. [0086] Predictive values, including negative and positive predictive values, are influenced by the prevalence of the disease in the population analyzed. In the methods, systems, and code of the invention, the statistical algorithms can be selected to produce a desired clinical parameter for a clinical population with a prevalence of MDD, MCI or ADRD. For example, statistical algorithms can be selected for MDD, MCI or ADRD prevalence of up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%), 60%), 65%), or 70%, which can be seen, e.g., in a clinician's office such as a

psychiatrist's, neurologist's or general practitioner's office.

[0087] The presence (or absence) or level of autoantibodies can be substantially equal to or similar to a control subject who has MDD. In certain instances, the control subject who has MDD is diagnosed using a Hamilton Depression Rating Scale or memory test. In these control subjects, the amount of autoantibody is measure and a standard curve generated. If the control subject with MDD and the test subject have similar levels of autoantibodies against FRa or the test subject has higher levels of autoantibodies than the control subject, and similar scores in the respective depression or cognitive scales, it is determined that the test subject has MDD. In some aspects, the level of autoantibodies is correlated to a scoring scale for MDD, such as HAMD-7, HAMD-17, HAMD-21, HAMD-28 and similar scoring scales. An algorithm or mathematical computation can be used to assign a specific level of autoantibodies to a particular score on a MDD assessment scale.

[0088] Similarly, the level of autoantibodies in patients with MCI or ADRD can be correlated to a scoring scale such as cognitive testing scales. Non-limiting examples of cognitive or memory tests include the Free and Cued and Selective Reminding Test, the Rey Auditory Verbal Learning Test, the California Verbal Learning Test, the Hopkins Verbal Learning Test (HVLT), the Wechsler Memory Scale, the Trail Making test, the Boston Naming Test, the Cambridge Neuropsychological Test Automated Battery (CANTAB), the Motor Control Task (MT) test, the Pattern Recognition Memory (PRM) test, the Paired Associates Learning (PAL) test, the Spatial Working Memory (SWM) test, and other tests to evaluate episodic memory (i.e., the ability to learn and retain new information), executive functions (e.g., set-shifting, reasoning, problem-solving, and planning), language (e.g., naming, fluency, expressive speech and comprehension), visuospatial skills, and/or attention control. An algorithm or mathematical computation can be used to assign a specific level of autoantibodies to a particular score on a MCI or ADRD assessment scale. [0089] In yet other aspects, the test subject has a higher level of autoantibodies compared to a control subject who is healthy or normal. If it is determined that the test subject has a higher level of autoantibodies against FRa compared to a normal subject (control subject), then the test subject is predicted to have a higher risk of having MDD or MCI or ADRD than not having the respective disease.

[0090] In some embodiments, a subject having MDD is more likely to have blocking or binding autoantibodies than a normal subject who does not have MDD. A subject with MDD can be at least two times more likely to express blocking or binding autoantibodies in blood, serum, plasma, saliva, urine or cerebrospinal fluid. The subject with MDD can be more likely to have blocking autoantibodies than a normal subject. The subject with MDD can be more likely to have IgM autoantibodies than a normal subject.

[0091] In some embodiments, a subject having MCI is more likely to have blocking or binding autoantibodies than a normal subject who does not have MCI. A subject with MCI can be at least two times more likely to express blocking or binding autoantibodies in blood, serum, plasma, saliva, urine or cerebrospinal fluid. The subject with MCI can be more likely to having binding IgG autoantibodies than a normal subject. The subject with MCI can be more likely to having binding IgM autoantibodies than a normal subject. [0092] In some embodiments, a subject having ADRD is more likely to have blocking or binding autoantibodies than a normal subject who does not have ADRD. A subject with ADRD can be at least two times more likely to express blocking or binding autoantibodies in blood, serum, plasma, saliva, urine or cerebrospinal fluid. The subject with ADRD can be more likely to having binding IgG autoantibodies than a normal subject. The subject with ADRD can be more likely to having binding IgM autoantibodies than a normal subject.

[0093] In some embodiments, the methods of the present invention may further comprise presenting the levels of the autoantibodies and/or the diagnosis of the subject to a user (e.g., a clinician such as a psychiatrist, neurologist or general practitioner). In some instances, the results of the methods are communicated or stored in a readable format or in a computer database or other suitable machine or device for storing such information. The method may include sending or reporting the results to a clinician.

[0094] In some aspects, the method also includes recommending or selecting a drug therapy for the subject at risk of having MDD. If the subject has a risk of MDD, a combination or adjunctive therapy of folate and an antidepressant drug can be recommended or administered to the subject in need thereof. In some cases, if the subject is resistant to an antidepressant drug such as a selective serotonin reuptake inhibitor (SSRI) or selective norepinephrine reuptake inhibitor (S RI), a different antidepressant drug is recommended or selected. If adjunctive folate therapy is recommended, a folate containing formulation can be Deplin ^® in the case of MDD. Deplin® is a medical food containing L-methylfolate, the fully-reduced, bioactive metabolic form of the vitamin B ₉ (folate).

[0095] If the subject has a risk of MCI or ADRD, a drug therapy comprising a folate containing formulation and optionally, an acetylcholinesterase inhibitor and/or memantine drug can be recommended or administered to the subject in need thereof. A subject diagnosed as having MCI or ADRD can be given a folate formulation monotherapy. In other cases, administration of a folate containing formulation and an acetylcholinesterase inhibitor is recommended. In some cases, administration of a folate containing formulation and a memantine drug is recommended. In yet other cases, administration of a folate containing formulation, an acetylcholinesterase inhibitor, and a memantine drug is recommended. The folate containing formulation can be CerefolinNAC ^® in the case of MCI. CerefolinNAC is a medical food containing L-methylfolate, methylcobalamin (the fully-reduced, bioactive metabolic form of the vitamin B _i2 , and N-acetyl-cysteine (an intracellular antioxidant). L- methylfolate is the only active form of folate taken up by the brain.

[0096] In other aspects, the method includes administering a drug therapy to a subject at risk of having MDD or MCI or ADRD to ameliorate at least one symptom of the disorder. As described above, neuropsychological, cognitive or mental tests can be performed to assess the symptoms or extent of MDD or MCI or ADRD in an individual. [0097] A drug therapy such as a combination drug regimen comprising a medically effective amount of a folate formulation and a therapeutically effective amount of an antidepressant drug can be administered to a patient having MDD.

[0098] In other instances, a medically effective amount of a folate formulation, and optionally, a therapeutically effective amount of an acetylcholinesterase inhibitor and/or memantine drug can be administered to a patient having MCI. For a patient with MCI, a medically effective amount of a folate formulation can be administered with a therapeutically effective amount of an acetylcholinesterase inhibitor. In some cases, the folate formulation is administered with a therapeutically effective amount of a memantine drug. In other cases, the folate formulation is administered with a therapeutically effective amount of an

acetylcholinesterase inhibitor and a therapeutically effective amount of a memantine drug.

[0099] In some embodiments, a medically effective amount of a folate formulation, and optionally, a therapeutically effective amount of an acetylcholinesterase inhibitor and/or memantine drug can be administered to a patient having ADRD. For instances, a medically effective amount of a folate formulation can be administered with a therapeutically effective amount of an acetylcholinesterase inhibitor. In some cases, the folate formulation is administered with a therapeutically effective amount of a memantine drug. In other cases, the folate formulation is administered with a therapeutically effective amount of an

acetylcholinesterase inhibitor and a therapeutically effective amount of a memantine drug. For ADRD, suitable therapies include acetylcholinesterase inhibitors. These therapies include, for example, donepezil (Aricept), rivastigmine (Exelon), and galantamine

(Razadyne), which are prescribed to treat mild to moderate AD symptoms. C. Antidepressant Therapy

[0100] If the subject has a risk of having MDD, a combination therapy or adjunctive therapy of a folate formulation and an antidepressant drug can be recommended, selected or administered. In some instances, the antidepressant drug is a selective serotonin reuptake inhibitor (SSRI) or a selective norepinephrine reuptake inhibitor (S RI). In some cases, the combination therapy or adjunctive therapy includes a folate formulation and a SSRI.

Alternatively, the combination therapy or adjunctive therapy includes a folate formulation and a SNRI.

[0101] Various types or classes of antidepressants are known and commercially available. Non-limiting examples of antidepressant include serotonin reuptake inhibitors (SRIs), serotonin reuptake inhibitors (SSRIs), serotonin and dopamine reuptake inhibitors (SDRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), serotonin-noradrenaline-dopamine reuptake inhibitors (SNDRIs), noradrenergic and specific serotonergic anti-depressants (NASSAs), norepinephrine-dopamine reuptake inhibitors (NDRIs), norepinephrine

(noradrenaline) reuptake inhibitors (NRIs), monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake enhancers (SSREs), melatonergic agonists, tryptamines, tricyclic antidepressants (TCAs), and atypical antidepressants.

[0102] SSRIs act to preventing the reuptake of serotonin by the presynaptic neuron, thereby maintaining high levels of serotonin in the synapse. Examples of SSRIs include, but are not limited to, the following (trade names in parentheses): alaproclate; amoxapine; citalopram (such as CELEXA®, CIPRAMIL®, EMOCAL®, SEPRAM® and SEROPRAM®);

clomipramine; dapoxetine; duloxetine (e.g., CYMBAL TA®); escitalopram oxalate (e.g., LEXAPRO®, CIPRALEX® and ESERTIA®); femoxetine; fenfluramine; fluoxetine (e.g. PROZAC®, FONTEX®, SEROMEX®, SERONIL®, SARAFEM®, FLUCTIN® (EUR), and FLUOX® (NZ)); fluvoxamine maleate (e.g., LUVOX®, FAVERIN®, and DUMYROX); indalpine; milnacipran; norfenfluramine; olanzapine; paroxetine (e.g., PAXIL®,

SEROXAT®, AROPAX®, DEROXAT®, REXETIN®, XETANOR®, and PAROXAT®); sertraline (e.g., ZOLOFT®, LUSTRAL® and SERLAIN®); trazodone (e.g., DESYREL®, MOLIPAXIN®, TRITTICO®, THOMBRAN®, TRIALODINE®, TRAZOREL®,

TRITICUM®, and TRAZO E®); venlafaxine and zimelidine. Additional examples of SSRIs and/or SRIs include, without limitations, citalopram, R-fluoxetine, nefazodone, imipramine, imipramine N-oxide, desipramine, pirandamine, dazepinil, nefopam, befuraline, fezolamine, cianoimipramine, litoxetine, cericlamine, seproxetine, WY 27587, WY 27866, imeldine, ifoxetine, tiflucarbine, viqualine, bazinaprine, YM 922, S 33005, F 98214TA, OPC 14523, cyanodothepine, tnmipramine, quinupramine, dothiepin, , nitroxazepine, McN 5652, McN 5707, 01 77, Org 6582, Org 6997, Org 6906, amitriptyline, amitriptyline N-oxide, nortriptyline, CL 255. 663, pirlindole, indatraline, LY 1 13.821, LY 214.281, CGP 6085 A, RU 25.591, napamezole, diclofensine, trazodone, EMD 68.843, BMY 42.569, NS 2389, sercloremine, nitroquipazine, ademethionine, sibutramine and clovoxamine.

[0103] Non-limiting examples of SNRIs include: venlafaxine (EFFEXOR XR®,

EFFEXOR®); desvenlafaxine (PRISTIQ®); sibutramine (MERIDIA®, REDUCTIL®);

levomilnacipran (FETZIMA®); nefazodone (SERZONE®); milnacipran (DALCIPRAN®, IXEL®' SAVELLA®); duloxetine (CYMBALTA®, YENTRENE®); and, bicifadine.

[0104] Bupropion, e.g., WELLBUTRIN®, is a non-limiting example of a serotonin and dopamine reuptake inhibitor (SDRI). Non-limiting examples of SNDRIs include:

tesofensine, brasofensine; NS2359; Nomifensine; Venlafaxine (EFFEXOR®) and

Sibutramine (MERID I A®/REDUC TIL® ) . A non-limiting example of a NASSA includes mirtazapine (AVANZA®, ZISPIN®, REMERON®). NRIs include, without limitation, atomoxetine, maprotiline, nisoxetine, reboxetine, viloxazine and TCAs/Tetras (such as AMITRIPTYLINE®, AMOXAPINE®, BUTRIPTYLINE®,

DESIPRAMINE®/LOFEPRAMINE®, DIBENZEPIN®, DOSULEPIN®, DOXEPIN®, FMIPRAMINE®, IPRINDOLE®, MELITRACEN®, NORTRIPTYLINE®, OPIPRAMOL®, PROTRIPTYLINE®, TRFMIPRAMINE®, and MAPROTILINE®). Non-limiting examples of MAOIs include: iproclozide, iproniazid, isocarboxazid, nialamide, pargyline, phenelzine, rasagiline, selegiline, toloxatone, tranylcypromine, RIMAs (brofaromine, beta-carbolines (harmaline) and moclobemide). A non-limiting example of a SSRE includes tianeptine (STABLON®, COAXIL®, and TATINOL®). A non-limiting example of a TCA includes desipramine (NORPRAMIN® and PERTOFRANEIS®).

[0105] Additional antidepressants that can be used in the invention described herein can include, but are not limited to, natural products such as Kava-Kava, and St. John' s Wort; dietary supplements such as s-adenosylmethionine; neuropeptides such as thyrotropin- releasing hormone; compounds targeting neuropeptide receptors such as neurokinin receptor antagonists; and hormones such as triiodothyronine.

[0106] One skilled in the art would be able to readily determine recommended dosage levels for known and/or marketed antidepressant drugs by consulting appropriate references such as drug package inserts, FDA guidelines, and the Physician' s Desk Reference. For example, the antidepressant drug dose can range from 0.1 mg/day to about 1000 mg/day, from about 0.5 mg/day to about 500 mg/day, from about 1 mg/day to about 400 mg/day, from about 5 mg/day to about 300 mg/day, or from about 10 mg/day to about 200 mg/day. One of skill in the art can readily adjust dosage for each different antidepressant drug, depending on a number of factors such as types and/or potency of antidepressants, severity of depression, physical condition of a subject (e.g., ages, genders, and weights), administration routes, other medications taken by a subject, and any combinations thereof.

[0107] In some aspects, combination therapy comprising the therapeutically effective amount of the folate containing formulation and the therapeutically effective amount of the antidepressant drug is sufficient to increase the degree of improvement in at least one neuropsychological test, e.g., as measured by HAMD-7, HAMD-17, HAMD-21, HAMD-28 or other efficacy measures, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%), at least about 80%, or at least about 90%, as compared to the degree of improvement obtained in the absence of the combination therapy. For instance, an improvement as measured by HAMD-7, HAMD-17, HAMD-21, or HAMD-28 represents a decreased score or a score closer to a score typicaly for a normal subject. In some aspects, the therapeutically effective amount of the antidepressant drug monotherapy is sufficient to increase the degree of improvement in at least one neuropsychological test, e.g., as measured by HAMD-7, HAMD-17, HAMD-21, HAMD-28, or other efficacy measures, by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold or more, as compared to the degree of improvement obtained in the absence of the combination therapy. [0108] In some embodiments, the therapeutically effective amount of the folate containing formulation is a higher dose compared to a dose administered to a subject who does not have autoantibodies to FRa. In some cases, an escalating dose of the folate containing formulation is used to treat MDD if the presence of blocking and/or binding autoantibodies is detected. In some cases, a subject with MDD and autoantibodies against FRa is given a higher dose of a folate compound than a subject with depression and without autoantibodies.

[0109] The presence or level of autoantibodies in a subject with MDD can indicate that the subject has a blockage of folate transport or passage across the blood-brain barrier. As such, the subject can be administered a therapeutically effective amount of the folate containing formulation such that the amount of folate in the subject' s cerebrospinal fluid is normal (or substantially equal to the amount in cerebrospinal fluid of a subject who does not have MDD).

[0110] In one aspect, provided herein is a method for treating major depressive disorder (MDD) in a subject in need thereof by administering to the subject having circulating autoantibodies to folate receptor alpha (FRa) a therapeutically effective amount of a folate containing formulation and a therapeutically effective amount of an antidepressant drug. In other aspects, the method is for prophylactically treating MDD in a subject in need thereof. The subject can have impaired or blocked folate transport across the blood-brain barrier. Alternatively, the subject can have a MTHFR SNP (e.g., a C677T heterozygous or homozygous SNP) or other SNPs involved or associated in one-carbon metabolism. In some embodiments, the subject is has a lower than normal level of MTHF in cerebrospinal fluid. Said subject may have normal levels of MTHF in whole blood. In additional embodiments, the subject is resistant to at least one antidepressant drug (e.g., a SSRI or a SNRI). In some cases, the subject exhibits at least one symptom of MDD prior to administering the folate containing treatment regimen.

D. ADRD and MCI Therapy

[0111] If the subject has a risk of having Alzheimer's disease and related disorders (ADRD), a suitable therapy such as an acetylcholinesterase inhibitor can be used,

recommended, selected or administered. These therapies include, for example, donepezil (Aricept®), rivastigmine (Exelon®), and galantamine (Razadyne®), which are prescribed to treat mild to moderate AD symptoms. Donepezil is approved to treat severe AD as well. The recommended dose for donepezil is 5 mg per day for 4 weeks, then increasing to 10 mg per day. Donepezil is available in pill form and also as an oral disintegrating tablet for those unable or unwilling to swallow a pill.

[0112] Another suitable therapy is memantine (Namenda ^® ), which is prescribed to treat moderate to severe AD symptoms or neramexane. One of skill in the art will recognize that any number of other pharmaceuticals, nutraceuticals, or supplements can be used. Agents used in the treatment of dementia, include, but not limited to, Thioridazine, Haloperidol, Risperidone, Cognex ^® , Aricept ^® , and Exelon ^® .

[0113] If the subject has a risk of having or has been diagnosed as having ADRD, a combination therapy of a folate formulation and an acetylcholinesterase inhibitor can be recommended, selected, or administered. In some embodiments, a combination therapy used to treat ADRD includes a folate formulation and a memantine drug. In other embodiments, a combination therapy used to treat ADRD includes a folate formulation, an

acetylcholinesterase inhibitor, and a memantine drug.

[0114] In some embodiments, the therapeutically effective amount of the folate containing formulation recommended, selected or administered to a subject having or at risk of having ADRD with autoantibodies against FRa is a higher dose compared to a dose administered to a subject who does not have autoantibodies to FRa. In some cases, an escalating dose of the folate containing formulation is used to treat ADRD if the presence of blocking and/or binding autoantibodies is detected. In some cases, a subject with ADRD and autoantibodies against FRa is given a higher dose of a folate compound than a subject with ADRD and without autoantibodies.

[0115] In one aspect, provided herein is a method for treating Alzheimer's disease and related diseases (ADRD) in a subject in need thereof by administering to the subject having circulating autoantibodies to folate receptor alpha (FRa) a therapeutically effective amount of a folate containing formulation. In other aspects, the method is for prophylactically treating ADRD in a subject in need thereof. In some embodiments, the subject is administered a therapeutically effective amount of a folate containing formulation in combination with an acetylcholinesterase inhibitor and/or memantine drug. The subject can be administered a therapeutically effective amount of a folate containing formulation in combination with an acetylcholinesterase inhibitor. The subject can be administered a therapeutically effective amount of a folate containing formulation in combination with a memantine drug. In other embodiments, the subject is administered a therapeutically effective amount of a folate containing formulation in combination with an acetylcholinesterase inhibitor and memantine drug. In some embodiments, the subject exhibits at least one symptom of ADRD. In other embodiments, the subject suffers from mild cognitive impairment due to Alzheimer's disease. In yet other embodiments, the subject suffers from impaired or blocked folate transport at the blood-brain barrier.

[0116] As MCI may represent a prodromal state to clinical Alzheimer's disease, treatments proposed for Alzheimer' s disease, such as antioxidants and acetylcholinesterase inhibitors, may be useful in the treatment of MCI. In addition, CerefolinNAC® is a medical food containing L-methylfolate, methylcobalamin (the fully-reduced, bioactive metabolic form of the vitamin B _i2 , and N-acetyl-cysteine (an intracellular antioxidant), which is useful in its treatment.

[0117] In the disclosed methods, preferably, both the 1-aminocyclohexane derivative (e.g., memantine) or the AChEI (e.g. donepezil) are present in a therapeutically effective amount. The optimal therapeutically effective amount should be determined experimentally, taking into consideration the exact mode of administration, form in which the drug is administered, the indication toward which the administration is directed, the subject involved (e.g., body weight, health, age, sex, and the like), and the preference and experience of the physician or veterinarian in charge. As disclosed herein, for human administration, the 1- aminocyclohexane derivatives or AChEIs are administered in suitable form in doses ranging from about 1 to 200 mg per day for each drug. More specifically, the 1-aminocyclohexane derivatives are preferably administered at doses 5-60 mg/day, and especially 10-40 mg/day; the AChEIs are preferably administered at doses 1-40 mg/day, and especially 5-24 mg/day.

[0118] In certain aspects, the 1-aminocyclohexane derivative which may be used in unit dosage amounts of the invention include, for example, 5 mg, 10 mg, 15 mg, and 20 mg for memantine and 5 mg, 10 mg, 20 mg, 30 mg, and 40 mg for neramexane. Preferred specific amounts of the AChEI which may be used in unit dosage amounts of the invention include, for example, 1.5 mg, 3 mg, 4.5 mg, and 6 mg for rivastigmine, 4 mg, 8 mg and 12 mg for galantamine, and 5 mg and 10 mg for donepezil.

[0119] If the subject has a risk of having or has been diagnosed as having MCI, a combination therapy of a folate formulation and an acetylcholinesterase inhibitor can be recommended, selected, or administered. In some embodiments, a combination therapy used to treat MCI includes a folate formulation and a memantine drug. In other embodiments, a combination therapy used to treat MCI includes a folate formulation, an acetylcholinesterase inhibitor, and a memantine drug.

[0120] In some embodiments, the therapeutically effective amount of the folate containing formulation recommended, selected or administered to a subject having or at risk of having MCI with autoantibodies against FRa is a higher dose compared to a dose administered to a subject who does not have autoantibodies to FRa. In some cases, an escalating dose of the folate containing formulation is used to treat MCI if the presence of blocking and/or binding autoantibodies is detected. In some cases, a subject with MCI and autoantibodies against FRa is given a higher dose of a folate compound than a subject with cognitive impairment and without autoantibodies.

[0121] In addition, numerous other therapies known in the art as useful in the clinical management of AD/MCI and its symptoms include herbal treatments such as Ginkgo Biloba, which can improve memory and cognitive function in people with memory problems or dementia. Examples of antioxidants useful to treat MCI include, but are not limited to, ubiquinone, aged garlic extract, curcumin, lipoic acid, beta-carotene, melatonin, resveratrol, Ginkgo Biloba extract, vitamin C, vitamin E or the like.

[0122] In another aspect, provided herein is a method for treating mild cognitive impairment (MCI) in a subject in need thereof by administering to the subject having circulating autoantibodies to folate receptor alpha (FRa) a therapeutically effective amount of a folate containing formulation. In other aspects, the method is for prophylactically treating mild cognitive impairment in a subject in need thereof. In some embodiments, the subject is administered a therapeutically effective amount of a folate containing formulation in combination with an acetylcholinesterase inhibitor and/or memantine drug. The subject can be administered a therapeutically effective amount of a folate containing formulation in combination with an acetylcholinesterase inhibitor. The subject can be administered a therapeutically effective amount of a folate containing formulation in combination with a memantine drug. In other embodiments, the subject is administered a therapeutically effective amount of a folate containing formulation in combination with an

acetylcholinesterase inhibitor and memantine drug. In some instances, the subject suffers from mild cognitive impairment. In other instances, the subject suffers from impaired or blocked folate transport at the blood-brain barrier. E. Folate Formulations

[0123] Any art-recognized folate formulation, e.g., folate-comprising compound can be selected and/or optionally administered to a human subject determined to have a risk of MDD or MCI or ADRD. In some aspects, the folate formulation comprises L-methylfolate. In some aspects, the folate formulation comprises 6(S)-5-methyltetrahydrofolate (also known as 6(S)-5-MTHF). In a preferred aspect, the folate comprising formulation is Deplin® for MDD and CerefolinNAC® for MCI. Deplin® is a medical food containing L-methylfolate, the fully-reduced, bioactive metabolic form of the vitamin B ₉ (folate). CerefolinNAC® is a medical food containing L-methylfolate, methylcobalamin (the fully-reduced, bioactive metabolic form of the vitamin B _i2 , and N-acetyl-cysteine (an intracellular antioxidant). L- methylfolate is the active form of folate that can be taken up by the brain. In certain instances, the folate disclosed in US Patent No. 6,441, 168 is used.

[0124] The folate formulation can include at least one (including at least two, at least three or more) alkaline metal or alkaline earth metal salt of folate, e.g., but not limited to, a calcium salt of folate. In some aspects, the folate formulation is methyl folate, also known as Me- THF, N5 -Methyl -THF, MTHF, 5-MTHF, L-methylfolate, and Levomefolic acid, or a medically acceptable salt thereof (e.g., sodium salt, potassium salt, magnesium salt, calcium salt, glucosamine salt, or galactosamine salt). Methyl folate calcium salt is available by prescription in the United States as DEPLIN® for MDD and CerefolinNAC® for MCI (L- methylfolate calcium salt). Methyl folate calcium salt is also available outside of the United States as METAFOLIN®, BODYFOLIN®, and NUTRIFOLIN®.

[0125] In some aspects, the folate formulation can include at least one (including at least two, at least three or more) glucosamine salt and/or galactosamine salt of folate (including, e.g., folic acid and reduced folate, e.g., but not limited to, tetrahydrofolate, and derivatives thereof). Examples of glucosamine-folate and/or galactosamine-folate and derivatives thereof, e.g., disclosed in U.S. Patent No. 7,947,662, can be administered to a human subject according to the methods described. In one embodiment, QUATREFOLIC® (Gnosis S.p.A, Milan, IT) or N-[4-[[[(6S)-2-amino-l,4,5,6,7,8-hexahydro-5-methyl-4-oxo-6- pteridinyl]methyl]amino]benzoyl]-L-glutamic acid, glucosamine salt can be administered to a human subject according to the methods described herein.

[0126] Additional examples of folates or folate-comprising compounds that can be administered to a subject according to the method provided herein include, but not limited to, the ones described in the U.S. Pat. Nos. 4,336, 185; 6,921,754; and 7,947,662; and U.S. Pat. App. Publication No. : US 2008/0064702, the disclosures of which are incorporated are herein incorporated by reference for all purposes.

[0127] The effective amount of folate for use in the treatment methods described herein can vary, depending upon the types of folate, severity of depression, cognitive symptoms, presence of FRa autoantibodies, physical conditions of a subject (e.g., ages, genders, weights), the presence or levels of unmetabolized folic acid, polymorphisms related to one- carbon metabolism inflammation and a combination thereof.

[0128] In some aspects, the therapeutically effective amounts of the folate formulation and antidepressant drug are sufficient to increase the degree of improvement in at least one neuropsychological test, e.g., as measured by HAMD-7, HAMD-17, HAMD-21 or HAMD- 28 or other efficacy measures, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%), at least about 80%, or at least about 90%, as compared to the degree of improvement obtained in the absence of either drug. In some aspects, the therapeutically effective amounts of the folate drug and the antidepressant drug are sufficient to increase the degree of improvement in at least one neuropsychological test, e.g., as measured by HAMD-7, HAMD- 17, HAMD-21, HAMD-28 or other efficacy measures, by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold or more, as compared to the degree of improvement obtained in the absence of either drug.

[0129] In other aspects, the therapeutically effective amounts of the folate formulation, and optionally, an acetylcholinesterase inhibitor and/or memantine drug are sufficient to increase the degree of improvement in at least one cognitive or memory test by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, as compared to the degree of improvement obtained in the absence of drug therapy. In some aspects, the therapeutically effective amounts of the folate formulation and optionally, the acetylcholinesterase inhibitor and/or memantine drug are sufficient to increase the degree of improvement in at least one cognitive or memory test by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold or more, as compared to the degree of improvement obtained in absence of drug therapy. F. Pharmaceutical Compositions and Administration Thereof

[0130] According to the methods of the present invention, the therapeutic drugs described herein to treat depression or mild cognitive impairment or Alzheimer's disease or related disorders are administered to a subject by any convenient means known in the art. The methods of the invention can also be used to select a suitable drug for the treatment of depression or MCI or ADRD. One skilled in the art will appreciate that the drug combination therapy or adjunctive therapy described herein can be administered alone or as part of a combined therapeutic approach with psychotherapy, psychodynamic therapy, cognitive behavior therapy and combinations thereof. [0131] The folate formulation or antidepressant or MCI or ADRD drug described herein can be administered via a single dosage form. For example, the single dosage form can be administered as a single tablet, pill, capsule for oral administration or a solution for parenteral administration. Alternatively, the drug can be administered as separate compositions, e.g., as separate tablets or solutions. The length of time between administrations of a sub-dose of a drug can be adjusted to achieve the desired therapeutic effect. In combination therapy, the drugs can be administered concomitantly or sequentially.

[0132] One skilled in the art would be able to readily determine recommended dosage levels for known and/or marketed antidepressant, MDD, AD or ADRD drugs by consulting appropriate references such as drug package inserts, FDA guidelines, and the Physician's Desk Reference. For example, the antidepressant drug dose can range from 0.1 mg/day to about 1000 mg/day, from about 0.5 mg/day to about 500 mg/day, from about 1 mg/day to about 400 mg/day, from about 5 mg/day to about 300 mg/day, or from about 10 mg/day to about 200 mg/day. One of skill in the art can readily adjust dosage for each different antidepressant drug, depending on a number of factors such as types and/or potency of antidepressants, severity of depression, physical condition of a subject (e.g., ages, genders, and weights), administration routes, other medications taken by a subject, and any

combinations thereof.

[0133] In some aspects, a dose of an antidepressant or a pharmaceutically acceptable salt thereof suitable for administration to a human is in the range of about 0.01 to 50 mg per kilogram body weight of the recipient per day, or in the range of 0.1 to 5 mg per kilogram body weight per day. In certain aspects, the desired dose can be presented as one single unit dosage form, e.g., containing about 1 mg to about 500 mg, or about 5 mg to about 300 mg. In other aspects, the desired dose can be presented in two, three, four, five or more sub-doses administered at appropriate intervals throughout the day. These sub-doses can be

administered in unit dosage forms, for example, containing about 0.1 mg to about 100 mg or about 1 mg to about 50 mg. [0134] In some aspects, the desired dose of folate can range of about 0.01 to about 50 mg per kilogram body weight of the recipient per day, in the range of about 0.05 to about 5 mg per kilogram body weight per day, or in the range of about 0.1 to about 1 mg per kilogram body weight per day. In certain aspects, the desired dose can be presented as one single unit dosage form, e.g., containing about 0.5 mg to about 500 mg, about 5 mg to about 250 mg, about 10 mg to about 100 mg, or about 10 mg to about 50 mg. In some aspects, one single unit dosage form can provide about 1 mg to about 70 mg folate, about 5 mg to about 60 mg folate, or from about 7 mg to about 50 mg folate. In other aspects, one single unit dosage form can provide about 15 mg to about 50 mg folate. In yet other aspects, one single unit dosage form can provide about 20 mg folate. In other aspects, the desired dose can be presented in two, three, four, five or more sub-doses administered at appropriate intervals throughout the day. These sub-doses can be administered in unit dosage forms, for example, containing about 0.1 mg to about 250 mg, about 1 mg to about 100 mg, about 2 mg to about 20 mg, or about 2 mg to about 10 mg.

[0135] The effective amount of folate administered to a selected human subject for the treatment of depression or MCI or ADRD as described herein is significantly higher than the typical amount taken as a dietary supplement (between 50-600 μg/day). In some aspects, the effective amount of folate administered to a selected human subject is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 1000-fold or more than the typical amount taken as a dietary supplement.

[0136] For oral administration, the therapeutically effective dose can be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders, and sustained-release formulations. Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

[0137] The pharmaceutical composition can be taken with or without ordinary foodstuffs. The pharmaceutically acceptable composition can be delivered via injection. These routes for administration (delivery) include, but are not limited to, subcutaneous or parenteral including intravenous, intraarterial, intramuscular, intraperitoneal, intramyocardial, inhalation, intranasally, and infusion techniques. In one embodiment, the pharmaceutical acceptable composition is in a form that is suitable for injection. In another embodiment, the pharmaceutical composition is formulated for delivery by a catheter.

[0138] When administering a pharmaceutical composition parenterally, it can be generally formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, cell culture medium, buffers (e.g., phosphate buffered saline), polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof. In some aspects, the pharmaceutical carrier can be a buffered solution (e.g. PBS). In some aspects, the pharmaceutical composition can be formulated in an emulsion or a gel.

[0139] In some aspects, combination therapy or adjunctive therapy (e.g., antidepressant or MCI drug or ADRD drug together with a folate formulation) is formulated in slow-release or sustained release composition. As used herein, the term "sustained release" or "sustained delivery" refers to continual delivery of a therapeutic agent in vivo over a period of time following administration. For example, sustained release can occur over a period of at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, at least about 16 hours, at least about 24 hours following administration. In some aspects, sustained release can occur over a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days following administration. In some aspects, the release of the drug monotherapy from a drug- delivery system can be steady state (zero-order kinetics) with at least about 30% (e.g., including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more) of the drug compound released between about 3-6 hours post administration, or between about 4-5 hours post administration. In one embodiment, the release of the drug compound from a drug-delivery system can be steady state (zero-order kinetics) with substantially full release (e.g., -100%) of the drug released between about 3-6 hours post administration, or between about 4-5 hours post administration. In some aspects, the drug compound can be released from a drug- delivery system at a rate that is slow enough not to overload the intestinal absorption capacity of a patient' s duodenum.

[0140] In some aspects, a drug delivery system can comprise the drug compound encapsulated in polymer-based particles. For example, the drug-containing polymer-based particles can be filled into capsules or single-dose sachets for additional control of release.

[0141] Controlled-release (e.g., sustained release) drug delivery systems for different administration methods (e.g., oral administration, injection, implantation, and inhalation) are known in the art and can be adopted to deliver a combination or adjunctive therapy for the treatment methods described herein. See, e.g., International Pat. App. Nos. WO 2012/1 1 1961 (oral formulation), WO 2012/131678 (injectable formulation); U. S. Pat. App. Nos. US

2012/0258161 (implantable formulation), US 2001/0038854, US 2001/0033866; and U. S. Pat. No. 8,268,347 (inhalation formulation), the disclosures of which are hereby incorporated by reference in their entirety for all purposes, for various types of drug-delivery systems to deliver an active agent via various administration routes. [0142] In certain instance, the modified release formulation of folate is disclosed in US Patent Application Publication No. 2015/0030676, the teachings of which are incorporated herein by reference.

[0143] A pharmaceutically acceptable carrier includes a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (i) sugars, such as lactose, glucose and sucrose; (ii) starches, such as corn starch and potato starch; (iii) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (iv) powdered tragacanth; (v) malt; (vi) gelatin; (vii) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (viii) excipients, such as cocoa butter and suppository waxes; (ix) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (x) glycols, such as propylene glycol; (xi) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (xii) esters, such as ethyl oleate and ethyl laurate; (xiii) agar; (xiv) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (xv) alginic acid; (xvi) pyrogen-free water; (xvii) isotonic saline; (xviii) Ringer's solution; (xix) ethyl alcohol; (xx) pH buffered solutions; (xxi) polyesters, polycarbonates and/or polyanhydrides; (xxii) bulking agents, such as polypeptides and amino acids (xxiii) serum component, such as serum albumin, HDL and LDL; (xxiv) C ₂ -Ci ₂ alcohols, such as ethanol; and (xxv) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. Pharmaceutically acceptable carriers can vary depending on the administration route and formulation.

[0144] Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it may be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like.

[0145] The compositions can also contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, colors, binders, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON' S PHARMACEUTICAL SCIENCE," incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. With respect to compositions described herein, however, any vehicle, diluent, or additive used should have to be biocompatible with the antidepressant or a pharmaceutically acceptable salt thereof. [0146] The pharmaceutical compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the composition described herein can be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. In one embodiment, sodium chloride is used in buffers containing sodium ions.

[0147] Viscosity of the compositions can be maintained at the selected level using a pharmaceutically acceptable thickening agent. In one embodiment, methylcellulose is used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose,

hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount which will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

[0148] Typically, any additives (in addition to the antidepressant compound) can be present in an amount of 0.001 to 50 wt % solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams to grams, such as about 0.0001 to about 5 wt %, about 0.0001 to about 1 wt %, about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, about 0.01 to about 10 wt %, and about 0.05 to about 5 wt %. For any therapeutic composition to be administered to a subject with compression, and for any particular method of administration, it is preferred to determine toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such

determinations do not require undue experimentation from the knowledge of the skilled artisan.

[0149] The compositions described herein can be prepared by mixing the ingredients following generally-accepted procedures. For example, the ingredients can be mixed in an appropriate pharmaceutically acceptable carrier and the mixture can be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally the pH can vary from about 3 to about 7.5. In some aspects, the pH of the composition can be about 6.5 to about 7.5. Compositions can be administered in dosages and by techniques well known to those skilled in the medical and veterinary arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., liquid).

[0150] A subject can also be monitored at periodic time intervals to assess the efficacy of a certain therapeutic regimen. For example, the activation states of certain signal transduction molecules may change based on the therapeutic effect of treatment with one or more of the drugs described herein to treat depression or mild cognitive impairment. The subject can be monitored to assess response and understand the effects of certain drugs or treatments in an individualized approach. Additionally, subjects who initially respond to a specific drug for depression may become refractory to the drug, indicating that these subjects have developed acquired drug resistance. These subjects can be discontinued on their current therapy and an alternative treatment prescribed in accordance with the methods of the present invention.

IV. EXAMPLES

[0151] The following examples are offered to illustrate, but not to limit, the claimed invention.

Example 1. Methods for Diagnosing a Subject with Major Depressive Disorder (MDD).

[0152] To assess to what extent blocking and binding antibodies moderate or modulate the effect on HAMD-28 scores in a Sequential Parallel Comparison Design (SPCD) trial (treatment-resistant depression 1/2 (TRD-1/2)), the following analyses were performed. The data is provided in Table 1. The level of blocking autoantibody and binding autoantibody (e.g., IgG autoantibody or IgM autoantibody) were measured according to the methods described herein. Of the patients previously diagnosed as having MDD, 70.3% were positive for a blocking antibody, binding antibody, or both. 77.8% of the patients in the TRD-1 study were positive for one or more of the autoantibodies and 64.3% of the patients in the TRD-2 study were positive as well.

Table 1. Correlation between MDD Diagnosis and Presence of autoantibodies to Folate

Receptor Alpha (FRa).

[0153] The results illustrate that a subject possessing at least one type of autoantibodies to FRa have a higher likelihood of having depression, particularly, major depressive disorder, than not having the disorder. Thus, these autoantibodies (blocking or binding) can be used as diagnostic markers for MDD. Example 2. Methods for Selecting Treatment for a Subject with Major Depressive Disorder (MDD).

[0154] Statistical analysis of the relationship between Delpin ^® (L-methylfolate) response in MDD patients and the presence of autoantibodies to FRa was performed. First, the effect of Deplin ^® versus placebo within the positive and negative subgroups (blocking+, blocking-, binding+, binding-) was analyzed. Seemingly unrelated regression models were fit to account for the potential correlation of observations in the two phases of SPCD design. Table 2 provides some of the results.

Table 2. Statistical Analysis of Drug Response and Autoantibodies.

[0155] The pooled effect represents a change in depression state on a validated depression scale of patients with binding autoantibodies (Deplin ^® group minus no Deplin ^® placebo group). The treatment effect represents a change in depression state on a validated depression scale of patients receiving Deplin ^® (patients having autoantibodies minus patient who do not have autoantibodies).

[0156] For binding antibodies, the effect (difference in reduction in HAMD28 between Deplin ^® and placebo arm) pooled across the two SPCD phases was -1.58 (95% CI: -5.93, 2.77), p-value 0.476 (not statistically significant) for those without binding antibodies and - 6.83 (95% CI: -9.87, -3.79), p-value < 0.001 (highly statistically significant) for those with binding antibodies. When a formal interaction between the presence of binding antibodies and treatment arm (Deplin ^® vs. placebo) was tested, the results showed statistical

significance: p-value = 0.038.

[0157] For blocking antibodies, the effect of Deplin ^® versus placebo was numerically similar between those without (-3.79) and with blocking antibodies (-3.07), however only the effect among those without blocking antibodies reached statistical significance (respective p- values 0.007 and 0.304). Consequently, the interaction between the presence of blocking antibodies and treatment arm (Deplin ^® vs. placebo) was not statistically significant (p-value = 0.958) in these studies. [0158] When the presence of binding or blocking antibodies was used to define a subgroup, the effect of Deplin ^® versus placebo was highly statistically significant (p-value < 0.001) with effect pooled across phases equal to -5.40 (95% CI: -8.32, -2.48).

[0159] This example illustrates that subjects with circulating binding autoantibodies to FRa are predicted to have a drug response to Deplin ^® . In addition, subject with either blocking autoantibodies or binding autoantibodies are predicted to positively respond to Deplin ^® .

Example 3. Method for Diagnosing a Subject with Mild Cognitive Impairment (MCI).

[0160] The relationship between mild cognitive impairment (MCI) and the presence of autoantibodies to folate receptor alpha (FRa) was evaluated in patients participating in the NAC-002 study. The presence or levels of blocking and binding autoantibodies against FR were detected as described herein in patients with mild cognitive impairment or Alzheimer's disease related disorders (ADRD). 65.3% of the patients with MCI had blocking

autoantibodies, IgG and/or IgM binding autoantibodies (Table 3).

Table 3. Correlation Between MCI and ADRD Diagnosis and Autoantibodies to FRa.

[0161] These examples illustrate that a subject possessing autoantibodies to FRa have a higher likelihood of having mild cognitive impairment (MCI) or Alzheimer's disease related diseases (ADRD), then not having the respective disorder. Thus, these autoantibodies (blocking or binding) can be used as diagnostic markers for MCI or ADRD. [0162] In a similar study, 57.9% of patients with MCI had binding autoantibodies.

Example 4. Matched Case-Control Analysis for the Prevalence of Folate Receptor alpha Autoantibodies in Patients with Depression or Major Depressive Disorder

(MDD).

[0163] This example provides an age- and gender-matched case-control analysis for the prevalence of autoantibodies to folate receptor alpha (FRa) in individuals with depression or MDD. The FRa autoantibodies included blocking antibodies or binding antibodies (binding IgG or IgM antibodies).

[0164] The case data was derived from the TRD-1 and TRD-2 (e.g., treatment-resistant depression) studies. The case-group included patients with depression/major depressive disorder at baseline (prior to drug administration), and the control group included age- and gender-matched normal individuals who did not have depression or major depressive disorder.

[0165] The main objective of the analysis was to test for any differences in prevalence of blocking and binding antibodies between cases and corresponding age- and gender-matched controls. Further, we tested for the difference in prevalence of either binding or blocking antibodies between the cases and the matched controls.

[0166] The main benefit for matching of cases and controls is the increase in efficiency in presence of possible confounders (age and gender). The test for differences in prevalence was carried out using a logistic regression model where the log-odds for the presence (or absence) of a particular antibody is modeled in terms of group (case/control) and adjusted for other covariates. The analysis steps carried out are as follows:

1. Test for possible confounding (or effect modification) by gender and age. This is done using a Chi-square test for the change in deviance between the target model (log-odds of presence of antibody in terms of group) and the full model (log-odds of presence of antibody in terms of group, age and gender).

2. Exact 1 : 1 matching is not possible for this data set. Matching is carried out using nearest sub-class matching with propensity scores (Ho et al., "Matchl Nonparametric Preprocessing for Parametric Causal Inference." In: Statistics Probability Letters, 2009, 1 14- 149). Conditional on the propensity score the distribution of the baseline covariates (like age at first visit and gender) will be similar between cases and control.

3. Chi-square test in step 1 is re-applied to the data derived using matching.

4. Unconditional logistic regression, adjusting for age and gender are then fitted to the matched data set for each antibody separately.

Results [0167] FIG. 1 shows the data of matching. Some older control subjects were left unmatched and not included in the analysis. After matching of cases and controls using propensity scores, there was still evidence of possible confounding (FIG. 2C). Next, an unconditional logistic regression was fitted to the matched data to test for the difference in prevalence (in terms of Odds-Ratios (OR)) of antibodies between cases and controls while adjusting for the age and gender. Prevalence for each antibody is given in Table 4.

Table 4. Raw Prevalence of Antibodies From the Matched Data.

Group N Ab IgG IgM Either control 101 0.18 0.20 0.12 0.43

Case 101 0.27 0.26 0.43 0.70

[0168] Results for the Chi-square test for deviance to test for effect-modification of age and gender are given in FIGS. 2B and 2C for the full data and the matched data, respectively. FIG. 2A provides matching case and controls in six sub-classes based on propensity scores. Results suggest that there may be some effect-modification in the case of binding-IgG (p- value = 0.069).

[0169] Data of the logistic regression are provided in Tables 5-8 in terms of Odds Ratios (OR) estimates and their 95% confidence intervals and corresponding p-values.

[0170] Odds of having blocking antibodies is significantly higher for the cases than the matched controls (OR = 2.16, 95% CI: 1.02-4.66, P-value= 0.004).

[0171] Odds of having binding IgG antibodies is not significantly different between the cases and the matched controls (OR = 1.28, 95% CI: 0.6-2.73, P-value = 0.518). [0172] Odds of having binding IgM antibody is significantly higher for cases than the matched controls (OR = 4.21, 95% CI: 1.98-9.46, P-value < 0.001).

[0173] Odds of having either blocking or binding antibodies is significantly higher in cases than in matched controls (OR = 3.24, 95% CI: 1.70-6.33, P-value < 0.001).

Table 5. Odds Ratio Estimates and 95% Confidence Intervals for Blocking FRA

Autoantibodies.

Parameter OR 2.5% 97.5% P-value

(intercept) 0.04 0.00 0.33 0.004 typecase 2.16 1.02 4.66 0.047

age 1.03 1.00 1.07 0.101 genderM 0.84 0.39 1.74 0.649

Table 6. Odds Ratio Estimates and 95% Confidence Intervals for Binding IgG FRA

Autoantibodies

Parameter OR 2.5% 97.5% P-value

(intercept) 0.69 0.10 4.38 0.697 typecase 1.28 0.60 2.73 0.518 age 0.98 0.95 1.01 0.140

genderM 2.00 0.99 4.05 0.054 Table 7. Odds Ratio Estimates and 95% Confidence Intervals for Binding IgM FRA

Autoantibodies.

Parameter OR 2.5% 97.5% P-value

(intercept) 0.70 0.11 4.31 0.698 typecase 4.21 1.98 9.46 0.000 age 0.97 0.94 1.00 0.068

genderM 0.94 0.44 1.97 0.880

Table 8. Odds Ratio Estimates and 95% Confidence Intervals for Either Blocking or

Binding (IgG or IgM) FRA Autoantibodies.

Parameter OR 2.5% 97.5% P-value

(intercept) 0.75 0.13 4.26 0.742 typecase 3.24 1.70 6.33 0.000 age 1.00 0.97 1.03 0.930

genderM 1.17 0.63 2.20 0.625

[0174] Matching based on propensity scores were carried out in order to balance the distribution of age and gender between the case and control groups. Assuming no other confounders are present, the data suggest that the prevalence of binding IgM autoantibodies significantly higher among patient with MDD than the matched controls. Also, the prevalence of blocking FRA autoantibodies is higher in MDD patients than the matched control. The odds of a MDD patient having either blocking or binding antibodies were also higher than for a matched control. Example 5. Matched Case-Control Analysis for the Prevalence of Folate Receptor alpha Autoantibodies in Patients with Cognitive Impairment.

[0175] This example provides an age- and gender-matched case-control analysis for the prevalence of autoantibodies (blocking antibodies or binding antibodies (e.g., binding IgG and IgM antibodies)) to folate receptor alpha in patients with mild cognitive impairment (MCI) or Alzheimer' s disease and related diseases (ADRD).

[0176] The case data was derived from the N-acetylcysteine (NAC) for improving cognitive dysfunction study. The case-group included patients with cognitive impairment at baseline (prior to drug administration), and the control group included age- and gender- matched normal individuals who did not have cognitive impairment.

[0177] The main objective of the analysis was to test for any differences in prevalence of blocking and binding antibodies between cases and age- and gender-matched controls.

Further, we tested for the difference in prevalence of either binding or blocking antibodies between the cases and the matched controls. [0178] The main benefit for matching of cases and controls is the increase in efficiency in presence of possible confounders (age and gender). The test for differences in prevalence was carried out using a logistic regression model where the log-odds for the presence (or absence) of a particular antibody is modeled in terms of group (case/control) and adjusted for other covariates. The analysis steps carried out are as follows: 1. Test for possible confounding (or effect modification) by gender and age. This is done using a Chi-square test for the change in deviance between the target model (log-odds of presence of antibody in terms of group) and the full model (log-odds of presence of antibody in terms of group, age and gender).

2. Exact 1 : 1 matching is not possible for this data set. Matching is carried out using nearest sub-class matching with propensity scores (Ho et al., "Matchl Nonparametric

Preprocessing for Parametric Causal Inference." In: Statistics Probability Letters, 2009, 1 14- 149). Conditional on the propensity score the distribution of the baseline covariates (like age at first visit and gender) will be similar between cases and control.

3. Chi-square test in step 1 is re-applied to the data derived using matching. 4. Unconditional logistic regression, adjusting for age and gender are then fitted to the matched data set for each antibody separately. Results

[0179] Results for the Chi-square test for deviance to test for effect-modification of age and gender are given in FIGS. 4B and 4C for the full data and the matched data, respectively. FIG. 4A provides matching case and controls in six sub-classes based on propensity scores. Matching prevented effect-modifications that were seen for the IgG binding variable in the full data.

[0180] FIG. 3 shows the data of matching. Some younger control subjects were left unmatched and not included in the analysis. Prevalence for each antibody is given in Table 9.

Table 9. Prevalence of the Presence of Antibodies.

Group N Ab IgG IgM Either control 120 0.19 0.20 0.11 0.44

case 120 0.15 0.44 0.38 0.66

[0181] Results of the logistic regression are given in Tables 10-13 in terms of Odds Ratios (OR) estimates and their 95% confidence intervals and corresponding p-values.

[0182] Odds of having blocking antibodies is not different between cases and controls (OR = 0.88, 95% CI: 0.37-2.09, P-value=0.772). [0183] Odds of having binding IgG antibodies is significantly higher in the cases than in the matched controls (OR = 3.16, 95% CI: 1.54-6.68, P-value = 0.002).

[0184] Odds of having binding IgM antibody is significantly higher for cases than the matched controls (OR = 6.73, 95% CI: 2.94-16.34, P-value < 0.001).

[0185] Odds of having either blocking or binding antibodies is significantly higher in cases than in matched controls (OR = 2.58, 95% CI: 1.32-5.12, P-value = 0.006).

Table 10. Odds Ratio Estimates and 95% Confidence Intervals for Blocking FRA

Autoantibodies.

Parameter OR 2.5% 97.5% P-value

(intercept) 0.66 0.02 19.71 0.809 typecase 0.88 0.37 2.09 0.772

age 0.99 0.94 1.04 0.602 genderM 0.75 0.38 1.48 0.405 Table 11. Odds Ratio Estimates and 95% Confidence Intervals for Binding IgG FRA

Autoantibodies.

Parameter OR 2.5% 97.5% P-value

(intercept) 0.21 0.01 3.26 0.264 typecase 3.16 1.54 6.68 0.002

age 1.00 0.96 1.04 0.899 genderM 1.88 1.06 3.40 0.033

Table 12. Odds Ratio Estimates and 95% Confidence Intervals for Binding IgM FRA

Autoantibodies.

Parameter OR 2.5% 97.5% P-value

(intercept) 0.76 0.04 15.72 0.858 typecase 6.73 2.94 16.34 0.000

age 0.97 0.93 1.02 0.238 genderM 0.87 0.46 1.64 0.662

Table 13. Odds Ratio Estimates and 95% Confidence Intervals for Blocking or Binding

(IgG or IgM) FRA Autoantibodies.

Parameter OR 2.5% 97.5% P-value

(intercept) 1.10 0.08 14.89 0.941 typecase 2.58 1.32 5.12 0.006

age 0.99 0.96 1.03 0.770 genderM 1.10 0.65 1.85 0.733

[0186] Matching based on propensity scores were carried out in order to balance the distribution of age and gender between the case and control groups. Assuming no other confounders are present, the data suggest that the prevalence of binding IgG or IgM FRA autoantibodies is significantly higher among patients with cognitive impairment than the age- and gender-matched controls. The odds of a cognitive impaired patient having either blocking or binding antibodies were also higher than for a matched control.

[0187] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.