DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Patent Application No. 63/071,035, filed August 27, 2020, and U.S. Provisional Patent Application No. 63/071,032, filed August 27, 2020, which are hereby incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. R56 AG062620 and Grant No. AG057457 awarded by National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to neurodegenerative diseases and conditions (e.g., Alzheimer’s disease) characterized with dysfunctional energetic function, unregulated microglia phagocytic activity and other related de-regulated biological functions. This invention further relates to methods and compositions for treating such neurodegenerative diseases and conditions with pharmaceutical compositions comprising one or more of phenyl isothiocyanate (PEITC), an analog of PEITC, oxidized nicotinamide adenine dinucleotide (NAD+), and reduced nicotinamide adenine dinucleotide (NADH), wherein such compositions are capable of protecting neurons from cell death and unregulated microglia phagocytic activity.

BACKGROUND OF THE INVENTION

There is an urgent need to develop novel therapies for neurodegenerative diseases and conditions such as Alzheimer's disease (AD). 10% of persons over age 65 and up to 50% over age 85 have dementia, with over 30 million people affected worldwide. AD affects over 26 million people worldwide and currently there is no cure for the disease. With the growing number of people living to older ages, there is an urgency to better understand elements of the pathogenic pathway, discover agents that target these elements, and establish their roles in the treatment and prevention of AD. As such, improved methods for treating neurodegenerative disorders (e.g., AD) are needed.

The present invention addresses this need.

SUMMARY

Alzheimer’s disease (AD) is the most common neurodegenerative disease with no effective treatment to date. The onset and development of AD pathology involves complex biological processes, leading to the formation of neurite plaques and neurofibrillary tangles (NFTs) and which are the pathological hallmarks of AD. Past AD drug developments have been overwhelmingly unsuccessful as the prevalence of the disease rises throughout the world, it becomes ever more crucial to develop disease-modifying drugs for AD.

In experiments conducted during the course of developing embodiments for the present invention, the inventors showed that by developing an integrative and translational drug repurposing pipeline to analyze multi-omics data generated from post-mortem brain tissues in AMP-AD, three natural compounds (NAD ⁺ , NADH, PEITC) appear to successfully perturb the pathological hallmarks of the disease by modulating multiple upstream cellular pathways linked to AD rather than targeting a singular pathway. The innovative pipeline centered on the construction of de-novo causal genetic regulatory network to discover key driver and pathways associated with AD by using a cutting-edge predictive (causal) network model. The in vitro experiments with human primary neurons and microglial cells demonstrate significant neuroprotective and phagocytosis-promoting effects of the three compounds and their combinations (NADH/PEITC and NAD '/PEITC) demonstrated strong synergistic therapeutic effects under AD condition. The brain proteomics data in 3xTg AD mice treated with individual compound further revealed different pathways, involving mitochondrial energy metabolism, immune responses, and phagocytosis, were shown to be significantly perturbed. Moreover, 3xTg mice treated with NADH, NAD+ and PEITC respectively showed significantly reduced the neurotoxic ratios of AP42 to 40 and p231-tau to total tau, and humanized-Tau (hTau) mice treated with PEITC showed significant reduction of neurotoxic ratio of p231-tau to total tau in the brain. These natural compounds individually or in combination are indicated as preventative and therapeutic agents for combating AD.

Accordingly, the present invention relates generally to neurodegenerative diseases and conditions (e.g., Alzheimer’s disease) characterized with dysfunctional energetic function, unregulated microglia phagocytic activity and other related de-regulated biological functions.

This invention further relates to methods and compositions for treating such neurodegenerative diseases and conditions with pharmaceutical compositions comprising one or more of phenyl isothiocyanate (PEITC) ( P ^hen Y' isothiocyanate ), an analog of PEITC, oxidized nicotinamide adenine dinucleotide (NAD+) reduced nicotinamide adenine dinucleotide (NADH) (

wherein such compositions are capable of protecting neurons from cell death and unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides a method of treating a mammal suffering from a neurodegenerative disorder comprising administering to the mammal a composition comprising one or more of PEITC, an analog of PEITC, NADH, and NAD+.

In certain embodiments, the present invention provides a method for preventing and/or inhibiting neuronal cell death in a mammal in need thereof, the method comprising administering to the mammal a composition comprising one or more of PEITC, an analog of PEITC, NADH, and NAD+.

In certain embodiments, the present invention provides a method for preventing and/or inhibiting unregulated microglia phagocytic activity in a mammal in need thereof, the method comprising administering to the mammal a composition comprising one or more of PEITC, an analog of PEITC, NADH, and NAD+.

In some embodiments, the composition is capable of protecting neurons from cell death and unregulated microglia phagocytic activity.

In some embodiments, the neurodegenerative disorder is selected from AD, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and mild cognitive impairment (MCI). In some embodiments, the AD is an early stage, prodromal phase of AD or late stage.

In some embodiments, the mammal is a human patient.

In some embodiments, wherein the composition is capable of protecting neurons from cell death and unregulated microglia phagocytic activity. In certain embodiments, the present invention provides a method for preventing and/or inhibiting neuronal cell death in a subject suffering from a neurodegenerative disorder (e.g., AD (e.g., early stage, prodromal phase, late stage), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI) comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more agents capable of preventing and/or inhibiting unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides for preventing and/or inhibiting unrelated microglia phagocytic activity in neuronal cells of a subject suffering from a neurodegenerative disorder (e.g., AD (e.g., early stage, prodromal phase, late stage), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI) comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more agents capable of preventing and/or inhibiting unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides a method of preventing the onset of a neurodegenerative disorder (e.g., AD (e.g., early stage, prodromal phase, late stage), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI) in a subject (e.g., a human subject) comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more agents capable of preventing and/or inhibiting unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides a method of treating and/or ameliorating the symptoms of a neurodegenerative disorder (e.g., AD (e.g., early stage, prodromal phase, late stage), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI) in a subject (e.g., a human subject) comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more agents capable of preventing and/or inhibiting unregulated microglia phagocytic activity.

Such methods are not limited to use of a particular agent capable of protecting neurons from cell death and unregulated microglia phagocytic activity. In some embodiments, the agent is selected from PEITC, an analog of PEITC, NADH, and NAD+. In some embodiments, the agent is a pharmaceutical composition comprising a therapeutically effective amount of one or more of

PEITC, an analog of PEITC, NADH, and NAD+.

The compositions, methods, systems, and kits described herein are not limited to a specific type or kind of PEITC analog. In some embodiments, a PEITC analog is any chemical moiety related to watercress and/or other cruciferous plant extraction and the structures related to PEITC.

In some embodiments, the PEITC analog is selected from:

In some embodiments, the PEITC analog is selected from:

• Allylglucosinolate (sinigrin) ( smigrm allyl isothiocyanate ( • Benzylglucosinolate (Glucotropaeolin) ( 3 ● benzyl isothiocyan ate ( _), ● Gluconasturtiin ( O- ((E -2- ^{yl)thio)propylidene)amino sulfate} ), ● (R)-4-(methylsulfinyl)butylglucosinolate (Glucoraphanin) ( glucoraphanin O 1 1 O S ² ₄ S ((E)-5- -2H- ), ● (R)-4-(methylsulfinyl)butyl isothiocyanate (sulforaphane) ( ● (R)-2-hydroxybut-3-enylglucosinolate (progoitrin) ( ● and (S)-5-vinyloxazolidine-2-thione (goitrin) ( ). In certain embodiments, the one or more agents capable of protecting neurons from cell death and unregulated microglia phagocytic activity may be comprised within any type or kind of composition. For example, in some embodiments, such a composition may be an over-the- counter composition, a pharmaceutical composition, or any kind of cosmetic composition. In certain embodiments, the present provides the following compounds: In certain embodiments, the present invention provides a composition comprising one or more of the following: PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises two or more of PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises three or more of PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the PEITC analog is selected from: isothiocyanate, Benzylglucosinolate (Glucotropaeolin), benzyl isothiocyanate, Gluconasturtiin, (R)-4-(methylsulfinyl)butylglucosinolate (Glucoraphanin), (R)-4-(methylsulfinyl)butyl isothiocyanate (sulforaphane), (R)-2-hydroxybut-3-enylglucosinolate (progoitrin), (S)-5- vinyloxazolidine-2-thione (goitrin), and any chemical moiety related to watercress and/or other cruciferous plant extraction. In some embodiments, the composition is an over-the-counter composition, or a pharmacological prescription.

In certain embodiments, the present invention provides an over-the-counter composition comprising one or more of the following: PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises two or more of PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises three or more of PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, a PEITC analog is any chemical moiety related to watercress and/or other cruciferous plant extraction and the structures related to PEITC. In some embodiments, the PEITC analog is selected from:

isothiocyanate, Benzylglucosinolate (Glucotropaeolin), benzyl isothiocyanate, Gluconasturtiin, (R)-4-(methylsulfinyl)butylglucosinolate (Glucoraphanin), (R)-4-(methylsulfinyl)butyl isothiocyanate (sulforaphane), (R)-2-hydroxybut-3-enylglucosinolate (progoitrin), and (S)-5- vinyloxazolidine-2-thione (goitrin). In some embodiments, the over-the-counter composition is a tablet, capsule, powder, suspension, or solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a translational drug-repurposing pipeline described herein (see, Example I).

FIG. 2 shows robust and significantly up- and down-regulated genes/proteins associated with AD (see, Example I).

FIG. 3 summarizes tested allelic loci-transcript and allelic loci-peptide correlation across brain regions/cell types and cohorts.

FIG. 4 depicts relationship between NADH, NAD+, PEITC and 138 key drivers (genes and peptides) for AD (see, Example I).

FIGs. 5-9 depicts graphical data demonstrating the calculations utilized to obtain the data described in Example I.

DEFINITIONS

For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

“About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.

The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as "including," "comprising/* or "having" certain elements are also contemplated as "consisting essentially of and "consisting of those certain elements.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise- indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

As used herein, the term "over-the-counter" means to provide by retail purchase without a prescription or license from a physician or medical practitioner (e.g., does not require a prescription from a physician in order to be administered to the human).

As used herein, the term "pharmaceutical compound" refers to any physical state of a material. Pharmaceutical compounds include but are not limited to capsules, tablets, liquids, topical formulations, and inhaled formulations.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. DETAILED DESCRIPTION

AD is a pressing global problem effecting over 40 million people currently living around the world (Collaborators GBDD, 2019). Furthermore, the number of individuals with AD continues to rise globally as the world’s population ages.

Pathological hallmarks of AD include neurofibrillary tangles (NFTs) and neurite plaques. NFTs result from abnormal hyperphosphorylation of Tau protein, which disrupts neuronal morphology and impairs short-term and long-term synaptic plasticity (Naseri et al. 2019). Neurite plaques are aggregates of amyloid beta (AP) peptides composed of 40 (A 40) or 42 (A 42) amino acids. A 42 is especially prone to aggregation due to its higher rate of fibrilization and insolubility (Lane et al. 2018). Elevated brain levels of A 42 can cause neurotoxicity and neuron death as evidenced by impaired learning and recognition memory (Jaeger et al. 2009). Additionally, formation of A plaques triggers microglia to polarize from a quiescent state to an activated phenotype. Microglia are myeloid cells that can perform proinflammatory/pro-killing or immunoregulatory functions in the brain (Ronaldson & Davis, 2020). They are also capable of phagocytosis, which contributes to the ability of microglia to remove cellular debris and contribute to neural repair (Akhmetzyanova et al. 2019; Ronaldson & Davis, 2020). These pathological processes suggest an opportunity for therapeutic targeting of neurons and microglia to promote neuroprotection in the setting of AD.

Efforts to develop disease-modifying AD drugs have so far overwhelmingly resulted in failure, and past developments have had little success in improving cognitive and functional ability in individuals affected by AD. Among these developments are large molecule therapeutics such as monoclonal antibodies and small molecules such as y-secretase inhibitors and P-site amyloid precursor protein cleaving enzyme (BACE) inhibitors. In preclinical trials, the monoclonal antibody bapineuzumab was shown to lower the amount of Ap present in the brain of mice, thus reducing AD pathology (Bard et al., 2000). Despite encouraging results, the drug failed to show significant improvement in functional or cognitive ability in treated patients in a phase three trial (Salloway et al., 2014). The drug also showed no significant difference in amyloid load or CSF phosphorylated tau levels between treatment and placebo groups in another phase three clinical trial (Vandenberghe et al., 2016). Other monoclonal antibodies that showed promising results but ultimately resulted in failure are solanezumab and gantenerumab. Another class of therapeutic developments currently being tested for treatment of AD are y-secretase inhibitors. Despite promising results in early trials, phase three clinical trials for the y-secretase inhibitor semagacestat were halted because of lack of efficacy and a worsening of function and cognition in patients (Doody et al., 2013). Another failed drug aimed at AD in this class is avagacestat. Other class of failed therapeutics include BACE inhibitors. In early studies, the BACE inhibitor atabecestat was shown to decrease CSF Ap levels in individuals with early-stage AD (Timmers et al., 2018). However, results from phase two clinical trials and a long-term safety and tolerability study showed a trend of worsening cognitive assessment scores in treated patients (Novak et al., 2020). Verubecestat is another BACE inhibitor that was shown to decrease CSF A levels in patients with AD (Kennedy et al., 2016) but was ultimately terminated because of failure in several later trials (Egan et al., 2019a; Egan et al., 2019b).

The multiple failures of therapeutics in clinical trials highlight a fundamental flaw of single-drug for single-target hypothesis for AD drug discovery and suggests a critical need to target multiple pathways simultaneously for a complex disease. Experiments conducted during the course of developing embodiments for the present invention resulted in the development of an integrative systems biology approach (Petyuk et al. 2018) to construct data-driven causal probabilistic models of gene and protein regulatory networks. These network models provided a systematic view of the molecular pathways and mechanisms to identify potential upstream master regulator genes and/or proteins (collectively named key drivers or therapeutic targets) that potentially modulate AD pathology. Based on the de-novo network-derived targets for AD, the inventors further developed a compound screening and prioritization process that repurposed FDA approved, natural and investigational compounds with known on-targets in the DrugBank against these therapeutic targets. The inventors identified three natural compounds (i.e., oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+, NADH) and phenethyl isothiocyanate (PEITC)) regulating total 97 AD key drivers and 212 significant (FDR<0.05) pathways identified by computational network analysis. The inventors then validated therapeutic effects of these individual compounds and their combinations in in vitro cell-based assays and two in vivo AD mice models (3xTg and hTau). The in vitro assays assessing neuroprotection and microglial phagocytosis demonstrated NAD+, NADH, PEITC individually and in combination significantly promoted neuron survival under AD condition and stimulated microglial antiinflammatory phagocytosis of neurotoxic AP42. In addition, these compounds both prevented and rescue neural injury. The 3xTg AD mice treated with NAD+, NADH, PEITC individually showed significant reduction in the levels of neurotoxic AP42 and ratio of AP42 to AP40. The humanized Tau mice treated with PEITC showed significant reduction in the level of hyperphosphorylated Tau (p231Tau) and ratio of p231Tau to Tau. The brain proteomic data generated from 3xTg AD mice further confirmed NAD+ and NADH significantly perturbed pathways related to mitochondrial metabolism and energy production, and PEITC significantly modulated pathways involved in immune responses, and phagocytosis, which validated the computational analysis. All the results point towards the effectiveness of these natural products as AD therapeutics.

Accordingly, the present invention relates generally to neurodegenerative diseases and conditions (e.g., Alzheimer’s disease) characterized with dysfunctional energetic function, unregulated microglia phagocytic activity and other related de-regulated biological functions. This invention further relates to methods and compositions for treating such neurodegenerative diseases and conditions with pharmaceutical compositions comprising one or more of PEITC, an analog of PEITC, NAD+, and NADH, wherein such compositions are capable of protecting neurons from cell death and unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides a method of treating a mammal suffering from a neurodegenerative disorder comprising administering to the mammal a composition comprising one or more of PEITC, an analog of PEITC, NADH, and NAD+.

In certain embodiments, the present invention provides a method for preventing and/or inhibiting neuronal cell death in a mammal in need thereof, the method comprising administering to the mammal a composition comprising one or more of PEITC, an analog of PEITC, NADH, and NAD+.

In certain embodiments, the present invention provides a method for preventing and/or inhibiting unregulated microglia phagocytic activity in a mammal in need thereof, the method comprising administering to the mammal a composition comprising one or more of PEITC, an analog of PEITC, NADH, and NAD+.

In some embodiments, the composition is capable of protecting neurons from cell death and unregulated microglia phagocytic activity.

In some embodiments, the neurodegenerative disorder is selected from AD, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI. In some embodiments, the AD is an early stage, prodromal phase of AD or late stage.

In some embodiments, the mammal is a human patient. In some embodiments, wherein the composition is capable of protecting neurons from cell death and unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides a method for preventing and/or inhibiting neuronal cell death in a subject suffering from a neurodegenerative disorder (e.g., AD (e.g., early stage, prodromal phase, late stage), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI) comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more agents capable of preventing and/or inhibiting unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides for preventing and/or inhibiting unrelated microglia phagocytic activity in neuronal cells of a subject suffering from a neurodegenerative disorder (e.g., AD (e.g., early stage, prodromal phase, late stage), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI) comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more agents capable of preventing and/or inhibiting unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides a method of preventing the onset of a neurodegenerative disorder (e.g., AD (e.g., early stage, prodromal phase, late stage), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI) in a subject (e.g., a human subject) comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more agents capable of preventing and/or inhibiting unregulated microglia phagocytic activity.

In certain embodiments, the present invention provides a method of treating and/or ameliorating the symptoms of a neurodegenerative disorder (e.g., AD (e.g., early stage, prodromal phase, late stage), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disease, subjective memory complaints, and MCI) in a subject (e.g., a human subject) comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of one or more agents capable of preventing and/or inhibiting unregulated microglia phagocytic activity.

Such methods are not limited to use of a particular agent capable of protecting neurons from cell death and unregulated microglia phagocytic activity. In some embodiments, the agent is selected from PEITC, an analog of PEITC, NADH, and NAD+. In some embodiments, the agent is a pharmaceutical composition comprising a therapeutically effective amount of one or more of

PEITC, an analog of PEITC, NADH, and NAD+.

The compositions, methods, systems, and kits described herein are not limited to a specific type or kind of PEITC analog.

In some embodiments, a PEITC analog is any chemical moiety related to watercress and/or other cruciferous plant extraction and the structures related to PEITC.

In certain embodiments, the PEITC analog is selected from:

(Glucotropaeolin), benzyl isothiocyanate, Gluconasturtiin, (R)-4- (methylsulfinyl)butylglucosinolate (Glucoraphanin), (R)-4-(methylsulfinyl)butyl isothiocyanate (sulforaphane), (R)-2-hydroxybut-3-enylglucosinolate (progoitrin), and (S)-5-vinyloxazolidine-2- thione (goitrin).

In certain embodiments, the one or more agents capable of protecting neurons from cell death and unregulated microglia phagocytic activity may be comprised within any type or kind of composition. For example, in some embodiments, such a composition may be an over-the- counter composition, a pharmaceutical composition, or any kind of cosmetic composition. In certain embodiments, the present provides the following compounds: pharmaceutically acceptable salts, solvates, and/or prodrugs thereof.

In certain embodiments, the present invention provides a composition comprising one or more of the following: PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises two or more of PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises three or more of PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, a PEITC analog is any chemical moiety related to watercress and/or other cruciferous plant extraction and the structures related to Allylglucosinolate (sinigrin), allyl isothiocyanate,

Benzylglucosinolate (Glucotropaeolin), benzyl isothiocyanate, Gluconasturtiin, (R)-4-

(methylsulfinyl)butylglucosinolate (Glucoraphanin), (R)-4-(methylsulfinyl)butyl isothiocyanate (sulforaphane), (R)-2-hydroxybut-3-enylglucosinolate (progoitrin), and (S)-5-vinyloxazolidine-2- thione (goitrin). In some embodiments, the composition is an over-the-counter composition, or a pharmacological prescription. In certain embodiments, the present invention provides an over-the-counter composition comprising one or more of the following: PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises two or more of PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises three or more of PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, the composition comprises PEITC, an analog of PEITC, NAD+, and NADH. In some embodiments, a PEITC analog is any chemical moiety related to watercress and/or other cruciferous plant extraction and the structures related to PEITC. In some embodiments, the PEITC analog is selected from: isothiocyanate, Benzylglucosinolate (Glucotropaeolin), benzyl isothiocyanate, Gluconasturtiin, (R)-4-(methylsulfinyl)butylglucosinolate (Glucoraphanin), (R)-4-(methylsulfinyl)butyl isothiocyanate (sulforaphane), (R)-2-hydroxybut-3-enylglucosinolate (progoitrin), and (S)-5- vinyloxazolidine-2-thione (goitrin). In some embodiments, the over-the-counter composition is a tablet, capsule, powder, suspension, or solution.

The methods and compositions of the present invention are useful in treating mammals. Such mammals include humans as well as non-human mammals. Non-human mammals include, for example, companion animals such as dogs and cats, agricultural animals such live stock including cows, horses and the like, and exotic animals, such as zoo animals.

Treatment can include administration of an effective amount of one or more of an agent capable of protecting neurons from cell death and unregulated microglia phagocytic activity (e.g., one or more of PEITC, an analog of PEITC, NAD+, and NADH). Administration can be by any suitable route of administration including buccal, dental, endocervical, intramuscular, inhalation, intracranial, intralymphatic, intramuscular, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, ophthalmic, oral, otic, biliary perfusion, cardiac perfusion, priodontal, rectal, spinal subcutaneous, sublingual, topical, intravaginal, transermal, ureteral, or urethral. Dosage forms can be aerosol including metered aerosol, chewable bar, capsule, capsule containing coated pellets, capsule containing delayed release pellets, capsule containing extended release pellets, concentrate, cream, augmented cream, suppository cream, disc, dressing, elixer, emulsion, enema, extended release fiber, extended release film, gas, gel, metered gel, granule, delayed release granule, effervescent granule, chewing gum, implant, inhalant, injectable, injectable lipid complex, injectable liposomes, insert, extended release insert, intrauterine device, jelly, liquid, extended release liquid, lotion, augmented lotion, shampoo lotion, oil, ointment, augmented ointment, paste, pastille, pellet, powder, extended release powder, metered powder, ring, shampoo, soap solution, solution for slush, solution/drops, concentrate solution, gel forming solution/drops, sponge, spray, metered spray, suppository, suspension, suspension/drops, extended release suspension, swab, syrup, tablet, chewable tablet, tablet containing coated particles, delayed release tablet, dispersible tablet, effervescent tablet, extended release tablet, orally disintegrating tablet, tampon, tape or troche/lozenge.

Intraocular administration can include administration by injection including intravitreal injection, by eyedrops and by trans-scleral delivery.

Administration can also be by inclusion in the diet of the mammal such as in a functional food for humans or companion animals.

It is also contemplated that certain formulations containing the compositions (e.g., compositions comprising one or more of PEITC, an analog of PEITC, NAD+, and NADH) capable of protecting neurons from cell death and unregulated microglia phagocytic activity are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms. Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated such as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art. The formulations can also contain substances that diminish proteolytic degradation and promote absorption such as, for example, surface-active agents.

The specific dose can be calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also depend upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity in assay preparations such as has been described elsewhere for certain compounds (see for example, Howitz et al., Nature 425:191-196, 2003). Exact dosages can be determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration.

The present invention also provides kits comprising one or more of agents capable of protecting neurons from cell death and unregulated microglia phagocytic activity (e.g., one or more of PEITC, an analog of PEITC, NAD+, and NADH) and instructions for administering the agent to an animal (e.g., a human patient suffering from a neurodegenerative disorder (e.g., AD)). The kits may optionally contain other therapeutic agents.

EXPERIMENTAL

The following examples are provided to demonstrate and further illustrate certain preferred embodiments of the present invention and are not to be construed as limiting the scope thereof. As used herein, the use of pronouns (e.g., “our”, “we”, etc.) refers to the inventors.

Example I.

The AD Population and Data Pre-processing

A translational discovery pipeline for drug discovery starts with integrative analysis of multi-layer omics data (genotyping, RNA-seq, proteomics) generated from three human cohorts sponsored by the Accelerating Medicines Partnership - Alzheimer's Disease (AMP-AD) consortium including i) the post-mortem dorsolateral prefrontal cortex (DLPFC) of 612 persons in the Religious Order Study and the Memory and Aging Project (ROSMAP) (Bennett et al., 2012b, 2012a; Jager et al., 2018), ii) the prefrontal cortex of 306 subjects in the Mount Sinai Brain Bank (MSBB) RNA-seq study (Wang et al., 2018), and iii) temporal cortex (TCX) of 266 patients in the Mayo RNA-seq study (Allen et al., 2016). In addition to AMP-AD data, we (the inventors) included the human BRAINOME dataset (RuiChangBrain2018) containing i) frontal and temporal cortical regions of 345 subjects in KRNONOSII cohorts and ii) frontal cortical regions of 409 subjects in RUSH cohort. For RNA-seq data pre-processing, we applied a consensus data processing pipeline including voom normalization, variance partition analysis (VP A) and covariate adjustment. For proteomics data pre-processing, we applied normalization, variance partition analysis and covariate adjustment. For genotyping data pre-processing, we performed imputation.

Besides brain region RNA-seq data, as brain tissue consists of various cell types, including neurons, endothelial and glial cells, we applied the computational framework population-specific expression analysis (PSEA)[23] to deconvolve bulk-tissue (brain region) RNA-seq data from post-mortem brain regions and isolate a neuron-specific and microglial- specific gene expression signal. We chose the PSEA method over other popular methods, such as Cibersort [24], dtangle [25], DSA [26], or NNLS [27], because these methods cannot directly estimate cell-type specific residuals from the bulk-tissue RNA-seq data, instead only estimating cell fraction in a bulk-tissue sample. We demonstrated the robustness of this deconvolution method using random selection of neuronal biomarkers derived from single-cell RNA-seq (scRNAseq) studies [28-32], Next, we applied a novel systems biology approach to integrate whole-genome genotype data with the brain regionl and single cell-type RNAseq, and to build causal network models of AD.

Translational Computational Systems Biology Approach to Repurpose Compounds for AD The computational drug repurposing pipeline (Figure 1) is centered on a cutting-edge computational systems biology model which integrates the multi-omics (genotyping, RNA-seq, proteomics) data of matched subjects in a cohort to construct network models and to identify therapeutic targets. For every cell type/brain region in each cohort, we identified AD-related (gene and protein) expression signature, single-nucleotide variants associated with gene expression and protein level, transcriptomic and proteomic co-expression modules associated with AD, data-driven systems biology network model and AD-associated pathways, and a set of potential therapeutic targets for AD (Online Method).

To evaluate robustness of AD-related analysis results across different datasets, we performed a meta-analysis of the above results across all cell types/brain regions, and cohorts. After identified the robust therapeutic targets, we repurposed both FDA approved and investigational compounds by matching the list of targets to known targets of compounds in DrugBank (version 5.0), which contains 9591 drug entries including 2037 FDA-approved small molecule drugs, 241 FDA-approved biotech (protein/peptide) drugs, 96 nutraceuticals and over 6000 experimental drugs. The repositioned compounds are then ranked according to the number of predicted AD targets a compound hit. Consequently, we prioritized three repurposed compounds which are validated to have neuroprotective effects on human primary neurons and promoting amyloid-beta uptake in human primary microglial cells under AD condition.

Meta-transcriptomic analysis revealed robust differential expression signature in Alzheimer’s disease

To identify robust expression signature significantly associated with AD, we performed dataset-specific differential expression (DE) analysis for all brain regions, cell types and cohorts. By comparing expression values from AD and pathologically confirmed controls (CN), we identified different number of significant (FDR<0.05) DE genes and proteins between AD and CN samples in each dataset.

To examine the robustness of DE signature, we performed a meta-analysis on the differential expression for total 19,119 variables (genes and proteins) across all datasets. We calculated the overall standardized mean difference (SMD) and its p-value for every variable with a random-effect model. A positive SMD value indicates a variable is overall up-regulated in AD across all datasets and a negative SMD value indicates a variable is overall down-regulated in AD across all datasets. Among the 19,119 variables, there are 4,463 significant up-regulated genes/proteins (SMD>0 and SMD’s p-value<0.05) and 3,796 significant down-regulated genes/proteins (SMD<0 and SMD’s p-value<0.05) across all the datasets. We further calculated p-value of heterogeneity for every variable across all datasets. A variable’s expression pattern is considered homogeneous/robust across all datasets if its heterogeneity p-value is larger than 0.05. As a result, we identified 3,344 variables which are significantly and robustly up-regulated in AD and 2,939 variables which are significantly and robustly down-regulated in AD. We highlighted the most robust and significantly up- and down-regulated genes/proteins associated with AD in Figure 2.

To evaluate the quality of DE signature derived from individual dataset, we calculated overlap significance between individual significant (FDR<0.05) DE signature with the significant (p-value<0.05) meta-DE signature. Out of the 10 individual DE lists, all 8 DE (gene) signatures are significantly overlapping with the meta-DE signature whereas none of the DE (protein) signature significantly overlapping with the meta-DE protein signature. The results indicated that meta-DE signature is a core AD-associated signature associated with AD across cohorts, brain regions, cell types and technical variance. In addition, we also identified individual DE signature sharing the common Meta-DE but also with unique signature specific to the cohort, brain region, and cell types. To retain the unique signature, we will proceed analysis with individual DE signature and residuals and integrate the therapeutic targets at last.

Robust genetic variants associated with AD expression traits across AMP -AD

We analyzed association of gene and peptide expression traits with genome-wide variants assayed in every dataset we collected. For genotype variants assayed by SNP arrays in ROSMAP and MAYO cohorts, we performed imputation (Online Methods). Overall, we tested allelic loci- transcript and allelic loci-peptide correlation across brain regions/cell types and cohorts which is summarized in Figure 3. We found that 5609, 9041, 4343, 3089, 5813, 3875, 3331, 6103, 2563, 5186, 5059, 4200, 5460 genes tested were significantly correlated with allele dosage (FDR < 0.05 or FDR<0.01) in the respective dataset: MAYO TCX CQN Nonimputed vl, MAYO TCX CQN Imputed vl, MAYO TCX CQN Imputed v2, MAYO TCX CQN Nonimputed v2, MAYO TCX CPM Imputed, MAYO CD68 CQN, MAYO ENO2 CQN, ROSMAP PFC CPM vl, ROSMAP PFC CPM v2, ROSMAP CD68 CPM, ROSMAP ENO2 CPM, KRONOS PFC TCX, RUSH PFC TCX, as well as 226 peptides tested were significantly corrected with allele dosage (FDR<0.05) in MSBB PFC Protein. Of these cis expression quantitative trait loci (cis-eQTL) detected in each dataset, 7 cis-eQTL were overlapping across all 14 datasets and 2093 cis-eQTL were overlapping across 13 out of 14 datasets in Figure 3 and 102,548 cis-eQTL were replicated in at least 50% of all datasets even though these datasets comprised different brain regions, cell types and cohorts. We examined the known association of total 829,970 significant cis-eQTLs across all datasets with diseases in GWAS catalog, which could help to identify co-morbid conditions with AD. We found that out of the 829,970 cis-eQTLs, 1,532 are significantly associated with at least one disease in GWAS catalog. Out of the 2093 robust cis-eQTLs, 16 are significantly associated with at least one disease in GWAS catalog (highlighted in Figure 3 with their associated diseases).

Identification of AD-related transcriptional modules by meta-coexpression module analysis

While DE analysis revealed AD-related expression signature, the power of such analysis to detect a small-to-moderate expression difference is small. On the other hand, gene and protein expression traits from coherent biological pathways are correlated and clustered into modules of traits which can be recapitulated by constructing co-expression modules.

To identify co-regulated human transcriptomic and proteomic modules that were robustly observed in a generalized manner across all datasets generated from brain regions, cell types and cohorts, we firstly constructed a co-expression network of the AD samples in each dataset by using WGCNA (Parikshak et al., 2016) and identified total 253 dataset-specific co-expression modules (MAYO TCX RNA: 23; MAYO ENO2 RNA: 21; MAYO CD68 RNA: 46; ROSMAP PFC RNA: 27; ROSMAP ENO2 RNA: 15; ROSMAP CD68 RNA: 47; KRONOS PFC TCX RNA: 27; KRONOS PFC TCX Protein: 6; RUSH PFC TCX RNA: 29; RUSH PFC TCX Protein: 8).

To compare the structure of all co-expression modules, we calculated overlap enrichment of module-molecule membership between all pairs of modules across all datasets and identified 1690 module pairs with significant overlap (FDR<0.05 for Fisher’s Exact Test). As expected, we found that co-expression modules from different datasets (regions, cell types, and cohorts) have good rate of replication indicating that there is no significant bias and/or batch effect in the processed residuals between the datasets.

Next, we characterized the functional relevance and AD-association of all 249 modules based on the following enrichment tests per module: 1) fold-enrichment for AD-related DE signature; 2) fold-enrichment for single cell-type biomarkers for single cell-type module network. In total, we selected 72 AD-associated modules (interchangeably AD-modules) that were significantly enriched for AD-related DE signature and single cell-type biomarkers. We evaluated the association significance with the AD pathological traits that is available in each dataset (MAYO: BRAAK, THAL; ROSMAP: BRAAK, CERAD, COGDX, KRONOS/RUSH: BRAAK, DET). Last, we repeated the structural comparison among AD-associated modules by overlapping enrichment test and identified 430 significant module-pairs (FDR<0.05) out of all AD-module pairs across datasets. Similar to all modules, we found that AD-associated modules from different datasets (regions, cell types, and cohorts) have good rate of replication indicating that module selection is not biased by datasets.

In next section, we employed predictive network model to infer causality between the variables (genes and proteins) from AD-modules in each dataset.

Data-driven Causal Regulation Inference by Predictive Network for AD

The ultimate goal of the experiments described herein was to identify upstream pathways and robust master regulators (key drivers) of AD pathology for drug repurposing. As last step of the translational drug repurposing pipeline, we built a causal network of transcriptomic and proteomic regulations by integrating analysis from previous steps with RNA-seq and proteomic residuals for every dataset. In total, we built 78 predictive networks over different versions of cis- eQTLs, cell types (neuron/microglia), brain regions (TCX/PFC) and cohorts (ROSMAP/MAYO/MSBB).

To build the predictive network, we first pooled all variables of AD-associated modules in every dataset limiting the causal network model to AD pathology only. Noting that the data- driven approach may not fully capture known signaling pathways because the data is generated and analyzed from a specific biological layer at a time, we expanded the AD-associated seeding variables by integrating prior signaling pathways in the knowledge databases via PathFinder algorithm (BioRXiv, CSC, Brain) (Online Method). Third, we incorporated cis-eQTLs as structural prior in the network where cis-eQTLs causally affect the expression levels of associated variables as a source of systematic perturbation to infer causal relations.

To compare network models, we first extracted all (causal) edges from each network model. Similar to module meta-analysis, we calculated overlap of edges between all pairs of predictive network models and identified network pairs with significant overlap (FDR<0.05 for Fisher’s Exact Test). As expected, most network structures that are significantly overlap with each other are within the same dataset, which reflects molecule and functional differences of regions and cell types related to AD pathology. Interestingly, we found that RNA network significantly overlaps with the multiscale (RNA-peptide) networks from KRONOS and RUSH cohorts, where these two multiscale networks significant overlap with each other confirming a replicated genetic regulatory mechanism associated with AD in the same brain region across the two cohorts. The two peptide networks from KRONOS and RUSH significantly overlap with each other indicating a robust PPI program associated with AD in the same brain regions across the two cohorts. The microglial and neuron-specific networks from ROSMAP significantly overlap with brain region network from ROSMAP indicating there might be a core subnetwork of pathways across neurons and microglia associated with AD pathology. To investigate the core mechanisms associated with AD across brain regions, cell types, and cohorts, we employed spine algorithm to derive a core subnetwork from each predictive network and evaluated the overlaps among these core structures. We found that the overlap significance among core subnetworks is consistent with the overlaps among entire predictive networks.

Repurposing Therapeutic Drugs Targeting Multiple Pathological Pathways in AD

To identify potential therapeutic compounds for Alzheimer’s disease, we firstly applied Key Driver Analysis (KDA) to the 78 predictive network models across brain regions, cell types and cohorts in the study. The KDA identified a list of key drivers/therapeutic targets in each network. Next, we repurposed total 13,682 drug entries in the latest DrugBank (version 5.1.7) including 2,646 approved small molecule drugs, 1,405 approved biologies (protein, peptide, vaccines, and allergenics), 131 nutraceuticals and over 6,402 experimental (discovery-phase) drugs, with additional 5,234 non-redundant protein (i.e. drug target, enzyme, transporter, carrier) sequences are linked to these drug entries, by matching the drugs with known targets to the key drivers derived identified from the 78 predictive network models. Based on the number of matched targets, we prioritized all 13,682 drugs compounds and selected three top compounds, i.e. NADH, NAD+, PEITC, hitting total 138 key drivers (genes and peptides) for AD in Figure 4. After mapping peptides to gene, 138 key drivers are mapped to 85 unique genes.

The translational drug-repurposing pipeline (Figure 1) has generated an integrated landscape of AD-associated mechanisms by different analyses including AD-associated differential expression signature, genetic variants, co-expression modules, causal network models, therapeutic targets/key drivers and repositioned drugs. To gain therapeutic insights, we (the inventors) extracted significant pathways enriched by this set of AD-associated molecule landscape, including 10 AD-related up-/down-regulated DE signatures derived from individual dataset, 1 AD-related up-/ down-regulated meta-DE signature, 14 lists of genes/peptides significantly associated with genetic variants in AD, 72 AD-associated modules, 78 AD- associated predictive networks, 78 AD-associated core subnetworks, and 1 set of 138 key drivers. Based on these 265 sets of AD-associated genomic phenotypes (see, Figure 5), we identified total 2,776 non-repeating pathways that are significantly enriched by at least one set of phenotype. Interestingly, we found that NADH and NAD+ which hits the largest number of key drivers modulates mitochondrial-related pathways, such as TCA-cycle, respiratory electron transport, oxidative phosphorylation, glycolysis and gluconeogenesis, indicating that mitochondrial functions and mitochondrial energy metabolism is an important therapeutic target for Alzheimer’s disease. Through the analysis, we nailed down 81 AD key driver genes and peptides in these pathways regulated by NADH and NAD+. In addition to NADH and NAD+, the third compound PEITC modulates chaperone-mediated protein folding pathways whose disruption is leading to accumulation of amyloid aggregates, and Tubulin folding pathway which forms the microtubules whose loss contributes to tau disassociation and abnormal tau accumulation in AD, parkin-ubiquitin in the proteasomal pathway. Parkin is E3-ubiquitin-protein ligase that ubiquitinates itself and specific substrate proteins playing a protective role by sequestering misfolded proteins and mutation in this gene will cause familial Parkison’s disease(ref). Other important PEITC-regulated pathways related to inflammation, cell cycle, cell death, apoptosis and survival, such as TNF-alpha signaling, mTOR signaling pathway, P38 signaling mediated by MAPKAP kinase, activation of BAD and translocation to mitochondria, EGF/EGFR pathway, and FAS pathway. In addition, PEITC regulates phagosome functions of the cells, translocation of Glut4 (glucose transporter) to the cell membrane which is critical for glucose uptake from bloodstream by the cells, and energy homeostasis pathways, such as Liver kinase Bl -mediated cellular events. PEITC also regulates cellular response to stress and DNA damage, such as regulation of HSF1 -mediated heat shock response, activation of BH3-only proteins, and chromatine remoding pathways via regulating MTA family of proteins, whose functions are only recently recognized in the brain, eye, circadian rhythm, mammary gland biology, spermatogenesis, liver, immunomodulation and inflammation, cellular radio-sensitivity, and hematopoiesis and differentiation. One of the nervous pathways directly regulated by PEITC is Trk receptors signaling mediated by PI3K and PLC-gamma. Trk receptors are a family of tyrosine kinases that regulates synaptic strength and plasticity in the mammalian nervous system, which affect neuronal survival and differentiation through several signaling cascades. Through the analysis, 57 AD-driving genes and peptides (27 unique genes) in these pathways regulated by PEITC were nailed down. Cytotoxic Effect of NADH, NAD+ and PEITC on Primary Cultures of Human Neurons and Human Microglia

A defining characteristic of the many failed AD therapeutics has been the focus on targeting single biological pathways in an effort to achieve therapeutic efficacy. This is particularly apparent with drugs that were designed to specifically target Ap plaques such as solanezumab (Doody et al. 2014) or avagacestat (Coric et al. 2015). This approach has identified three natural product compounds (i.e. , NADH, NAD+, PEITC) that interact with molecular targets associated with mitochondrial bioenergetics and/or antioxidant effects, which indicate an ability to interact with multiple AD-associated pathways and/or multiple CNS cell types relevant to AD pathology. Furthermore, cellular concentrations of NADH and NAD+ have been shown to be decreased in patients with late onset AD (Sonntag et al. 2017). This fact further emphasizes that a key characteristic of the AD phenotype is impaired mitochondrial energy metabolism and an abnormal shift towards glycolytic energy production (Sonntag et al. 2017).

To evaluate therapeutic effects on AD of these three repositioned compounds discovered by a translational computational systems biology pipeline, we first validated the cytotoxic effect of each compound, administered individually or in combination, on primary cultures of human neurons and on primary cultures of human microglia. To perform these critical experiments, we incubated cultured cells with 0 pM (control condition), 10 pM. 20 pM, 40 pM, 50 pM, 75 pM, and 100 pM of each compound and two combinations (NAD+/PEITC and NADH/PEITC). After 24 h incubation at 37°C in a humidified incubator, cell viability was measured using a 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Following treatment with NADH, NAD+, or PEITC and their combinations, cell viability in primary cultures of human neurons and microglia was not significantly altered as compared to control conditions (Dunnett’s T3 adjusted p-value>0.05), suggesting that the three compounds, when administered individually, did not have a cytotoxic effect (Figure 6A,B). Of particular importance to the therapeutic development of these compounds, combinations of NADH and PEITC or NAD+ and PEITC, when formulated in equimolar concentrations, did not significantly affect cell viability of both primary cultures of human neurons and primary cultures of human microglia (Figure 6A,B). Overall, our results demonstrated that NADH, NAD+, PEITC individually or in combination are non-toxic to human CNS cells in vitro, which is a critical step in the preclinical validation of these compounds. Neuroprotective Effect of Natural Compounds on Primary Cultures of Human Neurons Exposed to Aβ1-42 Aggregates. Next, we determined whether these compounds, administered independently or formulated in combination, could protect primary cultures of human neurons against neurotoxic effects of Aβ1-42 aggregates, an in vitro condition relevant to AD pathology. We evaluated both prevention and rescue of neuronal death by NADH, NAD+ and PEITC (Figure 7). In both experiments, the cultured human neurons were exposed to 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 20 μM, 40 μM, 50 μM, 75 μM, and 100 μM of each compound, either individually or in equimolar combinations. As a baseline parameter associated with Aβ1-42 exposure, primary cultures of human neurons challenged by Aβ1-42 experienced a 50% or greater reduction in cell viability (Figure 7). In the prevention experiment, cultures were exposed to fresh compounds every 24h over the 48h timeline of this experiment (i.e., 0h, 24h, and 48h) to resemble a pharmacological regimen of once daily dosing. At 24h, we added 1 μM of Aβ1-42 aggregates to the cell culture media and measured cell viability at 72h using the MTT assay (Online Method, Figure 7A). Our results showed that NADH, NAD+, PEITC either individually or in combination significantly prevented neuronal loss as compared to the negative control condition (i.e., cellular exposure to 1 μM Aβ1-42 without administration of any individual compounds or compound combinations). Additionally, we discovered that individual compounds have a wide effective dosage range from 100nM to 100 μM in prevention of neuron loss following Aβ1-42 exposure. Interestingly and surprisingly, we discovered that equimolar combinations of NADH/PEITC and NAD+/PEITC exhibited synergistic effects of neuroprotection that expanded the minimal effective concentration to 1nM and 10nM respectively as compared to the 100nM concentration for single compounds. To rule out false positive data points and experimental artifacts, we showed that sucrose did not prevent any neuronal death in response to Aβ1-42 aggregates (Figure 7A). In the rescue experiments, primary cultures of human neurons were exposed to 1 μM of Aβ1-42 aggregates at 0h for a period of 48h to model existing cellular damage under conditions relevant to AD. After 24h incubation with Aβ1-42, we added NADH, NAD+, PEITC, NADH/PEITC, and NAD+/PEITC directly to the culture media containing Aβ1-42 and incubated the cultured human neurons for an additional 24h when cell viability was measured using our MTT assay. Similar to the prevention experiments, NADH and NAD+ significantly rescued neuronal death caused by Aβ1-42 with a wide dosage range from 100nM to 100μM, and PEITC significantly rescued the neuron death from 1nM to 100μM (Figure 7B). The equimolar combination of NADH/PEITC and NAD+/PEITC exhibited synergistic neuroprotection with effective dosage ranges from 1nM to 100μM respectively. In summary, all five formulations of three repurposed natural compounds demonstrated significant effects in both preventing and rescuing neuronal loss caused by Aβ1-42 aggregate deposition, in vitro conditions that are characteristic of early-stage Alzheimer’s disease. Phagocytosis-promoting Effect of Natural Compounds in Primary Cultures of Human Microglia Exposed to Aβ1-42 Aggregates There is considerable interest in microglia-targeted therapies for AD, in part due to the phagocytotic functionality of these cells that can promote neuronal recovery and/or repair. Indeed, phagocytosis of insoluble Aβ by microglia can facilitate CNS clearance of Aβ aggregates and limit formation of amyloid plaques (Ronaldson& Davis, 2020). Indeed, Aβ can bind to receptors at the microglial cell surface, an event that can trigger release of proinflammatory cytokines such as TNF-⍺ and IL-1β, factors that are known to cause neuronal damage (Wang et al.2015). Additionally, Jiang and colleagues discovered that triggering receptor expressed in myeloid cells 1 (TREM1) can promote Aβ phagocytosis by microglia (Jiang et al.2016). This study was compelling since genetic knockdown of TREM1 resulted in increased CNS levels of Aβ1-42 in the APP/PSEN1 mouse model of AD. In contrast, selective overexpression of TREM1 on microglia attenuated AD neuropathological and promoted neurocognitive improvement (Jiang et al.2016). To determine if the natural compounds individually or in combination could promote phagocytosis in microglia, we designed prevention and rescue experiments to measure Aβ1-42 uptake in primary cultures of human microglia via intracellular fluorescence (Online method). Cultured human microglia were exposed to 1nM, 10nM, 100nM, 1μM, 10μM, 20μM, 40μM, 50μM, 75μM, and 100μM of each compound treatment either individually or in combination with equimolar in both prevention and rescue experiment settings. In the prevention setting, we treated the cultured microglial medium with compounds at 0h. After 24h, we added 1μM of Aβ1-42 aggregates to the treated media and measured cell viability at 72h using the Aβ uptake assay (Online Method, Figure 8A). The results showed that 27 out of total 50 treatment conditions (5 formulations x 10 dosages) significantly increased Aβ1-42 uptake by microglia as compared to the negative control (i.e., microglial cells that were exposed to Aβ1-42 without any compounds). Of particular significance and interest, we observed that PEITC alone stimulated microglial phagocytosis of Api-42 under all tested dosages from InM to lOOpM. NADH significantly increased microglial phagocytosis of Api-42 at lOOnM, IpM, 20pM and 50pM. NAD+ significantly increased microglial Api-42 uptake at lOnM, I pM. 20pM and 75pM. Interestingly, the equimolar combination of NADH and PEITC enabled a lower minimal effective dosage at lOnM comparing to NADH alone. The equimolar combination of NAD+ and PEITC showed a range of effective dosage ranges from lOOnM, IpM, lOpM and 20pM. In the rescue experiment settings, primary cultures of human microglia were first challenged with Api-42 for 24h. At 24h, compound formulations were added to the culture media containing Api-42. Uptake of Api-42 was then measured 24h later (i.e., at the 48h experimental time point, Figure 8B). The results showed that three and eight dosages of NADH and NAD+ significantly rescued microglial phagocytosis of Api-42 respectively comparing to the negative control condition(NADH: 40, 50, 75pM, and NAD+: InM to 50pM). PEITC significantly increased the microglial phagocytosis of Api-42 at lOnM, IpM, lOpM, 20pM and 40 pM. The equimolar formulation of NADH/PEITC and NAD+/PEITC significantly stimulated microglial phagocytosis at 40, 50, 75 and lOOpM, and 50, 75, lOOpM respectively.

The two equimolar formulations of NADH/PEITC and NAD+/PEITC showed synergistic effects to prevent and rescue neuronal death caused by insoluble Api-42. To determine the optimal dosage of the two formulations for neuronal rescue and microglial phagocytosis stimulation, we treated the primary cultures of human neurons and primary cultures of human microglia in the prevention settings respectively with exhaustive combination of individual dosage of lOpM, 20pM, 40pM, 50pM, 75pM, and lOOpM. The results showed that the maximal prevention of neuronal loss can be achieved by the combination of 20pM PEITC with 20pM NADH (mean=96.4%, Figure 9A), and 50pM PEITC with 40pM NAD+ (mean=95%, Figure 9B) respectively, comparing to the negative control (mean=58.1%). The maximal stimulation of microglial phagocytosis of Api-42 can be achieved by the combination of lOpM PEITC with 20pM NADH (mean=70.6%, Figure 9C) and 40pM PEITC NAD+ with 20pM NAD+ (mean=72.8%, Figure 9D) respectively, comparing to the negative control (mean=50.3%).

Experimental Conclusion

Taken together, we have used unique translational systems biology drug discovery pipeline to identify natural product compounds (i.e., NADH, NAD+, PEITC) that could be directed towards preclinical evaluation as AD therapeutics. In vitro assays for the chosen compounds have demonstrated that they have positive effects against Api-42-mediated cytotoxicity in primary cultures of human neurons and can promote phagocytosis of Api-42 by primary cultures of human microglia. The experimental design included both pre- and postcompound exposure conditions, which effectively modelled preventative and therapeutic situations that are encountered during aging and/or AD pathology. Indeed, the identification of these natural product compounds demonstrated the power of the computational approach. Compared to previous treatment strategies for AD that have failed when subjected to the rigors of clinical trials, we are confident that the repurposing strategy will yield effective therapeutics due to the consideration of several biological pathways and the ability to modify the physiology of multiple CNS cell types.

Example II.

This example describes the synthesis of PEITC analogs (see scheme 1). me 1 EITC analogs and synthesis

The structures contain isothiocynate and aromatic ring connected by (CH2)n (n = 0-12). Aromatic refers to: phenyl and fused phenyl ring and the rings tethering substituent groups such as alkyl, aryl, Cl, Br, F, I, amino, OH, alkoxy, etc and heteroaromatics such as pyridine, indole, quinoline etc and these heteroaromatics attaching substituent groups such as alkyl, aryl, Cl, Br, F, I, amino, OH, alkoxy, etc. Some selected examples are provided shown below. We have purchased and/or made some of these compounds for biological activities. They can be prepared by reaction of corresponding amines with CS2 in the presence of acetyl chloride (AcCl) and TEA (tri ethyl amine).

Additional experiments will be conducted with these PEITC analogs using high- throughout screening with MTT and Abeta uptake assays to assess the therapeutic efficacy of such PEITC analogs in the treatment of neurodegenerative disorders as described herein.

Additional in vivo experiments will be conducted with these PEITC analogs to assess the therapeutic efficacy of such PEITC analogs in the treatment of neurodegenerative disorders as described herein.

Example III.

Experiments will be conducted with a purpose of investigating, in vivo, effects of therapeutic combinations of NAD+/PEITC and NADH/PEITC on biomarkers associated Alzheimer’s disease (i.e. , amyloid beta deposition, accumulation of phosphorylated tau) and neurocognitive performance. These studies will be conducted in two transgenic mouse models of Alzheimer’s disease (i.e., 3xTg mice and hTau mice). The 3xTg mouse model contains three mutated human transgenes that are associated with familial Alzheimer’s disease (i.e., APP Swedish, MAPT P301L, PSEN1 M146V). These mice display progressive amyloid beta deposition that can be detected in some brain regions by 3-4 months of age. Additionally, tau hyperphosphorylation is present in the hippocampus of these mice by 12-15 months of age. The hTau mouse model has been designed to express only human tau isoforms. As these mice age, they express six isoforms of human tau and develop tau pathology in the CNS. In such experiments, transgenic Alzhemer’s disease mice will be treated with increasing dose combinations of NAD+/PEITC and NADH/PEITC via intraperitoneal and oral routes via an once-daily dosing paradigm for 28 consecutive days. Levels of amyloid beta and hyperphosphorylated tau as well as biomarkers associated with neuroinflammation and oxidative stress will be measured using state-of-the-art ELISA technology. Neurocognitive performance in these mice will be evaluated using our conditioned place preference (CPP) methodology. Additionally, pharmacokinetic studies will be performed on NAD+/PEITC and NADH/PEITC dosage combinations following oral and intravenous dosing. Brain and plasma concentrations of NAD+, NADH, and PEITC will be measured using LC/MS-MS. It is expected that both NAD+/PEITC and NADH/PEITC will decrease brain expression of protein biomarkers relevant to Alzheimer’s disease and improve neurocognitive performance as measured by the CPP paradigm. It is also expected that brain permeation of NAD+, NADH, and PEITC will occur at therapeutically effective concentrations as shown by the pharmacokinetic studies.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

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EQUIVALENTS

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.