A METHOD OF TREATING ESSENTIAL TREMOR BY TARGETING LEAKY RyR1

CHANNELS

INVENTORS

Andrew R. Marks, Phyllis L. Faust, Sheng-Han Kuo, Elan D. Louis, and Regina T. Martuscello

A METHOD OF TREATING ESSENTIAL TREMOR BY TARGETING LEAKY RyR1 CHANNELS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 63/270,652 filed on October 22, 2021, the contents of which are incorporated here by reference in its entirety.

FIELD

The present subject matter relates to a method for treating essential tremor, and particularly, to a method for treating essential tremor by targeting leaky RyR1 channels. BACKGROUND

The cerebellum is a brain region that is thought to be involved in a number of neurodegenerative diseases, including essential tremor (ET), spinocerebellar ataxias, multiple system atrophy, Parkinson’s disease, and dystonia, yet detailed studies of the pathology of the cerebellum in many of these diseases are either very limited or have never been performed.

ET stands out as one of the most prevalent neurological diseases. Its most recognizable feature is an 8 - 12 Hz action tremor (i.e., tremor occurring during volitional movement) of the arms, head tremor, as well as limb and gait ataxia in some patients. ET is present in 4% of individuals aged >40 and as many as 22% - 23% aged >65 years, among whom it may be as prevalent as Alzheimer’s disease and osteoarthritis. Although the condition is sometimes labeled "benign," this term is misleading. The tremor is chronic and usually progressive, producing disabilities with basic daily activities such as eating, writing, and body care. More than 90% of patients who seek medical care report disability, and in severely affected end-stage patients, the tremor results in substantial incapacitation. Between 15 and 25% of patients are forced to retire prematurely, and 60% choose not to apply for a job or promotion because of uncontrollable shaking.

While the traditional belief had been that ET was not associated with mortality, recent data show a 45% increased risk of mortality in patients with ET. In particular, death from pneumonia was six times more common in ET cases than controls (p = 0.02), suggesting that ET is a disease associated both with morbidity and mortality. There is no cure for ET, and first- line medications, of which there are only two, are ineffective in 50% of patients. This leaves a sizable population of ET patients untreated and disabled. Deep-brain stimulation surgery provides partial relief for one feature of ET (arm tremor), but its use is generally restricted to severe, pharmacologically intractable cases; it is invasive; and the surgery itself carries risks of stroke, infection, and mortality. There is also emerging evidence that its effectiveness wanes over time as the underlying disease progresses.

Accordingly, a method for treating essential tremor by targeting leaky RyR1 channels is needed.

SUMMARY

A method of treating essential tremor (ET) can include administering a calcium channel stabilizer to a subject in need thereof. The Ry cal compounds (Ryanodine receptor calcium release channel stabilizers) restore binding of leaky Ryanodine Receptor 1 (RyR1) to calstabinl in a brain cell of the subject (e.g., a cell in the cerebellum) to stabilize the leaky RyR1 and, thereby, reduce or inhibit endoplasmic reticulum (ER)-calcium leak. Inhibiting ER-calcium leak can ameliorate ET and reduce cerebellar oscillatory activity. In an embodiment, the calcium channel stabilizer is Ry cal S107, shown below: or a salt thereof, for example an HC1 salt.

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments will now be described in detail with reference to the accompanying drawings.

Fig. 1 is a diagram depicting how RyR1 interacts with calstabin-1 to modulate its channel function.

Fig. 2A depicts immunoprecipitation analysis of RyR1 of the postmortem ET cerebellum.

Fig. 2B is a graph showing site-specific phosphorylation of RyR1 in ET cerebellum compared to controls.

Fig. 2C is a graph showing nitrosylation of RyR1 in ET cerebellum compared to controls.

Fig. 2D is a graph showing oxidation of RyR1 in ET cerebellum compared to controls.

Fig. 2E is a graph showing dissociation of RyR1 from calstabin-1 in ET cerebellum compared to controls. Fig. 2F depicts RyR1 immunoprecipitation analysis of RyR1 of the postmortem ET cerebellum, Parkinson’s disease cerebellum and control cerebellum.

Fig. 3 A depicts immunoprecipitation analysis of point mutation in RyR1 mouse model that mimics site-specific phosphorylation of serine 2844.

Fig. 3B is a schematic depicting the freely moving tremor measurement setup.

Fig. 3C is a time/frequency spectrum graph showing tremor severity of heterozygous RyR1-S2844D mice and homozygous RyR1-S2844D mice.

Fig. 3D is an intensity/frequency graph depicting severity of tremor in homozygous and heterozygous mice.

Fig. 3E is an intensity/frequency graph depicting severity of tremor in homozygous and heterozygous mice.

Fig. 3F is a time/frequency spectrum graph showing tremor severity of homozygous RyR1-S2844D mice in action and at rest.

Fig. 3G is an intensity/frequency graph depicting severity of tremor in homozygous RyR1-S2844D mice in action and at rest.

Fig. 3H is a graph showing age-dependent increase in tremor intensity of RyR1-S2844D mice.

Fig. 4A is a time/frequency spectrum graph showing robust cerebellar oscillatory activity, coherent with tremor in RyR1-S2844D mice using a freely moving setup.

Fig. 4B is a time/frequency spectrum graph showing perturbed cerebellar oscillatory activity and suppressed tremor upon cerebellar microinfusion of dantrolene in RyR1-S2844D mice.

Fig. 5 A shows an immunostain of a 6-month-old RyR1-S2844D mouse cerebellum with calbindinD28k, demonstrating focal axonal swellings (arrows).

Fig. 5B shows an immunostain of a 6-month-old RyR1-S2844D mouse cerebellum with VGlut2, demonstrating extension of climbing fibers into outer 20% of molecular layer.

Fig. 5C depicts graphs showing slice physiology of 3-month-old RyR1-S2844D cerebellum demonstrating reduced CV of simple spikes, showing enhanced regularity, but no change in firing rate (n =14 cells in each group) (CV: coefficient of variation, ISI inter-spike intervals. : not significant).

Fig. 6A shows a time/frequency spectrum graph showing impact of caffeine on tremor in a RyR1-S2488D mouse and a corresponding time/frequency line graph showing impact of caffeine on tremor in a RyR1-S2488D mouse.

Fig. 6B is blot indicating enhanced binding calstabin-1 to RyR1 in the cerebellum of RyR1-S2488D mice that are administered Ry cal SI 07 for 1 month.

Fig. 6C is an image of PCs (calbindin, green) of the motor cerebellum of a WT mouse expressing hMG3qmCherry (red).

Fig. 7A is a schematic depicting binding partners and regulators of RyR1 channel phosphorylation and stabilization

Fig. 7B is a diagram showing in vitro microsomal Ca ²⁺ leak assay.

Fig. 8A is a blot showing protein phosphatase-1 (PPI), but not protein kinase A (PKA), depleted from RyR1 macromolecular complex in ET cerebellum.

Fig. 8B is a representative fluorescence-time plot of microsomal calcium release and corresponding graph of calcium leak (% of uptake) showing increased ER microsomal calcium release in ET vs. controls (n = 3 each, and reversed by in vitro Rycal S107 treatment in ET cases .

Fig. 8C is a graph showing that total tremor scores and PC counts/mm correlate with RyR1 calstabin-1 depletion in ET cases.

Fig. 9 depicts string network clusters of dysregulated genes in ET cerebellar cortex for highly relevant pathways of axon guidance (red), kinesins/microtubule motor activity (dark blue), ER-golgi transport (green), and calcium signaling (purple).

Fig. 10 depicts differential gene expression analysis in PCs isolated by laser capture microdissection for 34 ET cases and 16 age-matched control samples.

Fig. 11 depicts string network clusters dysregulated in ET PCs for highly relevant gene ontology (GO) pathways.

Figs. 12(A)-12(F) are graphs depicting 12(A) principal component analyses of the z scored raw data across all seven diagnostic categories; 12(B) the Pearson’s correlation between each morphologic metric and the first principal component; 12(C) the Pearson’s correlation between each morphologic metric and the second principal component; 12(D) principal component analyses of the z scored raw data across select diagnoses; 12(E) the Pearson’s correlation between each morphologic metric and the first principal component; and 12(F) principal component analyses of the z scored raw data across select diagnoses.

Figs. 13(A)-13(C) depict 13(A) a heatmap showing metrics (rows) and diagnoses (columns), with each cell in the heatmap representing the average log2-fold-change (disease vs. control) for each metric; 13(B) hierarchical clustering of the correlation coefficients between all pairwise combinations of metrics, with colored scale of red (positive correlation), white (no correlation) and blue (negative correlation), prominent positively correlated (red and orange asterisks) or negatively correlated (blue asterisk) (observables drive many of the significant differences across disease categories, and are designated as “ Key Drivers’" in 13(A)); 13(C) three scores which were computed across disease categories by combining the average fold-change (disease/control) for selected metrics and plotted on a log-transformed scale, including a “Severity Score” (panel a), a “Purkinje Cell Loss Score” (panel b), and a score reflecting climbing fibers

(CFs) in the outer 20% of the molecular layer (panel c) (within each violin, the dashed line shows the median value, and the dotted lines indicate outer quartiles in the data distribution).

DETAILED DESCRIPTION

Definitions

The following definitions are provided for the purpose of understanding the present subject matter and for constructing the appended patent claims.

It is noted that, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges, percentage ranges, or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the described subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described subject matter.

As used herein, the term “salt” has the same meaning as commonly understood to one of ordinary skill in the art. Specifically, a salt is a chemical compound consisting of an ionic assembly of positively charged cations and negatively charged anions.

As used herein, the term “hydrate” has the same meaning as commonly understood to one of ordinary skill in the art. Specifically, a hydrate is a compound with extra water molecules that are part of its structure.

As used herein, the term “solvate” has the same meaning as commonly understood to one of ordinary skill in the art. Specifically, a solvate is a compound formed by the interaction of a solvent and a solute.

As used herein, the term “complex” has the same meaning as commonly understood to one of ordinary skill in the art. Specifically, a complex is a molecular entity formed by loose association involving two or more component molecular entities (ionic or uncharged), or the corresponding chemical species. The bonding between the components is normally weaker than in a covalent bond.

As used herein, the term “prodrug” has the same meaning as commonly understood to one of ordinary skill in the art. Specifically, a prodrug is a precursor of a drug - a compound that, on administration to a subject, undergoes metabolic processes that convert the compound to the drug.

Throughout the application, descriptions of various embodiments use “comprising” language. However, it will be understood by one of skill in the art, that in some specific instances, an embodiment can alternatively be described using the language “consisting essentially of’ or “consisting of’.

For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Embodiments

A method of treating essential tremor (ET) can include administering a calcium channel stabilizer to a subject in need thereof. In one embodiment, the subject can be a patient. In an embodiment, the calcium channel stabilizer is orally administered to a patient to treat essential tremor. In an embodiment, a pharmaceutical composition comprises the calcium channel stabilizer and a pharmaceutically acceptable carrier.

Provided herein is a method of treating essential tremor, comprising administering a therapeutically effective amount of a calcium channel stabilizer to a patient in need thereof, the calcium channel stabilizer including 1,4-benzothiazepine, whereby the calcium channel stabilizer enhances a binding affinity of calstabin-1 to RyR1 in Purkinje cells of the patient. In some embodiments, the calcium channel stabilizer is orally administered to the patient. Provided herein is a method of treating essential tremor, comprising administering a therapeutically effective amount of a calcium channel stabilizer to a subject in need thereof, the calcium channel stabilizer comprising a 1,4-benzothiazepine moiety whereby the calcium channel stabilizer enhances or restores binding of calstabin-1 to RyR1 in a brain cell of the subject. In an embodiment, the brain cell is a cerebellum cell. In an embodiment, the brain cell is a Purkinje cell.

In an embodiment, the calcium channel stabilizer enhances or restores binding of (ryanodine receptor type 1) RyR1 with FKBP12 (calstabin-1) in a brain cell, for example a cell in the cerebellum to stabilize the closed state of RyR1 channels and, thereby, reduce, ameliorate, or inhibit endoplasmic reticulum (ER)-calcium leak into the cytoplasm. As described herein, RyR1 are calcium channels localized in the ER membrane that control calcium release into the cytoplasm. Disturbances in calcium handling by the ER of PCs can be linked to ET. Thus, inhibiting ER-calcium leak can ameliorate ET and reduce cerebellar oscillatory activity.

In an embodiment, the calcium channel stabilizer is a small molecule compound that can restore binding of FKBP12 (calstabin-1) to RyR1 (e.g., PKA hyperphosphorylated RyR1). In an embodiment, the calcium channel stabilizer can restore the binding of leaky RyR1 with calstabin-1 in brain cells. In other embodiments, the brain cell comprises a cerebellum cell, or the brain cell comprises a Purkinje cell. In an embodiment, the calcium channel stabilizer can penetrate the blood/brain barrier. The calcium channel stabilizer can include compounds commonly referred to as “Ry cals,” such as 1,4-benzothiazepines and related structures, described in U.S. Patent No. 8,710,045, issued on April 29, 2014, and U.S. Patent No. 8,022,058, issued on September 20, 2011, the contents of each of which are incorporated by reference herein.

In an embodiment, the Rycal calcium channel stabilizer comprises the following structural formula: wherein, n is 0, 1, or 2;

R is located at one or more positions on the benzene ring; each R is independently selected from the group consisting of H, halogen, — OH, — NH2, — NO2, — CN, — N3, — SO3H, acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, — SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R1 is selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, — SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero- )cycloalkyl, and (hetero-)cyclyl;

R2 is selected from the group consisting of — C=O(R5), — C=S(R6), — SO2R7, — POR8R9, — (CH2)m-R10, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero- )cycloalkyl, and (hetero-)cyclyl;

R3 is selected from the group consisting of H, CO2Y, CONY, acyl, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, — SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl; and wherein Y is selected from the group consisting of H, alkyl, aryl, cycloalkyl, and heterocyclyl;

R4 is selected from the group consisting of H, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, — SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R5 is selected from the group consisting of — NR16, NHNHR16, NHOH, — OR15, CONH2NHR16, CO2R15, CONR16, CH2X, acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero- )cyclyl;

R6 is selected from the group consisting of — OR15, NHNRie, NHOH, — NRie, CH2X, acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R7 is selected from the group consisting of — OR15, — NR16, NHNHR16, NHOH, CH2X, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R8 and R9 independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, — S — , — CN, — N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

Rio is selected from the group consisting of NH2, OH, — SO2R11, — NHSO2R11, C=O(Ri2), NHC=O(R ₁₂ ), — OC=O(R ₁₂ ), and — POR ₁₃ R ₁₄ ;

R11, R12, R13, and R14 independently are selected from the group consisting of H, OH, NH2, NHNH2, NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, , nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl;

X is selected from the group consisting of halogen

R15 and R16 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl; or a salt, hydrate, solvate, complex, or prodrug thereof.

In an embodiment, the calcium channel stabilizer is selected from the group consisting

or a salt, hydrate, solvate, complex, or prodrug of these compounds.

In an embodiment, the calcium channel stabilizer can include the following general structural formula: wherein, n is 0, 1, or 2; q is 0, 1, 2, 3, or 4; each R is independently selected from the group consisting of H, halogen, — OH, — NH ₂ , — NO2, — CN, — CF ₃ , — OCF ₃ , — N ₃ , — SO ₃ H, — S(=O) ₂ alkyl, — S(=O)alkyl, — OS(=O) ₂ CF ₃ , acyl, — O-acyl, alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl, heteroaryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, — O-acyl, alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl, heteroaryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)arylthio, and (hetero-)arylamino may be optionally substituted;

Ri is selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;

R ₂ is selected from the group consisting of H, — C(=O)R5, — C(=S)R6, — SO ₂ R7, — P(=O)R8R9, — (CH ₂ ) _m — R10, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be optionally substituted;

R ₃ is selected from the group consisting of H, — CO ₂ Y, — C(=O)NHY, acyl, — O-acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted; and wherein Y is selected from the group consisting of H, alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl, and wherein each alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;

R4 is Selected from the group consisting of H, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;

R5 is selected from the group consisting of — NR15R16, — (CH ₂ ) _t NR15R16, — NHNR15R16, — acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted, and wherein t is 1, 2, 3, 4, 5, or 6;

R ₆ is selected from the group consisting of CH ₂ X, acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

R ₇ is selected from the group consisting of CH ₂ X, alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

R ₈ and R ₉ independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

Rio is selected from the group consisting of

R ₁₁ , R ₁₂ , R ₁₃ , and R ₁₄ independently are selected from the group consisting of H, OH, NH ₂ , — NHNH2, — NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; X is selected from the group consisting of halogen, — CN, — CO ₂ R ₁₅ , — C(=O)NR ₁₅ R ₁₆ , — NR ₁₅ R ₁₆ , — OR ₁₅ , — SO ₂ R ₇ , and — P(=O)R ₈ R ₉ ; and R15 and R16 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, OH, NH2, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; and optionally R15 and R16 together with the N to which they are bonded may form a heterocycle which may be substituted; the nitrogen in the benzothiazepine ring may optionally be a quaternary nitrogen; or an enantiomer, diastereomer, tautomer, pharmaceutically acceptable salt, hydrate, solvate, complex, or prodrug thereof.

In an embodiment, the calcium channel stabilizer can include the following general structural formula: wherein, n is 0, 1, or 2; q is 0, 1, 2, 3, or 4; each R is independently selected from the group consisting of H, halogen, acyl alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl, heteroaryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)arylthio, and (hetero-)aryl amino; wherein each acyl, — O-acyl, alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl, heteroaryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)arylthio, and (hetero-)arylamino may be optionally substituted;

R1 _i s selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;

R ₂ is selected from the group consisting of alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be optionally substituted;

R ₃ is selected from the group consisting of H, — CO2Y, — C(=O)NHY, acyl, — O-acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted; and wherein Y is selected from the group consisting of H, alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl, and wherein each alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;

R ₄ s selected from the group consisting of H, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;

R ₅ is selected from the group consisting of acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted, and wherein t is 1, 2, 3, 4, 5, or 6;

R ₆ is selected from the group consisting of CH2X, acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; R ₇ is selected from the group consisting of — alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

R ₈ and R ₉ independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;

R10 is selected from the group consisting of

R11, R12, R13, and R14 independently are selected from the group consisting of H, OH, NH2, — NHNH2, — NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; X is selected from the group consisting of halogen, and

R15 and R16 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, OH, NH2, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optiona7ly substituted; and optionally R ₁₅ and R ₁₆ together with the N to which they are bonded may form a heterocycle which may be substituted; the nitrogen in the benzothiazepine ring may optionally be a quaternary nitrogen; or an enantiomer, diastereomer, tautomer, pharmaceutically acceptable salt, hydrate, solvate, complex, or prodrug thereof; provided that when q is 0 and n is 0, then R ₂ is not H, Me, Et, — C(=O)NH2, — C(=O)NHPh, — C(=S)NH-nButyl, — C(=O)NHC(=O)CH ₂ C1, — C(=O)H, — C(=O)Me, — C(=O)Et, — C(=O)CH=CH ₂ , — S(=O) ₂ Me, — S(=O) ₂ Et, — C(=O)OC(CH ₃ ) ₃ , or 9-β-D- ribofuranosyl-9H-purin-6-yl or — C(=O)Ph; further provided that when q is 0 and n is 1 or 2, then R ₂ is not H, — C(=O)Me, — C(=O)Et, — S(=O) ₂ Me, — S(=O) ₂ Et or, — C(=O)OC(CH ₃ ) ₃ ; further provided that when q is 1, and R is Me, Cl, CN or F at the 6 position of the benzothiazepine ring, or Br at position 7 of the benzothiazepine ring, then R ₂ is not H, Me, — C(=O)H, — C(=O)Me, — C(=O)Et, — C(=O)Ph, — S(=O) ₂ Me, or — S(=O) ₂ Et; further provided that when q is 1, n is 0, and R is OH, C ₁ -C ₃ alkoxyl at the 7 position of the benzothiazepine ring, then R ₂ is not H, — C(=O)CH=CH ₂ , — C(=O)CH ₂ Br, — (CH ₂ ) ₃ - 4-benzylpiperidine, further provided that when q is 0, n is 0 or 2, R1 is H or oxo, R ₃ is H or Me and R4 is H, then R ₂ is not — C=ONHPh, — C=ONHCOCH ₂ C1, — C=ONH ₂ , — C=ONH(n-Bu), — C=S(NHPh), — C=S(NHCOCH ₂ C1), — C=S(NH ₂ ), — C=SNH(n-Bu), — CH ₂ CH ₂ N(Me) ₂ , — CH ₂ CH ₂ NH ₂ or — C=OCHC1 ₂ ; further provided that when q is 2, each R is methoxy at positions 7 and 8 of the benzothiazepine ring, R ₃ and R4 are each H and n is 0 or 2, then R1 is not methyl, — CH ₂ Ph or 3,4- dimethoxybenzyl, and R ₂ is not — C(=O)Me; further provided that when q is 0, R1, R ₂ and R4 are each H, then R ₃ is not H or CH ₃ ; further provided that when q is 0, R ₂ is H, — CH ₂ C(=O)OCH ₃ , — CH ₂ C(=O)NH ₂ , — C(=O) — C6H4 — C1, — CH ₂ — C6H4 — C1, — (CH ₂ ) ₃ -morpholino, — (CH ₂ ) ₃ -4-methylpiperazino, — (CH ₂ ) ₂ — C(=O)OCH ₃ , 2,2',3,3'-tetrahydro-4(5H)-l,4 benzothiazepine, or — CH ₂ -Ph, R ₃ and R4 are either H or CH3 but not both CH3, then R1 is not oxo; further provided that when q is 2, each R is methoxy at positions 7 and 8 or 7 and 9 of the benzothiazepine ring, R1, R2 and R4 are each H and n is 0, then R3 is not H; further provided that when q is 0, Ri, R3 and R4 are each H and n is 0, then R2 is not methyl, benzotriazolylmethyl, 4-methoxybenzyl, Ph — C=C — CH2 — , 4-chlorobenzyl, ethyl, pentyl, — CH ₂ P(O)(OCH ₂ CH3) ₂ , Ph-CO— CH2CH2— , C(=O)CH=CH ₂ and C(=O)CH ₂ Br; and further provided that when q is 1, R is CH3 at position 9 of the benzothiazepine ring, R1, R3 and R4 are each H and n is 0, then R2 is not methyl, benzotriazolylmethyl, pentyl, — CH2P(O)(OCH2CH3)2, or 4-methoxybenzyl. In an embodiment, the calcium channel stabilizer enhances or restores binding of calstabin-1 to RyR1 in a brain cell, wherein the brain cell may comprise a cerebellum cell or a Purkinje cell.

In an embodiment, the calcium channel stabilizer is selected from the group consisting of:

. In an embodiment, the calcium channel stabilizer is administered to a subject and enhances or restores binding of calstabin-1 to RyR1 in a brain cell of the subject. In further embodiments, the brain cell comprises a cerebellum cell, or the brain cell comprises a Purkinje cell.

In an embodiment, the calcium channel stabilizer is Ry cal SI 07, shown below:

, or a salt thereof, such as the HCl salt.

An embodiment of the present subject matter is directed to a pharmaceutical composition comprising a calcium channel stabilizer and a pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical composition includes Ry cal SI 07 or one or more salts, hydrates, solvates, complexes, or prodrugs thereof, and a pharmaceutically acceptable carrier. In an embodiment, the calcium channel stabilizer enhances or restores binding affinity of calstabin-1 to RyR1 in a brain cell of the subject. In further embodiments, the brain cell comprises a cerebellum cell, or the brain cell comprises a Purkinje cell.

To prepare the pharmaceutical composition, the calcium channel stabilizer, as the active ingredient, is intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques. Carriers are inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorings, sweeteners, preservatives, dyes, and coatings. In preparing compositions in oral dosage form, any of the pharmaceutical carriers known in the art may be employed. For example, for liquid oral preparations, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like. Further, for solid oral preparations, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like.

The present compositions can be in unit dosage forms such as tablets, pills, capsules, powders, granules, ointments, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampules, auto-injector devices or suppositories, for oral parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. The composition can be presented in a form suitable for daily, weekly, or monthly administration. The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful, suppository and the like, an amount of the active ingredient necessary to deliver an effective dose. A therapeutically effective amount of the Calcium channel stabilizer or an amount effective to treat a disease, such as ET, may be determined initially from the Examples described herein and adjusted for specific targeted diseases using routine methods.

It is believed that dysfunction of Purkinje cells (PC)s, the principal neurons in the cerebellar cortex, is a core biologic feature of ET. Morphological changes in PCs have previously been identified in ET, including excitatory synaptic organization abnormalities, dendritic spine alterations, moderate PC loss, and a broad range of PC axonal changes, including proximal axonal swellings called torpedoes. It was further found that a mouse model with ET-like CF-PC synaptic pathology developed ET-like tremor, supporting that PC synaptic pathology contributes to tremor. Indeed, there is mounting evidence that ET can be conceptualized as a disease of the cerebellum, with dysfunction linked to morphologic changes associated with PCs.

While calcium handling in the endoplasmic reticulum (ER) is a basic cellular function, the cerebellum is particularly vulnerable to disturbances of this pathway. PCs heavily depend on ER calcium channels for their intrinsic pacemaking activity. PCs have two unique physiological features: 1) very high basal firing rate at 50-100Hz, and 2) dependence on calcium signaling to control the precision of the firing rate and rhythmicity. These key features make PCs among the neurons with the highest metabolic demand and intracellular calcium loads in the brain. Intracellular calcium levels of PCs are tightly controlled by ER calcium handling, and PCs use two main channels to gate ER calcium release: 1,4,5-inositol trisphosphate receptor type 1 (IP3R1) and ryanodine receptor type 1 (RyR1). The RyR1 axis is regulated by the influx of extracellular calcium through plasmalemmal T-type calcium channels, which increase the RyR1 channel opening probability to allow further release of ER calcium into the cytoplasm, a process that is termed calcium induced calcium release. T-type calcium channel genetic mutations lead to SCA42. While these human genetic mutations in ER handling are known to be important for cerebellar ataxia, it is becoming increasingly clear that RyR1 and ER calcium handling may play a central role in ET.

RyRs are a class of calcium channels localized in the ER membrane that control calcium release and thereby regulate intracellular calcium levels. RyR1 is predominantly expressed in PCs in the brain and in skeletal muscles, whereas RyR2 is expressed in hippocampus and cerebral cortex in the brain and in the heart. An RNAseq analysis of postmortem ET cerebellum conducted by the present inventors identified RyR1 transcript level alterations and dysregulation of ER calcium handling pathway. RyR1 is also an ER calcium channel with unique biophysical properties. Upon cellular stress, RyR1 undergoes site-specific phosphorylation at serine 2844 by protein kinase A (PKA). This phosphorylation of RyR1 leads to a subsequent cascade of biochemical events: 1) oxidation and nitrosylation of the channel, which eventually lead to 2) the displacement of its native binding partner, calstabin-1.

As the binding to calstabin-1 is critical to stabilize the closed state of RyR1 channels, loss of this interaction causes calcium release from the ER to cytoplasm, creating a chronic “leaky” state (Fig. 1). As shown in Fig. 1, under cellular stress, RyR1 undergoes protein kinase A (PKA)-mediated phosphorylation, oxidation and nitrosylation. These posttranslational modifications collectively cause conformation changes and displacement of calstabin-1, which in turn destabilizes the closed state and renders RyR1 chronically “leaky.”

As described herein, ET cerebellum has increased levels of phosphorylated RyR1, along with other post-translational modifications of the channel (i.e., oxidation and nitrosylation) and loss of binding with calstabin-1, which together provide evidence of a RyR1 leaky state. These data further support the notion that ET could be a disease of genetic predispositions with environmental influences, converging at RyR1, a unique channel situated at the center of the ER calcium handling pathway.

As described herein, a genetically modified mouse model with a point mutation in RyR1 mimics constitutive site-specific phosphorylation (S2844D), was used to determine whether leaky RyR1 contributes to tremor. In this mouse model, RyR1 becomes oxidized and nitrosylated, with loss of binding to calstabin-1, indicating a RyR1 leaky state (Fig. 1). Both heterozygous and homozygous RyR1-S2844D mouse models developed robust 10Hz tremor, at the frequency range of human ET. Moreover, homozygous RyR1-S2844D mice displayed more severe tremor than heterozygous RyR1-S2844D mice, demonstrating a gene dosedependent effect. The RyR1-S2844D mice had action tremor with minimal rest tremor, recapitulating the key clinical feature of ET patients. The results are summarized in (Table 1).

Table 1

Using in vivo recording of cerebellar physiology in a freely moving setup in homozygous RyR1-S2844D mice, it was further found that robust cerebellar oscillatory activity, coherent with tremor, can be blocked with cerebellar micro-infusion of dantrolene, a selective RyR1 blocker. These results suggest that cerebellar leaky RyR1 causes abnormal cerebellar physiology that contributes to tremor.

As described herein, a study is conducted to show that dysfunctional ER calcium handling leads to structural and physiological alterations in the cerebellum that drive tremor using both mouse models and postmortem human ET brains. Another study is conducted to determine whether bi-directional modulation of ER calcium handling alters PC physiology and tremor in RyR1 -leaky mice and to examine the PC specificity of these alterations. Another study is conducted to determine whether the upstream regulators for RyR1 biochemical remodeling and the degree of RyR1 leakiness are altered in the postmortem human ET cerebellum. These metrics are correlated with tremor severity and PC pathologic changes. The present teachings are illustrated by the following examples.

EXAMPLES

EXAMPLE 1 RyR1 in the postmortem ET cerebellum is biochemically remodeled ET is a disease associated with various environmental stressors and the key regulator of ER calcium handling, RyR1, undergoes a variety of post-translational modifications in response to cellular stress. These post-translational modifications of RyR1 in the postmortem ET cerebellum by RyR1 were examined by co-immunoprecipitation analysis.

It was found that RyR1 in ET cerebellum undergoes extensive post-translational modifications: site-specific phosphorylation, nitrosylation, oxidation, and dissociation from its native stabilizing binding partner, calstabin-1 (Figs. 2A-2E). These biochemical changes impair the ability of RyR1 to stably close, causing them to be “leaky”. Disease specificity was studied next by comparing with Parkinson’s disease, the second most common tremor disorder. It was found that, unlike ET, Parkinson’s disease cerebellum does not have biochemical remodeling of RyR1, demonstrating the specificity to ET (Fig. 2F).

EXAMPLE 2

A mouse model with leaky RyR1 develops progressive, ET-like action tremor To determine whether leaky RyR1 may contribute to tremor or is merely a non-specific consequence of cellular stress in ET, a mouse model was established with a point mutation in RyR1 that mimics constitutive site-specific phosphorylation of serine 2844 (RyR1-S2844D), which leads to subsequent oxidation and nitrosylation of RyR1 and displacement of calstabin- 1 (Fig. 3 A) and recapitulates the biochemical leaky phenotypes of RyR1 in ET (Fig. 2A). A freely moving setup (Fig. 3B), was used to determine whether this mouse model develops tremor. Both heterozygous and homozygous RyR1-S2844D mice developed tremor at 10 Hz, and homozygous mice exhibited more severe tremor than heterozygous mice, suggesting genedose effects (Figs. 3C-3E). Since ET patients have predominant action tremor, tremor in homozygous RyR1-S2844D mice (“RyR1-S2844D mice”, hereafter) in moving or rest state was investigated, by coupling tremor measurement with movement detection (Fig. 3B). It was found that RyR1-S2844D mice develop tremor during action but minimal tremor at rest (Figs. 3F-3G). ET is also a progressive disorder and tremor becomes more severe with aging. As such, the mouse model also exhibited progressive tremor with aging (Fig. 3H). These data show that RyR1 leaky mice recapitulate key features of ET (Table 1).

Cerebellar physiology of RyR1 leaky mice. The in vivo recording setup was used next to measure tremor with simultaneous recording of cerebellar physiology in freely moving RyR1- S2844D mice.

Robust cerebellar oscillatory activity was found, by local field potential (LFP) recording, coherent with tremor (Fig. 4A). To determine whether leaky RyR1 in the cerebellum plays a role in such oscillatory activity and tremor, cerebellar microinfusion of the selective RyR1 blocker, dantrolene, was performed. In a RyR1-S2844D mouse, dantrolene shifted cerebellar oscillatory activity to a lower frequency and also caused tremor suppression. Both cerebellar oscillatory activity and tremor gradually returned in the washout period (Fig. 4B). These data support the role of cerebellar leaky RyR1 in tremor.

EXAMPLE 3

Structural and Physiological Changes of the Cerebellum in Tremor RyR1 leaky mice are used as a model to determine cerebellar anatomic structure (PC synaptic organization and degeneration), physiology (PC firing patterns in slice and cerebellar oscillatory activity I freely moving mice), and biochemistry (in vitro assays of RyR1 channel properties) in tremor emergence and progression.

Overall strategy and choice of time points: Tremor and long-term recording of in vivo cerebellar physiology with chronic headstages are followed in RyR1-S2844D mice. Three (3) time points (3, 6, and 12 months) are chosen for slice physiology, morphological analyses, and biochemical studies. RyR1-2844D mice exhibit robust action tremor at 6-month-old, and tremor becomes progressively worse with aging (Fig. 3H). For these experiments, 3-month-old mice are used. After sacrificing the mice, the cerebellum is cut in half along the sagittal midline. Half of the cerebellum is fixed in 4% paraformaldehyde for immunohistochemistry, providing sufficient material for neuropathological assessments described below. The remaining half of the cerebellum is further divided into two portions, with midline vermal sections of cerebellum used to prepare acute slices for physiology, and the remaining cerebellum is frozen and used for biochemical assay of RyR1 leak.

Tremor and in vivo cerebellar physiology measurement: Tremor is measured with Convuls-1, a platform (Columbus Instruments) with a load sensor beneath, which is connected to an AC amplifier (BlackRock) with the filters set at a bandpass of l-50Hz, 81-83 to allow mice to move freely on the platform. This platform has been well characterized to measure tremor in rodents.81-83 A real time video capture system, NeuroMotiveTM, is adapted to synchronize the measurement of tremor frequency and amplitude and mouse movements (Figs. 3F, 3G, and 4B), which allows reliable separation of action tremor and rest tremor. Cerebellar oscillatory activity is measured using LFP recording (Fig. 4A). LFP is measured because cerebellar oscillatory activity 1) is the physiological correlate for tremor, 2) exists in RyR1- S2844D mice (Fig. 4A), and 3) has been observed in ET patients by electroencephalogram, providing translatability for future clinical trials. Surface electrodes (California Fine Wire Company) are implanted in the mouse cerebellum with chronic headstages at 1 -month old age and tremor and cerebellar oscillatory activity is measured bi-weekly until 12-month-old age (Fig. 4A).

Cerebellar slice physiology: PC activity in sagittally sectioned acute cerebellar slices is measured at different time points. PCs in lobules V and VI, the motor cerebellum, are recorded in artificial cerebral spinal fluid as previously described. PCs have large cell bodies and are readily visually identified in these tissue sections. Recordings of neuronal activity is performed with electrodes made from glass capillaries. Data are acquired with a HEKA EPC 10 amplifier, and spike sorting is performed offline using Phython 2.7 with SciPy package. The spontaneous firing frequency of simple spikes and the coefficient of variation (CV) of interspike intervals are analyzed, which have been shown to be tremor correlates. This method has been extensively used to study physiological underpinning in several SCA animal models, and PC slice recording is feasible in the mature cerebellum in adult mice beyond 4- to 6-month-old age.

Neuropathological evaluation: Since a wide range of cerebellar pathology has been identified in ET, the rewiring of cerebellar circuitry could occur at different temporal sequences, contributing to tremor emergence and progression. To further understand the anatomical alterations in tremor, four ET-relevant readouts are measured to study PC pathology in the lobules V and VI, in 7m thick paraffin sections. These readouts that have been identified in the postmortem ET cerebellum were chosen: 1) abnormal PC synaptic organization (i.e., climbing fibers form abnormal synaptic connection with thin, spiny branchlets of PC dendrites, which should have been in the parallel fiber territory), 2) reduced PC dendritic spine density, 3) modest PC loss, and 4) PC axonal swelling (i.e., torpedoes).

PC synaptic organization: Dual immunofluorescence of vesicular glutamate transporter 2 (VGlut2) is performed to visualize climbing fiber synapses (SYSY 135404) and calbindin to visualize PCs (Swant CB38) in cerebellar parasagittal sections. The percentage of VGlut2 puncta on the thin, spiny PC dendritic branchlets, identified as more than 6 dendritic spines in 1 pm length of PC_dendrites, and the percentage of climbing fibers in the outer 20% of the cerebellar molecular layer is analyzed, 2) PC dendritic spine density: Calbindin immunofluorescence is performed to visualize PC dendritic spines in parasagittal sections and to measure linear density of PC dendritic spines, as previously described, 3) PC number: PC number is counted in the motor cerebellum in Hematoxylin and Eosin stained parasagittal sections and the counts adjusted to PC layer length, as previously described. Although complex stereological analyses will not be performed, this method can detect as low as a 15% loss of PCs 4) PC axonal torpedoes: PC axonal torpedoes are counted, as previously described. ER calcium leak assay: RyR1 leakiness is determined with a cerebellar microsomal calcium flux assay, which is described in detail (see Fig. 7B).

Sex as a biological variable, sample size, and scientific rigor: 12 mice per group are studied at 3 different time points of anatomical, physiological, and biochemical assessment (3-, 6- and 12- month-old). With this sample size, the mean outcome for a group with a margin of error of about +/- 0.63 in units of SD can be estimated, which allows detection of a standardized effect size of about 1.20 in units of SD with 80% power, setting the significance level at 0.05. From preliminary data of tremor measurement (Fig. 3H), standardized effects sizes of 4.8 in 6- month-old compared to 9-month-old RyR1-S2844D mice can be observed.

A total of 96 mice are used. The mice are randomized into each age group. While sex differences in RyR1-S2844D mouse tremor were not observed, gender may play some role in cerebellar pathology in ET patients. Therefore, equal numbers of male and female mice are allocated in each group to explore sex differences Research staff are blinded to age and genotype of the mice during the tremor, cerebellar physiology, and pathology assessments.

EXAMPLE 4

Determine if bi-directional modulation of ER calcium handling alters PC physiology and tremor

To demonstrate that manipulations of ER calcium handling bi-directionally improves or worsens tremor and corresponding cerebellar physiology, RyR1 leaky mice are utilized as a model to test the effects of manipulating cerebellar ER calcium handling on tremor and cerebellar physiology, using in vivo pharmacology and chemogenetics. Mouse genetics are used to test the PC specificity of leaky RyR1 in tremor.

Tremor and in vivo cerebellar physiology measurement: Caffeine, a potent activator for RyR1, is used to determine whether aggravating RyR1 leakiness will worsen cerebellar physiology and tremor. An immunostain of a 6-month-old RyR1-S2844D mouse cerebellum with calbindin (Fig. 5A) does not show marked PC loss, but axonal torpedoes are seen (Fig. 5 A, arrows and inset). With VGlut2 and calbindin staining (Fig. 5B), increased climbing fibers in outer 20% of the molecular layer are seen (arrows, beyond dotted line). Slice physiology of 3-month-old RyR1-S2844D cerebellum (Fig. 5C) demonstrates reduced CV of simple spikes, showing enhanced regularity, but no change in firing rate (n = 14 cells in each group). CV: coefficient of variation, ISI: inter-spike intervals. **/?<0.01, n.s.: not significant.

Stereotactic surgery is performed to place infusion probe and chronic headstages into the cerebellum of RyR1-S2844D mice. Two weeks later, microinfusion of caffeine into freely moving RyR1 -S2844D mice is performed to examine the effects of aggravating RyR leakiness for cerebellar physiology and tremor (Fig. 6A). To determine whether fixing RyR1 leakiness will improve cerebellar physiology and tremor, Ry cal SI 07 (a small molecule that can be administered orally) was administered to 6- month-old RyR1-S2844D mice with chronic headstage implanted. Ry cal SI 07 and has been shown to restore the binding of leaky RyR1 with calstabin-1 to fix RyR1 leakiness and has good penetration of the blood brain barrier. Ry cal SI 07 was orally administered to the mice in drinking water for 1 month, a time period known to fix RyR1 leakiness in mice. The effects of Ry cal SI 07 on tremor and cerebellar physiology in RyR1-S2844D mice are evaluated and tremor after 1 -month washout are assessed, as the effect of this compound is reversible. In addition, brain specificity is tested by feeding another group of mice with a similar compound, called S36; which has identical effects of restoring calstabin-1 binding and fixing leaky RyR1 but does not penetrate the blood brain barrier.

To determine whether activating IP3R axis to induce further ER-calcium leak worsens cerebellar physiology and tremor: Other than RyR1, the IP3R axis is an important ER calcium handling pathway for PCs, and IP3R in cerebellar cortex is only expressed in PCs. Taking advantage of these unique features of IP3R, more ER leakiness in PCs is created. To activate IP3R, an artificial G protein coupled receptor, hMG3q, is used. Upon binding to the exogenous ligand, clozapine Noxide (CNO), hMG3q activates downstream IP3R to activate ER calcium efflux. AAV8-CaMKIIa-hMG3q-mCherry (Addgene 50476) are stereotactically injected into lobules V and VI of the cerebellums of RyR1-S2844D mice. CaMKIIa promoter is used because PCs are the only neuronal population in the cerebellar cortex that express CaMKIIa, and this promoter provides robust PC expression. In the same surgery, a chronic headstage for measurement of cerebellar oscillatory activity is implanted. After 3 -weeks of recovery, a single dose of either CNO to activate the hMG3q-IP3R axis or saline as control is intraperitoneally injected to investigate the effects on cerebellar physiology and tremor. After two weeks of recovery, the treatment (CNO vs. saline) is reshuffled to minimize mice used.

To determine the PC-specificity of ER calcium leak in tremor: To determine whether RyR1 leakage in PCs is sufficient to create tremor, a PC-specific RyR1 leaky state in mice is created. Since PC-specific knockout of RyR1 will not simulate the PC-RyR1 leaky state because lack of RyR1 will only create less ER calcium leak, an RyR1 native binding partner are used to control the leakiness. Calstabin-1 binds to RyR1 on its cytoplasmic domain to prevent the leaky state (Fig. 1). PC-specific calstabin-1 knockout is generated to create a PC- specific RyR1 leaky state. To achieve this, Pcp2Cre mice (JAX 004146) are crossed with calstabin-1 flox/flox mice (readily available in Mark’s lab) to generate Pcp2Cre:calstabin- Iflox/flox mice. Tremor and cerebellar oscillatory activity are examined bi-weekly in this mouse line and compared with calslabin-lflox flox mice. If these mice develop tremor, neuropathological analysis are performed.

Sex as a biological variable, sample size, and scientific rigor: With 10 mice in each group and the significance level at 0.05, this sample size allows detection of a standardized effect size of 1.54 (in units of standard deviation [SD]) with 90% power. A power analysis was performed based on a prior study of viral-mediated expression of dominant negative connexin to manipulate gap junctions in inferior olives in harmaline-treated mice, which showed the SD of tremor coherence are 1.5. Assuming this SD, a standardized effect size of 1.54 corresponds to being able to detect a mean change (comparing premanipulation vs. post-manipulation) of about 2.3 (=1.54*1.5). An equal number of male and female mice are allocated in each group, so that potential sex differences can be explored. If preliminary sex differences are observed, the sample size is further doubled to determine the sex differences. Tremor and cerebellar physiology are analyzed offline by staff blinded to treatment and genotyping.

EXAMPLE 5

Human ET cerebellum Alterations in Levels of RyR1 regulators and RyR1 Leakiness RyR1 is a macromolecular complex that includes kinases and phosphatases, which regulate channel function and may serve as additional therapeutic targets for ET. To test whether levels of RyR1 phosphorylation regulators and the degree of RyR1 leakiness are altered in the ET cerebellum and correlate with tremor severity and PC pathologic changes, levels of RyR1 complex binding partners and biochemically measured RyR1 leakiness are determined in the postmortem human ET vs. control cerebellum. These measurements are correlated with ET cerebellar pathologic features and clinical tremor severity.

To determine whether there are alterations in levels of RyR1 associated kinases and phosphatases in ET cases vs. controls: The large cytoplasmic domain of RyR1 is a scaffold for a kinase (PKA), a phosphatase (protein phosphatase 1 [PPI]) and a phosphodiesterase (PDE4D3) by specific targeting proteins, including spinophilin (for PPI) and mAKAP (for PKA and PDE4D3)(Fig. 7A). To determine whether binding of kinases, phosphatases, and their targeting proteins to RyR1 are altered in ET cerebellum, leading to increased RyR1 phosphorylation and a leaky channel state, RyR1 are immunoprecipitated from cerebellar cortex lysates of 60 ET cases and 30 age-matched controls, and Western blot analysis is performed on immunoprecipitates for RyR1, PKA, PPI, PDE4D3, spinophilin 1 and mAKAP. Signals are quantified with Odyssey system (LLCOR Biosciences) and the ratio of each phosphorylation modulatory protein/RyR1 determined. To determine whether cerebellar RyR1 mediated ER calcium handling differs between ET cases vs. controls: While it was discovered that an ER calcium handling abnormality might contribute to ET, a direct measurement of this ER calcium defect in the postmortem human brain is still lacking. To determine whether ET cerebellum has abnormal ER calcium handling, a microsomal calcium leak assay (Fig. 7B) in microsomes isolated from frozen postmortem cerebellum of 60 ET cases and age matched controls can be mixed with ATP and a Fluo4 calcium indicator fluorescent dye, which loads calcium-Fluo4 dye into microsomes through the ER SERCA pump. After reaching saturation, further calcium uptake can be inhibited by thapsigargin. Calcium leaking out of ER microsomes is detected as increased Fluo4 fluorescence in a spectrophotometer. In separate incubations, Ry cal SI 07 is added to stabilize leaky RyR1 and to confirm the calcium efflux is due to leaky RyR1. Changes of fluorescence intensity are recorded using FeliX software, v2 (PTI). The percentage of total calcium taken up into microsomes that leaks into the solution is determined.

To determine whether the extent of RyR1 phosphorylation and calstabin-1 depletion, levels of RyR1 bound kinases and phosphatases, and microsomal RyR1 calcium leakiness correlate with tremor severity and PC morphological changes in ET: While it has been determined that cerebellar RyR1 is important for tremor, the correlation of the RyR1 biochemically leaky phenotype with tremor severity and PC morphological changes in ET is still not known. The present disclosure provides several quantitative metrics in human ET cerebellum that biochemically reflect a leaky state of RyR1, including: 1) RyR1 phosphorylation status (p-RyR1/RyR1 ratio), 2) depletion of calstabin-1 binding (calstabin- 1/RyR1 ratio), 3) altered levels of phosphorylation regulators bound to RyR1 (e.g., PPl/RyR1 ratio, and 4) a direct microsomal calcium flux assay to measure RyR1 leakiness (calcium leak, % of uptake). Here, RyR1 phosphorylation and calstabin-1 depletion in 60 ET cases is assessed, and then these 4 biochemical measurements are correlated with clinical metrics (total tremor scores, tremor disability scores, disease duration, early onset vs. late onset ET, familial vs. sporadic ET, and men vs. women), and PC morphological metrics (PC count, PC axonal torpedoes), which have already been determined.

Patient selection. Existing ET and control brains are used from the New York Brain Bank (NYBB) at Columbia University. A movement disorders specialist confirms all ET diagnoses with detailed clinical questionnaires, medical records, videotaped neurological exams, and Archimedes spirals. About 30 mild-moderate ET cases (total tremor scores < 25) and 30 severe ET cases (total tremor scores >25), are used to assess correlation with tremor severity. 30 age-matched controls are selected from the NYBB. Sex as biological variables, Sample size, and Scientific rigor. With this sample size, the mean outcome for a group with a margin of error of +/- 0.37 in units of standard deviation (SD) are estimated. When comparing groups with the significance level set at 0.05, a standardized effect size of about 0.74 in units of SD with 80% power are detected.

In a prior study examining PC synaptic pathology, standardized effect sizes ranging from 0.78-1.5 were observed. Thus, the sample size used herein is adequate to detect possible differences. Since there is no predilection of gender in ET, 112 equal numbers of men and women among ET cases and controls are chosen to explore gender differences, and also factor in gender as a variable in correlation analyses. All pathological assessments are performed blinded to diagnosis, tremor severity, and other clinical features.

Studies to demonstrate feasibility have been performed. The present inventors found increased RyR1 phosphorylation and calstabin-1 depletion in ET cerebellum (Fig. 2 A) and a reduction of RyR1 -bound PPI levels but not PKA levels in 3 ET cerebellum compared to 3 control cerebellum, indicating a potential disturbance in the phosphatase axis (feasibility for Fig. 8A). In addition, increased ER calcium leak in microsomes isolated from 3 ET cerebellum vs. 3 controls was demonstrated. Adding Rycal S107, which by stabilizing calstabin-1 binding to RyR1 and suppressing the calcium leak in ET cases, demonstrates the calcium leak is via RyR1 (feasibility for Fig. 8B). Preliminary analysis in a cohort of ET cases (n = 11) shows correlations between depletion of calstabin-1 bound to RyR1 and total tremor scores (r =0.70, , and the morphologic PC count (r =0.65, p =0.04) (Figs. 8C-8D). These findings support that greater depletion of calstabin-1 from RyR1, indicating chronically leaky RyR1, leads to more severe tremor and loss of PCs in ET.

One consideration for ER calcium handling in ET is that activity of the SERCA protein that pumps cytoplasmic calcium into the ER may be increased to compensate for ER calcium leak. Therefore, SERCA activity is tested in a subset of ET and control cerebellum (n = 10 in each group). If SERCA defects are seen, SERCA activity in all ET cases and controls are tested.

Another consideration is that there are other RyR subtypes such as RyR2 and RyR3, which are not significantly expressed in PCs. Nonetheless, RyR2 and RyR3 in the cerebellum could still play a modulatory (or compensatory) role. Therefore, an investigation is conducted to determine whether changes in RyR2 and RyR3 levels and post-translational modifications are seen in a small subset of ET cases vs. controls (n = 10). If differences are detected, RyR2 and RyR3 modifications are determined in all ET and controls and these levels are incorporated as variables into correlation analyses. EXAMPLE 6

Morphologic and Molecular Events Underlying ET

Over the past decade, a cluster of morphological changes have been identified in the essential tremor (ET) cerebellum, predominantly centered in/around the Purkinje cell (PC). T Several of these pathologies have also been observed in primary cerebellar neurodegenerative diseases such as spinocerebellar ataxias (SCAs) and multiple system atrophy (MSA), but the degree to which these changes occur has not been formally studied or compared with that in ET. The cerebellum is increasingly being implicated in tremor generation in other diseases such as Parkinson’s disease (PD) and dystonia, yet their cerebellar pathology is presently unexplored. Hence, there is a large morphologic data gap. On a more primary level, RNA sequencing-based transcriptome analyses (RNAseq) was recently performed in cerebellar cortex of ET versus control patients and dysregulation was identified in four main biological processes, including 1) axon guidance, 2) microtubule motor activity, 3) endoplasmic reticulum (ER) to Golgi transport and 4) calcium signaling/synaptic transmission. To further explore the molecular basis of ET, laser-capture microdissection (LCM) was performed to specifically target PCs, thereby facilitating a precise evaluation of cell-specific changes associated with ET (R21 NS077094, 2011-13, Faust PI). A highly novel differential gene expression profile was obtained by direct sequencing of RNA (RNAseq) isolated from cerebellar PCs of 10 ET vs. 10 control brains. Differentially expressed transcripts were identified, which code for proteins that regulate neuronal function. However, these are initial/exploratory analyses involving 10 ET cases and 10 controls and they must be confirmed using a replicate dataset. Furthermore, a parallel set of LCM-RNA-seq studies, exploring the molecular transcriptome of PCs in PD, dystonia, MSA and SCA, have yet to be performed. Hence, there are several molecular data gaps. A 5-year study, which uses post-mortem brain tissue from patients with ET and a range of other neurodegenerative diseases involving cerebellum, is conducted to (1) determine whether there is a distinct profile of pathological changes in the cerebellum of ET patients as well as distinct profiles in patients with four other neurodegenerative diseases and (2) explore the molecular biology of PCs across neurodegenerative diseases characterized by cerebellar involvement and/or tremor.

Major causative genes have to be identified for ET and the molecular events within PCs that underlie their degeneration in ET remain largely unknown. Thus, gene expression profiling is the best approach to explore molecular alterations in PCs from ET patients vs. controls. More recently, significant advances in DNA sequencing technologies enable whole transcriptome analysis by RNA-seq. RNA-seq has a low frequency of false-positive findings and is highly reproducible and more sensitive for detecting differential expression compared with microarray technology.

To begin exploration of an underlying molecular source of ET disease pathogenesis, the present inventors performed the first transcriptomic analysis by direct sequencing of RNA from frozen cerebellar cortex tissue in 33 ET patients compared to 21 normal controls. Principal component analysis showed a heterogenous distribution of the expression data in ET patients that only partially overlapped with control patients. Differential expression analysis identified 231 differentially expressed gene transcripts (‘top gene hits’), a subset of which has defined expression profiles in the cerebellum across neuronal and glial cell types but a largely unknown relationship to cerebellar function and/or ET pathogenesis. Gene set enrichment analysis (GSEA) identified dysregulated pathways of interest and stratified dysregulation among ET cases. By GSEA and mining curated databases, the present inventors compiled major categories of dysregulated processes and clustered string networks of known interacting proteins. These ‘top gene hits’ contribute to regulation of four main biological processes, which are 1) axon guidance, 2) microtubule motor activity, 3) endoplasmic reticulum (ER) to Golgi transport and 4) calcium signaling/synaptic transmission. The results of this transcriptomic analysis suggest there is a range of different processes involved among ET cases, and draws attention to a particular set of genes and regulatory pathways that provide an initial platform to further explore the underlying biology of ET.

Fig. 9 shows a string network cluster of dysregulated genes in ET. Among the differentially expressed genes, highly relevant pathways for axon guidance (Red), kinesins/microtubule motor activity (Dark Blue), ER-Golgi transport (Green), and calcium signaling (Purple) are highlighted. RyR1 is present in the purple calcium signaling node.

Transcriptional profiles from entire brain tissue regions are representative of a heterogeneous mixture of cell types that effectively mask the expression of low abundance transcripts, or molecular changes that occur only in a small population of affected neurons, such as PCs in cerebellar cortex. To overcome this limitation, the present inventors have perfected LCM to specifically target PCs. In this technique, a UV laser is used under microscopic guidance to remove (i.e., cut out) neurons of interest directly from histologic sections. With this technique, gene expression profiles targeted to cerebellar PCs can further elucidate how molecular dysfunction in ET PCs contribute to disease pathogenesis.

The amount of RNA obtained from the PC LCM requires use of low-input RNA-seq methods, where RNA is first amplified to prepare cDNA for sequencing library preparation. PC RNA samples were extracted using a RNeasy Micro Kit and eluted in small volumes with RNase inhibitors. RNA samples underwent library preparation for sequencing using a SMARTer Stranded Total RNA-Seq kit V2 - Pico input. Briefly, samples were fragmented and underwent first-strand synthesis, followed by addition of an adapter and unique barcode for each sample. Samples were cleaned with AMPure beads prior to rRNA depletion with ZapR, to enrich mRNA for sequencing. The final mRNA library was amplified and purified again with AMPure beads. cDNA library quality was assessed via High Sensitivity DNA Agilent Bioanalyzer assay. Final libraries were quantified and pooled for sequencing to ensure equal loading across samples. Samples were sequenced using a NextSeq550 sequencer with a High- Output 75-cycle kit. An average of 20 million single-end 75bp reads per sample was obtained.

Reads were mapped to the human transcriptome (hgl9, iGenomes annotation) using Tophat 2.0 without novel junction detection. Cufflinks/Cuffdiff 2.0 was then used to calculate the expression level of each gene in fragments per kilobase per million mapped reads (FPKM) along with p-values and false discovery-corrected q-values for differential expression between the ET and control groups.

The present inventors performed RNA-seq on PCs isolated by LCM from 34 ET and 16 age-matched control samples. Differential expression analysis identified 36 differentially expressed genes (padj < 0.05), enriched for RNA splicing and collagen basement membrane proteins (FIG. 10). Principal component analysis showed a heterogenous distribution of the expression data in ET patients that only partially overlapped with that in control patients. As in a prior transcriptome study of whole cerebellar cortex, significant heterogeneity among ET patients was found, and ET cases were more enriched in positive principal component 2 space (PC2+).

To investigate biological sources of variability across these samples and better understand the potential biological relevance of these data, the present inventors employed a Gene Set Enrichment Analysis (GSEA) to analyze gene expression in all samples based on the principal components. ET samples are more enriched in positive PC2 space compared to controls, and gene ontology (GO) pathways for positive PC2 correlations are enriched for a number of pathways important for neuronal functioning, including golgi-endoplasmic reticulum trafficking, kinase signaling, transcription regulation, RNA processing, axon guidance, and calcium signaling and synapse organization (Fig. 11). RyR1 is present in the yellow calcium signaling pathway.

Onset of disease also varies among ET patients, and many of those with early onset of disease (age < 50 years) also have a family history of ET, suggesting a strong genetic component in disease pathogenesis. Principal component 3 further segregated ET cases into those with early onset of disease (age <50 years) versus those with later onset disease.

In summary, there are overlapping findings between the transcriptome studies on whole cerebellar cortex tissue and those obtained from PCs isolated by LCM. Both studies identified dysregulated pathways in Golgi -endoplasmic reticulum trafficking, axon guidance and calcium signaling in ET patients. Additional pathways of dysregulation identified in the LCM study point to a role for alterations in RNA splicing and collagen basement membrane formation for PC function in ET.

The present disclosure investigates whether there is a distinct profile of pathological changes in the cerebellum of ET patients as well as distinct profiles in patients with four other neurodegenerative diseases. Using postmortem cerebellar tissue from ET, SCAs, MSA, PD, and dystonia cases and neurologically normal controls, each of the morphological changes itemized in Table 2 along with additional recently developed approaches itemized in Table 3 are quantified. 160 brains (50 ET, 25 SCA, 15 MSA, 30 PD, 15 dystonia, 25 controls) are used for an exploratory-discovery data set (planned analysis at mid-point of Year 3) and to validate the findings with 160 additional brains as a replicate data set (analysis at end of Year 5). There may be a particular profile of quantifiable morphological changes in the cerebellum (i.e., a disease signature), distinguishing ET from other neurodegenerative diseases that are characterized by cerebellar involvement and/or tremor (SCAs, MSA, PD, dystonia). Furthermore, each of these other diseases may have its own distinctive morphological pattern, referred to as the “pathologomic” profile.

Table 2

Several morphological changes, centered in/around Purkinje cells (PCs), have been identified in the cerebellum of essential tremor (ET) patients. These changes have not been contextualized within a broader degenerative disease spectrum, limiting their interpretability. To address this, the present inventors compared the severity and patterning of degenerative changes within the cerebellar cortex in patients with ET, other neurodegenerative disorders of the cerebellum (spinocerebellar ataxias (SCAs), multiple system atrophy (MSA)], and other disorders that may involve the cerebellum [Parkinson’s disease (PD), dystonia]. Using a postmortem series of 156 brains [50 ET, 23 SCA (6 SCA3; 17 SC A 1, 2 or 6), 15 MSA, 29 PD, 14 dystonia, 25 controls], data on 37 quantitative morphologic metrics was generated, which were grouped into 8 broad categories: (1) PC loss, (2) heterotopic PCs, (3) PC dendritic changes, (4) PC axonal changes (torpedoes), (5) PC axonal changes (other than torpedoes), (6) PC axonal changes (torpedo-associated), (7) basket cell axonal hypertrophy, (8) climbing fiber-PC synaptic changes. In analyzing the data, the present inventors used z scored raw data for each metric across all diagnoses (5772 total data items). Dystonia and PD each differed from controls in only 2/37 metrics, whereas ET differed in 21, SCA3 in 8, MSA in 19, and SCA1/2/6 in 26 metrics. Comparing ET with primary disorders of cerebellar degeneration (i.e., SCAs), a spectrum of changes was observed reflecting differences of degree, being generally mild in ET and SCA3 and more severe in SCA1/2/6. Comparative analyses across morphologic categories demonstrated differences in relative expression, defining distinctive patterns of changes in these groups. Thus, the degree of cerebellar degeneration in ET aligns ET with a milder end in the spectrum of cerebellar degenerative disorders, and a somewhat distinctive signature of degenerative changes marks each of these disorders.

Principal component analysis revealed that diagnostic groups were not uniform with respect to cerebellar pathology (FIGS. 12A-12F). When analyzed across all diagnostic categories, the two major axes of variation in the data segregated individual patients into distinct groupings (FIG. 12A). To a large extent, the first axis of variation (principal component 1) separated MSA and SCA1/2/6 from the remaining diagnoses, with the data points in MSA and SCA1/2/6 distinctly situated to the right of the main cluster. Metrics that were highly correlated to principal component 1 (i.e., they were major influencers of principal component 1) were torpedo counts (green bars), several torpedo-associated changes in PC axons (dark blue bars) and PC dendritic swellings (yellow bars) (FIG. 12B). These metrics had higher values in MSA and SCA1/2/6. In contrast, metrics that were anti-correlated with principal component 1 reflect less PC loss (purple bars) and better-preserved climbing fiber synapses (light blue bars). The second axis of variation (principal component 2) mainly separated MSA from SCA1/2/6, with most data points for SCA1/2/6 located below those of MSA (FIG. 12A). Metrics that were highly correlated to principal component 2 (i.e., they were major influencers of principal component 2) were most calbindin PC axonal changes (orange bars) except for PC puncta, torpedo-associated changes in PC axons (dark blue bars), torpedo counts (green bars) and better-preserved PC counts (purple bars) (FIG. 12C). Principal component 2 anti -correlated values included greater percentage of empty baskets (purple bar, i.e., more PC loss), dendritic swellings (yellow bars), and calbindin PC puncta (orange bars). Thus, this initial analysis predominantly highlights distinct differences in morphologic changes between SCA1/2/6 and MSA vs. other disease categories and between SCA1/2/6 and MSA.

While controls, ET, Parkinson’s disease, and dystonia form a dense cluster (FIG. 12A), there is not complete overlap, and variable segregation is apparent predominantly along the axis of principal component 1 when MSA and all SCAs are excluded from the analysis (FIG. 12D). All but one control sample (black dots) has a principal component 1 value < - 0.50, and the median of all controls centers at principal component 1 = - 2.37. Compared to controls, dystonia (light blue dots, median principal component 1 = - 0.97) and Parkinson’s disease (green dots, median principal component 1 = - 0.78) patients are shifted to the right, and ET patients (red dots) are even further shifted to the right (median principal component 1 = 1.53). Metrics correlating strongly with principal component 1 include torpedo counts (green bars), calbindin torpedo-associated changes with thickened-, recurrent-, and branching-PC axons (dark blue bars), dendritic swellings (yellow bars) and calbindin thickened PC axons (orange bar) (FIG. 12E). These metrics had higher values in ET than controls. There is also a notably stronger positive correlation for VGlut2 outer 20% and basket cell rating, consistent with their enrichment in the ET cerebellum vs. controls. Anti-correlated metrics were preserved PC counts (purple bars) and climbing fiber synaptic density (light blue bar). The present inventors also compared the principal component data for ET and all SCAs, as disorders whose primary identifiable pathology is most evident in the cerebellum, vs. controls (FIG. 12F). The distribution of data for ET (red dots) and SC A3 (orange dots) are shifted to the right vs. controls (black dots) along the principal component 1 x -axis, with median values at - 2.45 (controls), - 1.05 (ET) and - 0.47 (SC A3). Notably, there is significant overlap in data points for SCA3 and many ET cases. SCA1/2/6 patients (blue dots) are shifted to a much greater extent along both principal component 1 (median = 6.16) and principal component 2 axes. The distribution of SCA1/2/6 cases along the principal component 1 x -axis reflects varying disease severities that correlated with severity of PC loss, centered at 4.20 for SCA1, 6.98 for SCA2 and 9.78 for SCA6. In summary, these analyses showed that these diagnoses were not uniform with respect to cerebellar pathology, with the greatest (though not identical) changes observed in MSA and SCA and lesser changes in the other groups (FIGs. 12A-12E). In terms of primary disorders of cerebellar degeneration, the changes lay along a spectrum, with those observed in ET and SCA3 being the mildest and those in SCA1/2/6 being the most severe relative to controls (FIG. 12F). As a whole, each of these disorders occupies a distinctive space along a spectrum of cerebellar degeneration.

The change in each metric across diagnoses was next quantified, using controls as a reference group (FIG. 13 A). Metrics (rows) and diagnoses (columns) are shown. Each cell in the heatmap is the average log2 -fold-change (disease vs. control) for each metric. Dark purple cells indicate that there is a high mean value for the metric relative to controls and green indicates the opposite. Elements labeled with an asterisk indicate that the difference from controls is statistically significant, with false discovery rate < 0.01. Several observations may be made. First, when compared to controls, dystonia and Parkinson’s disease differed significantly in only 2/37 metrics (see two asterisks). In contrast, the number of differing metrics was 21 for ET, 8 for SCA3, 19 for MSA, and 26 for SCA1/2/6 (FIG. 13A).

Second, in terms of categories of pathological change, dystonia and Parkinson’s disease significantly differed from controls in 2/8 categories; SCA3 differed in only 3/8 categories (FIG. 13 A). ET and SCA1/2/6 each significantly differed from controls in 7/8 categories, and MSA from controls in 6/8 categories (FIG. 13A). Third, in 17 metrics across 6 categories (i.e., PC loss, heterotopic PCs, PC dendritic changes, PC axonal changes [torpedoes], PC axonal changes [other than torpedoes], and PC axonal changes [torpedo-associated]), neither dystonia nor Parkinson’s disease differed from controls, yet there was a spectrum of change, with ET generally at the low end and SCA1/2/6 or MSA at the high end of severity (FIG. 13 A) .

Fourth, for three metrics, including the percentage climbing fibers in the outer 20% of the molecular layer (VGlut2) and two metrics involving PC puncta (calbindinD28k), the pattern observed for ET was not merely on the cerebellar degeneration spectrum. The mean percentage of climbing fibers in the outer 20% of the molecular layer (VGlut2) was distinctly increased in ET compared to controls, whereas in SCA1/2/6 and MSA there was a decrease compared with controls (FIG. 13 A). The density of PC puncta (calbindin) and PC puncta per PC (calbindin) was only significantly increased in SCA1/2/6 and involved a larger percentage of PCs when seen in a sample. In control, ET or MSA samples, these structures were rare.

Next, scores were computed for each patient as a fold-change relative to control averaged over selected observables and plotted on a log2-transformed scale (FIG. 13C), including (1) a “Severity Score” containing the core set of 20 highly correlated observables (FIG. 13B, red asterisks), (2) a “PC Loss Score” [inverse PC body/mm, inverse PC nucleolus/mm, percentage empty baskets], and (3) climbing fibers in the outer 20% of the molecular layer. The “Severity Score” differed significantly across all diagnoses, with a gradual increase from dystonia to Parkinson’s disease to ET, was intermediate for SCA3, and high for MSA and SCA1/2/6. The PC loss score was higher in ET than in control, dystonia, Parkinson’s disease or SCA3, was widely variable in MSA, and highest in SCA1/2/6. Along with the climbing fiber outer 20% score, which shows a distinct increase in ET vs. all other diagnoses, these data demonstrate that ET had both common and distinctive combinations of morphologic patterns across a spectrum of cerebellar degeneration.

In summary, the degree of observed changes in the cerebellar in ET aligns ET with disorders of cerebellar degeneration, albeit at the milder end of the spectrum. Yet these disorders do not blandly express the same generic pattern of degeneration, with their only distinguishing feature being the degree to which they express that pattern. In fact, there is evidence that a somewhat distinctive signature of degenerative changes marks each of these disorders.

The present subject matter being thus described, it will be apparent that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the present subject matter, and all such modifications and variations are intended to be included within the scope of the following claims.