Novel Treatment For Wolfram Syndrome

Related Applications and Grant Support

This application claims the benefit of priority of United States Provisional

Application s.n. 62/860,033, of identical title, filed June 1 1, 2019, the entire contents of which application is incorporated by reference in its entirety herein.

This invention was made with government support under P01DK05775 I, DM 12921 , DK020579, TR002065 and F30DK111070 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

The present invention is directed to a novel treatment for Wolfram Syndrome, to methods of treatment in order to delay, inhibit, ameliorate and/or reduce the likelihood of symptomology of a patient with Wolfram Syndrome.

Background and Overview of the Invention

Wolfram syndrome is an orphan, autosomal recessive neuroendocrinological disease in which patients experience loss of physical and psychological functions. The cardinal manifestations include diabetes mellitus, diabetes insipidus, bilateral optic atrophy, hearing and vision loss along with progressive motor, autonomic and psychiatric abnormalities. The course of Wolfram syndrome is progressive, and the prognosis is poor and typically fatal by mid-adulthood. Only palliative treatments exist; there are no therapies to slow progression.

More specifically, Wolfram syndrome is an aggressive multisystem

neurodegenerative and endocrinological disease, also known by the acronym DIDMOAD (diabetes insipidus, insulin-deficient diabetes mel!itus, optic atrophy and deafness). (14) Wolfram syndrome is caused by mutations in the protein wolframin and is inherited in an autosomal recessive pattern. Patients typically are first identified around age 6 by glucosuria and diabetes mellitus, followed by marked loss of peripheral vision and color perception due to optic nerve atrophy around age 11 (15). 70% of Wolfram syndrome patients suffer from central diabetes insipidus and neuron-based hearing loss. Urinary tract manifestations are another mam clinical finding for patients affecting the majority of this population. More than half of patients develop neurological or psychiatric disorders, most commonly manifested as problems with balance and coordination (ataxia), seizures, and peripheral neuropathy beginning in early adulthood ( 16). Psychiatric disorders associated with Wolfram syndrome include psychosis, episodes of severe depression, and impulsive and aggressive behavior. Brain stem atrophy is also a prominent feature and it is this effect that leads to death, secondary to central apnea (15) Wolfram syndrome is often fatal by mid-adulthood ( 17) due to complications arising from the many features of the condition (3).

The inventor of the present application previously identified drags that inhibit NCSI function as part of a study of chemotherapy induced peripheral neuropathy (CIPN). Examples from all classes of the drugs that are effects in CIPN were tried. The inventor was completely surpri sed that only one of the compounds was effective in maintaining cell viability and function in cells with protein mutations that cause Wolfram Syndrome This drug has never been considered as a candidate for therapy in Wolfram Syndrome

Using the inventor’s extensive knowledge of the calcium signaling complex that includes neuronal calcium sensor 1 (NCSI), the current novel therapy for Wolfram Syndrome has been identified. Hie present invention represents the first therapy for Wolfram

Syndrome. No other therapies exist.

Wolfram Syndrome is a rare genetic disorder affecting people who are homozygous for mutations in wolfrarnin. Given the present invention, this may be expanded to patients who are carriers in wolfrarnin (heterozygous). Heterozygous individuals have a 25 fold or higher incidence of mood disorders, another condition that needs new improved therapies.

Brief Description of the Invention

The present invention is directed to a novel treatment for Wolfram Syndrome and to methods for treating, inhibiting, ameliorating, reducing the likelihood and/or delaying the onset of symptoms which occur as a consequence of Wolfram Syndrome and its progression . In addition, in embodiments, the present invention is directed to methods of treating, inhibiting ameliorating, reducing the likelihood and/or delaying the onset of symptoms which are associated in patients who are heterozygous carriers in wolframin, particular mood disorders, bipolar disorder, depression, severe depression and impulsive verbal and physical aggression which are often exhibited by these individuals.

In an embodiment, the present invention is directed to the use of Ibudilast (AV41 i) or an analog or derivative thereof, including AV1013, in the treatment of Wolfram Syndrome in a patient or subject in need, the method comprising administering to a patient with Wolfram Syndrome an effective amount of Ibudilast (AV411) or analog or derivative thereof, including AV1013 or a related analog to treat, inhibit, ameliorate, reduce the likelihood and/or delay the onset one or more symptoms associated with Wolfram Syndrome in an individual including premature death, diabetes mellitis, diabetes insipidus, visual impairment, including optical atrophy, color blindness, slow reacting ins, high frequency hearing loss and tonal deafness, emotional agitation, tremors, seizures, peripheral neuropathy, autonomic dysfunction, ataxia, ptosis, nystagmus, endocrinopathies, brainstem atrophy, gastrointestinal disorders, including dysmotility (diarrhea/constipation), urinary tract atony, urinary incontinence, recurrent urinary infections, hydronephrosis, primary gonadal atrophy

(especially in men), menstrual irregularities and delayed menarehe, among others, including psychiatric disorders such as severe depression, bipolar disorder, impulsive verbal and physical aggression.

In an embodiment, the present invention is directed to the use of Ibudilast (AV41 1 ) or an analog or derivative thereof, including AV1013 in the treatment of heterozygous wolframin in a patient or subject in need to treat, inhibit, ameliorate, reduce the likelihood and/or delay the onset of one more symptoms associated with heterozygous wolframin including psychiatric disorders, especially depression or severe depression.

These and/or other embodiments are described in the detailed description of the invention which follows.

Brief Description of the Figures

FIGURE l shows a model for the effects of mutations to wolframin on NCS1, calcium (Ca ²⁺ ) signaling, secretion, and cell survival.

FIGURE 2 shows that NCS1 and wolframin bind in a calcium dependent manner. NCSl and the cytoplasmic domain of wolframin (wfsl-GST) were expressed in bacteria and NCS1 was purified. The cytoplasmic domain of wolframin had a GST tag that was used to pull down the protein complex. Increased free calcium, increased pull down ofNCSl Blot was probed with an antibody against NCS1. p.d ^:: = pull down, i.b. = immunoblot

FIGURE 2B shows the docking ofNCSl to the cytoplasmic domain of woiframin. NCS1 (green) is the crystal structure (1G81). The woiframin structure (blue) was deduced using the Roberta Server software. The red arrow points to red residues on NCS1 that is the calpain cleavage site

FIGURE 3 show's the ratio ofWFSl to NCS1 is constant despite very different levels ofNCSl expression in four different cell lines.

FIGURE 4 shows WFS1-KO mouse brain cortex has low NCS1 (right lane) compared to WT tissue b-actin is shown as a loading control.

FIGURE 5 show's that glucose induces calcium oscillations in INS-1 cells. At low and zero glucose, oscillations are minimal in WT cells and absent in WFS1 KO cells. At 30 niM glucose, WT cells display robust calcium oscillations, but the magnitude and shape of the calcium transients in WFSl KO Insl cells are decreased.

FIGURE 6 show's NCS1 levels in INS-1 cells after glucose treatment. Compared to cells treated with 0 mM glucose, NCS1 level w'as increased in the CTRL cells, but decreased hr WFSl KO cells treated with 30 mM glucose.

FIGURE 7 shows that Woiframin (WFSl) KO decreases phosph-Akt in INS-1 cells. In the resting state (5 mM glucose) Phosphorylation of AKT (pAKT) was lower than WT.

FIGURE 8 show's colony formation in woiframin (WFSl, left) orNCSl (right) KO cells. Cells were plated and maintained in an incubator. After 2 weeks plates were stained and percent coverage by cells were compared. Note that loss of either protein leads to fewer colonies.

FIGURE 9 show's cell survival is diminished in INS-1 cells lacking woiframin (WFSl) after treatment with 30 mM glucose for 48 hours. Survival is normalized to % survival at 0 mM glucose.

FIGURE 10 shows glucose tolerance in WFSl (left) or NCS1 (right) KO and WT mice. Mice were injected with 2 g glucose and blood samples were collected over time and glucose measured. Note that both WFS1 (left) or NCSi (right) KO mice achieved higher glucose levels than the WT mice. WFSl data adapted from reference (49).

FIGURE 11 shows that INS-1 cells were transfected with scrambled siRNA or siRNA against WFSl, pretreated with or without 10 mM dantrolene for 48 h, then incubated in media containing 0.5 mM thapsigargin for 6 h. Thapsigargin treatment induces cell stress. Protein levels of cleaved spectrin were analyzed by immunoblotting and quantitated relative to GAPDH levels. Taken from (56).

FIGURE 12 shows the cell survival (WT) after treatment with 31 mM glucose for 48 hours. Treatment with valproate, lithium, or chlorprmazine had no effect on survival, but calpain inhibitor or ibudilast did. Survival is normalized to % survival at 1 mM glucose.

FIGURE 13 shows cell survival (WFSl KO) after treatment with 31 mM glucose for 48 hours. Treatment with valproate, lithium, or chlorprmazine had no effect on survival, but calpain inhibitor or ibudilast did. Survival is normalized to % survival at 1 mM glucose.

FIGURE 14 shows that treatment with ibudilast does not alter cell survival (WT) at low (“normal”) glucose levels.

FIGURE 15 shows that cell survival (WT) after treatment with 31 mM glucose for 48 hours is decreased. Treatment with ibudilast did protect cell survival. Nearly full protection occurs at 1 uM ibudilast. Survival is normalized to % survival at 1 mM glucose.

FIGURE 16 shows that WFSl regulates intracellular calcium homeostasis in INS1 cells. (A) Western blot confirming the loss of WFSl protein in WFS1-KO cells and the re- expression ofWFSl in WFS1-KO cells (WFS1-OE). (B) WFS1-KO cells exhibited significantly elevated resting cytosolic calcium compared to WFSl WT cells, which could he rescued by re-expressing WFSl . (C) WFS1-KO cells showed significantly higher calpain activity than WFS1-WT cells. (D) Panel show's averaged traces of 8-18 coverslips for each ceil type in response to 50 nM ATP. (E-G) Compared to WFS1-WT ceils, WFSl-KO cells showed decreased max amplitude, area under the curve, and rate of rise for the cytosolic calcium traces shown in D. Re-expressing WFSl fully rescued max amplitude and rate of rise and partially rescued area under the curve. (H) Panel shows averaged traces of 15 coverslips for each cell type (both WFSl-WT and WFSl-KO cells stably expressing mito-gCaMP6F) in response to 100 nM ATP. (I-K) Compared to WFSI-WT cells, WFS 1-KO cells showed decreased max amplitude, area under the curve, and rate of rise for the mitochondrial calcium traces shown in H.

FIGURE 17 shows that WFS 1 -KO cells show more severely impaired calcium signaling due to hyperglycemia. Cells were incubated for 24 hours with normal medium (CTRL) or medium with 15 mM or 30 niM additional glucose (15G and 30G, respectively) prior to imaging. (A) WFSI-WT cells showed elevated resting cytosolic calcium after incubation with 30G, whereas WFS 1 -KO cells showed no change. (B) Panel shows averaged traces of 12-25 coverslips for each condition in response to 50nM ATP. Glucose toxicity caused impairments of ATP -evoked calcium transients in a concentration-dependent manner. However, WFS 1-KO cells showed a more impaired response at CTRL and 15G. At 30G, both WFSI -WT and WFS 1-KO cells were equally impaired. (C-E) Quantification of max amplitude, area under the curve, and rate of rise for the cytosolic calcium traces shown in B.

FIGURE 18 shows that overexpression of WFS l’s interacting partner NCS1 rescues calcium homeostasis in WFS1-KO cells. (A) For co-immunopreeipitation, mouse brain lysate was incubated with NCS1 antibody or rabbit IgG as control. Tmmunoblots were incubated with antibodies as indicated. (B) Representative blot showing protein abundance of NCS 1 in WFS I-WT and WFS1-KO cells treated for 48 h with additional 30 niM glucose (30G). (C) Quantification of B (10-12 independent preparations for each condition), values normalized to CTRL. Whereas WFSI-WT cells showed an increase in NCS 1 level, WFS 1-KO cells show'e-H-d a decrease. (D) Western blot confirming the overexpression of NCS1 in WFS 1- KO cells. (E) Panel shows averaged traces of 9-32 coverslips for each cell type in response to 50nM ATP. Overexpression of NCS 1 rescued ATP -evoked cytosolic calcium response in WFS 1-KO cells. (F-I) Quantification of max amplitude, area under the curve, and rate of rise for the cytosolic calcium traces shown in E. (I) Overexpression of NCSl rescued elevated resting cytosolic calcium in WFS 1-KO cells.

FIGURE 19 shows that calpain inhibitor XI and ibudilast rescue cell viability and resting cytosolic calcium in WFS l-KO cells. (A) Measurement of cell viability of WFSI-WT and WFSl-KO cells showed no difference under control conditions on day 1. (B)

Measurement of growth over 7 days showed no difference between WFS I-WT and WFS1- KO cells (C) Measurement of cell viability, normalized to CTRL conditions. Treatment combinations as indicated for 48 hours, 30G = 30 mM additional glucose, C.I. ^::: calpain inhibitor XI (10 mM), IBU = ibudilast (10 pM). WFSl-KO cells showed a significantly larger reduction in cell viability compared to WFS1-WT cells. Cell viability in both cell lines were rescued by C.I. and IBU. 24 h treatment with (D) calpain inhibitor XI (10 mM) or (E) ibudilast (10 mM) reversed elevated cytosolic calcium in WFSl-KO cells without affecting WFS 1-WT cells.

FIGURE 20 shows that WFS1-KO cells exhibit decreased insulin secretion, which can be reversed by calpain inhibitor XI and ibudilast. (A) Measurement of glucose-stimulated insulin secretion using insulin ELISA assays, at baseline (2.5 niM glucose) and after stimulation (9 mM glucose). WFS1-WT cells showed significantly higher insulin secretion than WFS1-KO ceils under control conditions. Treatment with 10 mM calpain inhibitor XI or 10 mM ibudilast ameliorated the difference between WFS1-WT and WFS1-KO cells. (B) Representative blot showing protein abundance of the insulin signaling pathway. (C-D) Quantification of A (3-10 independent preparations). Compared to WFS1-WT cells, WFS1- KQ showed a significant reduction in rIIIb (Y1150/1151) and pAKT (S473). Re-expressing WFS 1 in WFS 1 -KG cells increased levels of rIKb and pAKT expression at least to WFS 1 - WT cells. (E) Representative blot showing protein abundance of pAKT (T308). (F)

Quantification of D (7 independent preparations). pAKT (T308) was significantly downregulated in WFS1-KQ cells. (G) Proposed model. Loss of WFS 1 results in global calcium dysregulation which impairs cell viability and insulin secretion. Calpain inhibitor XI and ibudilast can restore proper b-cell function, suggesting them as drug candidates for the treatment of Wolfram syndrome and similar diseases.

FIGURE SI shows that WFS1-WT control and WFS1-KO cells were generated using CRISPR-Cas by tire Genome Engineering and iPSC Center at Washington University in St. Louis. (A) gRNA was designed to target an early, conserved exon. (B-C) Sequencing results for the cell lines obtained. WFS1 -KO clone #1 was used for all experiments shown in the main figures. Experiments using WFSl-KO done #2 to validate the findings in clone 1 were included in FIGURE! S3. In both clones, insertion and deletion mutations resulted in immature stop codons before a.a. 230 in both alleles. The WT cell line was obtained from a clone that did not show CRISPR modification. FIGURE S2 shows that WF SI regulates intracellular calcium homeostasis in INS1 cells. (A) WFS1-KO cells showed a significant elevation of cytosolic calcium compared to WFS1-WT cells. (B) Panel shows averaged traces of 5-6 coverslips for each cell line in response to 1 mM thapsigargin. The Fluo-4-AM signal was normalized to intensity at 10 s (C, D) Quantification of area under the curve and max amplitude for cytosolic calcium traces shown in B, no difference was observed. (E) Panel shows averaged traces of 9-10 coverslips for each cell type in response to 50nM ATP. The fluo-4-AM signal was normalized to intensity at 10 s. (F-H) Compared to WFS1-WT cells, WFS1-KO cells showed decreased area under die curve, max amplitude, and rate of rise for the cytosolic calcium traces shown in E. (1) Representative blot of InsPSRl and InsP3R3 protein abundance in WFS1-WT and WFS1-KO cells. (J-K) Quantification of I (from 4-7 independent preparations) showed no difference in InsPSRl and InsP3R3 expression in both cell lines. (L) Representative blot of siibceiliiiar fractionations isolated obtained from HEK293 ceils, c.m. = crude mitochondria, containing mitochondria and MAMs. Tubulin was used as a marker for the cytosolic proteins, VDAC for mitochondrial proteins and calreticulin for non-MAM ER-proteins. (M)

Representative blot of subceiluiar fractionations obtained from INS1 WFS1-WT and WFS1- KO cells. Same markers as m L.

FIGURE S3 show's the Validation of key findings in a second CRISPR-WFS1-KO INS1 clone. (A) Representative blot confirming loss of WFS1 in WFS1-KO clone 2, and showing protein abundance of pAkt (S473) and t.Akt. (B) Quantification of B (from 7 independent preparations). (C) WFS1-KO clone #2 cells exhibited significantly elevated cytosolic calcium compared to WFS1-WT cells. (D) High glucose -induced loss of cell viability in WFS1-KO clone #2 ceils was dose-dependently reversed by ibudilast.

FIGURE S4 shows NCS1 protein and mRNA levels in WFS1-WT and WFS i -KO ceils. (A) Representative blot showing NCS1 expression. (B) Quantification of A (from 6-8 independent preparations), normalized to WT, no difference was observed between cell lines (C) Quantification of 5 independent qPCR experiments, no difference was observed between the different conditions.

FIGURE S5 show's a drug screen for compounds that rescue cell viability in WFSI- KO cells. Compounds were selected to target WFS1, NCSi, and/or calcium signaling. (A) Preliminary screening of various drugs to prevent glucose toxicity. Following treatment with 30 rriM additional glucose (30G, 48h), both WFS1-WT and WFSI-KO cells showed significant cell death as compared to cells cultured in normal medium . However, WFS 1 -KO ceils showed more severe ceil death compared to WFS1-WT and WFS1-OE cells at high glucose. Of the 7 compounds tested, only calpain inhibitor XI and ibudilast rescued cell viability back to normal level. (B) Calpain inhibitor XI did not affect cell viability in WFS1- KO cells at baseline. (C) Calpain inhibitor XI dose-dependently reversed high glucose- induced loss of ceil viability in WFS1-KO cells. (D) Ibudilast slightly raised cell viability in WFS 1 -KO cells at baseline. (D) Ibudilast dose-dependently reversed high glucose-induced loss of cell viability in WFS1-KO cells. (F) Lithium did not reverse hyperglycemia-induced loss of cell viability in WFS1-KO cells.

FIGURE 6S show's total IR, total Akt, and PP2Ac protein levels in WFS1-WT and WFS i-KO cells. (A) Total IR and (B) total Akt protein levels were not changed, representative blot shown in FIGURE 5SA. (C) Representative blot showing protein abundance of PP2Ac in WFS 1-WT and WFSI-KO cells. (D) Quantification of C (from 4 independent preparations), no significant difference was observed between cell lines.

Detailed Description of the Invention

The following terms shall be used throughout the specification to describe the present invention. Where a term is not specifically defined herein, that term shall be understood to be used in a manner consistent with its use by those of ordinary' skill in the art.

Where a range of values is provided, 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 invention. The upper and lower limits of these smaller ranges that may independently be included in the smaller ranges are also

encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. In instances where a substituent is a possibility in one or more Markush groups, it is understood that only those substituents which form stable bonds are to be used. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set out below.

The term“patient” or“subject” is used throughout the specification within context to describe an animal, generally a mammal, especially including a domesticated animal and preferably a human, to whom treatment, including prophylactic treatment (prophylaxis), with the compounds or compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the patient or subject of the present invention is a human patient of either or both genders.

The term“compound” or“agent”, as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein and includes tautomers, regioisomers, geometric isomers as applicable , and also where applicable, optical isomers (e.g.

enantiomers) thereof, as well as pharmaceutically acceptable salts (including any

pharmaceutically acceptable salt) thereof. Within its use in context, the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds as well as diastereomers and epimers, where applicable in context. The term also refers, in context to prodrug forms of compounds which have been modified to facilitate the administration and delivery^ of compounds to a site of activity.

The term“Wolfram’s syndrome” is used to describe a rare genetic disorder affecting people who are homozygous for mutations in wolframin. Wolfram syndrome is an inherited condition that is typically associated with childhood-onset insulin-dependent diabetes meliitus and progressive optic atrophy. In addition, many people with Wolfram syndrome also develop diabetes insipidus and sensorineural hearing loss. An older name for the syndrome is DIDMOAD, which refers to diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Some people have mutations in the same gene that causes Wolfram syndrome but they do not get all the features of the syndrome, so they are said to have WFS1 -related disorders. For example, this name would be used to describe someone with severe sensorineural hearing loss caused by WFS1 gene mutations but without diabetes or other features. WFS1 -related disorders include sensorineural hearing loss, diabetes mellitus, psychiatric illness/disorders, and variable optic atrophy.

Symptoms/secondary effects of Wolfram’s syndrome include premature death, diabetes mellitis, diabetes insipidus, visual impairment, including optical atrophy, color blindness, slow reacting iris, high frequency hearing loss and tonal deafness, emotional agitation, tremors, seizures, peripheral neuropathy, autonomic dysfunction, ataxia, ptosis, nystagmus, endocrinopathies, brainstem atrophy, gastrointestinal disorders, including dysmotility (diarrhea/constipation), urinary' tract atony, urinary incontinence, recurrent urinary infections, hydronephrosis, primary gonadal atrophy (especially in men), menstrual irregularities and delayed menarche, among others, including psychiatric disorders such as severe depression, bipolar disorder, impulsive verbal and physical aggression. Therapy pursuant to the present invention involves inhibiting, delaying the onset of and/or ameliorating at least one and preferably numerous symptoms/secondary effects associated with Wolfram’s syndrome. Often diabetes mellitis and/or diabetes insipidus or one or more psychiatric disorders are ameliorated pursuant to treatment according to the present invention.

The present invention is also directed to treating individuals who suffer from “heterozygous w'olframin”, rather than homozygous or full Wolfram syndrome (described above). Individuals with heterozygrous woifraniin, far more numerous in numbers than individuals with Wolfram syndrome, often suffer from psychiatric disorders, especially depressive disorders including bipolar disorder, depression, severe depression and impulsive verbal and physical aggression.

The term“ibudilast” or AV411 or a derivative or analog thereof is used to describe certain pyrazolo[l ,5a]pyridine compounds which find use in the present invention. Thus, ibudilast compounds useful in the present invention include Ibudilast (AY411), AV1013 (w'hich substitutes a free amine for one of the two methyl groups in the 2-methyibutanone side chain of Ibudilast) as well as pyrazolo[l,5a]pyridine phosphosdiesterase inhibitors disclosed in international application publication W02007146087, the entire contents of which are incorporated by reference herein.

Preferred ibudiiast derivati ve or analogs thereof include ibudiiast and related compounds according to the chemical structure:

Where R ¹ is a C ₁ -C ₆ alkyl, preferably a C ₂ or C ₃ alkyl, most often isopropyl; and

R ² is C ₁ -C ₆ alkyl (preferably a C ₂ -C ₄ alkyl, most often isopropyl) where each R ^c is independently H or C ₁ -C ₃ alkyl (preferably H or methyl); and

i is 0, 1, 2 or 3 (preferably 0 or 1 ), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

In preferred compounds, R ^] is isopropyl and R2 is isopropyl (ibudiiast AV411) or

= ( )(P b)M 1.· (i is 1 and R ^c is methyl) (AV1013) or a pharmaceutically acceptable salt or en -antiomer thereof.

The term“pharmaceutically acceptable salt” or“salt” is used throughout the specification to describe a salt form of one or more of the compositions herein which are presented to increase the solubility of the compound in saline for parenteral delivery or in the gastric juices of the patient’s gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids well known m the pharmaceutical art. Sodium and potassium salts rnay be preferred as neutralization salts of carboxylic acids and free acid phosphate containing compositions according to the present invention. Salts of amines are often prepared by acidifying the free amine with an acid, especially a weak acid such as an organic acid to form an ammonium salt (e.g. ammonium lactate, ammonium acetate, ammonium chloride, ammonium sulfate, etc.), among other methods. The term“salt” shall mean any salt consistent with the use of the compounds according to the present invention. In the case where the compounds are used in

pharmaceutical indications, including the treatment of Wolfram’s syndrome, including heterozygous wolframin, the term“salt” shall mean a pharmaceutically acceptable salt, consistent with the use of the compounds as pharmaceutical agents.

The tern“coadministration” shall mean that at least two compounds or compositions are administered to the patient at the same time, such that effective amounts or concentrations of each of the two or more compounds may be found in the patient at a given point in time. Although compounds according to the present invention may be co-administered to a patient at the same time, the term embraces both administration of two or more agents at the same time or at different times, provided that effective concentrations of all coadministered compounds or compositions are found in the subject at a given time. Compounds according to the present invention may be administered with one or more additional bioactive agents, especially including an additional agent for purposes of treating one or more symptoms or second disease states of Wolfram’s syndrome or heterozygroups wolframin.

Pharmaceutical compositions comprising combinations of an effective amount of at least one compound disclosed herein, often according to the present invention and one or additional compounds as otherwise described herein, all in effective amounts, in combination with a pharmaceutically effective amount of a carrier, additi ve or excipient, represents a further aspect of the present invention. These may be used in combination with at least one additional, optional bioactive agents, especially antibiotics as otherwise disclosed herein.

The compositions of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers and may also be administered in controlled-release formulations. Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium earboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.

The compositions of the present invention may be administered orally, parenteraily, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, among others. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional, intraperitoneal and intracranial injection or infusion techniques. Preferably, the compositions are administered orally (including via intubation through the mouth or nose into the stomach), intraperitoneal!y or intravenously.

Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or weting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injeetabies, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, earners which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule fomi, useful diluents include lactose and dried com starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may he administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also be administered topically, especially to treat symptoms which occur in or on the skin. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-acceptable transdermal patches may also be used.

For topical applications, the phamiaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol,

polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.

Alternatively, the phamiaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more

pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the phamiaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

The amount of compound in a pharmaceutical composition of the instant in vention that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host and disease treated, the particular mode of administration. Preferably, the compositions should be formulated to contain between about 0.05 milligram to about 750 milligrams or more, more preferably about 1 m illigram to about 600 milligrams, and even more preferably about 10 milligrams to about 500 milligrams of active ingredient, alone or in combination with at least one additional compound which may be used to a symptom of Wolfram syndrome or heterozygous wolframin or a secondary symptom or condition thereof.

Methods of treating patients or subjects in need for a symptom or condition of Wolfram syndrome or heterozygous wolframin as otherwise described herein, comprise administration of an effective amount of a pharmaceutical composition comprising therapeutic amounts of one or more of the compounds described herein and optionally at least one additional bioactive agent according to the present invention. The amount of active ingredient(s) used in the methods of treatment of the instant invention that may be combined with the carrier materials to produce a single dosage form will vary- depending upon the host treated, the particular mode of administration. For example, the compositions could be formulated so that a therapeutically effective dose of between about 0.01, 0.1, 1, 5, 10, 15,

20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 mg/kg of patient/day or in some embodiments, greater than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/kg of the novel compounds can be administered to a patient receiving these compositions. It should also he understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of adm inistration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease or condition being treated.

A patient or subject (e.g. a human) suffering from Wolfram syndrome or heterozygous wolframin can be treated by administering to the patient (subject) an effective amount of a compound according to the present invention including pharmaceutically acceptable salts, solvates or polymorphs, thereof optionally in a pharmaceutically acceptable carrier or diluent, either alone, or in combination with other known antibiotic or pharmaceutical agents, preferably agents which can assist m treating the bacterial infection or ameliorate the secondary effects and conditions associated with the infection. This treatment can also be administered in conjunction with other conventional therapies known in the art.

The present compounds, alone or in combination with other agents as described herein, can be administered by any appropriate route, for example, orally, parenterally, intravenously, intraderm ally, subcutaneously, or topically, in liquid, cream, gel, or solid form, or by aerosol form.

The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication, without causing serious toxic effects in the patient treated. A preferred dose of the active compound for all of the herein-mentioned conditions is in the range from about 10 ng/kg to 300 mg/kg, preferably 0.1 to 100 mg/kg per day, more generally 0.5 to about 25 mg per kilogram body weight of the recipient/patient per day A typical topical dosage will range from about 0.01-3% wt/wt in a suitable carrier.

The compound is conveniently administered in any suitable unit dosage form, including but not limited to one containing less than Irng, 1 mg to 3000 rng, preferably 5 to 500 mg of active ingredient per unit dosage form. An oral dosage of about 25-250 mg is often convenient. The active ingredient is preferably administered to achieve peak plasma

concentrations of the active compound of about 0.00001-30 mM, preferably about 0.1-30 mM. This may be achieved, for example, by the intravenous injection of a solution or formulation of the active ingredient, optionally in saline, or an aqueous medium or administered as a bolus of the active ingredient. Oral administration is also appropriate to generate effecti ve plasma concentrations of active agent.

The concentration of active compound in the drug composition will depend on absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or superv ising the administration of the compositions, and that the concentration ranges set forth herein are exemplary' only and are not intended to limit the scope or practice of the claimed

composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.

Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative can be incorporated with excipients and used in the fonn of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragaeanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primoge!, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Wren the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents.

The active compound or pharmaceutically acceptable salt thereof can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as other anticancer agents, antibiotics, antiiungals, antiinflammatories, or antiviral compounds. In certain preferred aspects of the invention, one or more chimeric antibody-recruiting compound according to the present invention is coadministered with another anticancer agent and/or another bioactive agent, as otherwise described herein.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or oilier synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite: chelating agents such as

ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can he enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled and/or sustained release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poiyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions or cholestosomes rnay also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound are then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.

Examples

First Set

Wolfram Syndrome was previously considered a mitochondrial disease because of its symptoms and the presence of several studies reporting mitochondrial dysfunction.

However, new evidence now suggests that Wolfram syndrome is caused primarily by endoplasmic reticulum (ER) dysfunction. The ER is a membrane-delineated intracellular compartment that is involved in many cellular processes including protein and lipid synthesis, calcium storage, detoxification, cell signaling, and apoptosis. It has been shown that pancreatic b cells (18) and neurons are exquisitely sensitive to ER dysfunction, probably because their high rates of protein synthesis. In Wolfram syndrome, pancreatic b cells and neuronal cells are selectively damaged by mutations in the WFS1 gene. This gene encodes a transmembrane protein, wolframin, localized to the ER. We have recently reported that ER calcium depletion leads to ele vated ER stress levels, calpain activation, and the initiation of ER stress-associated pancreatic b cell death and neurodegeneration (19).

Several protein binding partners for wolframin have been proposed (20), but more information is needed to appreciate the functional consequences of these interactions. One of these binding proteins, neuronal calcium sensor 1 (NCS1), not only binds to wolframin, but a recent report suggests that nonfunctional wolframin loses its ability to bind to NCSi (21) making NCSI susceptible to ealpain-dependent proteosomal degradation. A loss of NCSI would lead to decreased calcium signaling (FIGURE 1) which makes cells more susceptible to ER stress (22) and is essential for insulin secretion and cell survival. In Wolfram syndrome, these altered processes lead to changes in glucose tolerance and atrophy of neurons needed for hearing and vision.

Neuronal calcium sensor 1 (NCS1) regulates calcium dependent cell functions.

NCS1 is a multi-functional calcium-binding protein found in virtually all cell types, especially epithelial cells and neurons (7) Binding of calcium to NCS 1 induces a conformational change (23) that exposes a hydrophobic crevice for protein binding. NCSl has a number of protein partners (7, 24 ) and these interactions affect a range of cellular processes including those related to transduction (7) and membrane trafficking (24, 25). Most relevant to this project, NCSl regulates secretion (6), neurotransmitter release (26) and cell survival (27).

Calpain activation and Wolfram syndrome.

The inventor identified the calpain cleavage site on NCS 1 (31) and showed that mutating residues in the calpain cleavage site prevents loss of NCS 1 and maintains calcium homeostasis (32). Calpains, with calpains 1 and 2 being ubiquitously expressed and most well-characterized, form a family of promiscuous proteolytic enzymes that are essential for many biological functions (38). Calpains are found in almost all eukaryotes and some bacteria. Normally inactive, calpains are activated by high calcium levels following insults that trigger calcium flow through plasma membrane-associated channels or release from intracellular stores which includes both the ER and the mitochondria. Once activated, calpains cleave numerous protein substrates including membrane receptors, ion channels, structural proteins, kinases and phosphatases, ultimately leading to calcium dysregulation and cell death (39). Calpain hyperactivation is a major factor underlying neurodegeneration in traumatic brain injury (40), Alzheimer’s disease (41), ischemia (42) and other

neurodegenerative conditions (39).

After calpain is activated there is cleavage and degradation of a number of substrates, including NCS 1 , which then diminishes In sP3R -mediated calcium signaling. In addition to changes in calcium homeostasis, calpain hyperactivation facilitates a pro-inflammatory, pro- neurodegenerative environment (43-45). Normally, calpain activity is atenuated by activation of its endogenous inhibitor, calpastatin (CAST). We and other have shown that inhibition of caipain activity with caipain inhibitors or through overexpressing of CAST is effective in rescuing neurodegeneration at the molecular and behavioral levels (46-48). We also showed that overexpressing CAST protected NCS! level and rescued calcium signaling in neuroblastoma cells treated with paclitaxel (1 1) However, the lack of inhibitor specificity and the broad range of caipain substrates present a major challenge for using direct caipain inhibitors to rescue neurodegeneration. We have searched for specific agents that prevent caipain activation (e.g., by decreasing calcium elevations) or protect critical proteins needed to prevent Wolfram syndrome.

Significance. Currently, no therapies for Wolfram syndrome exist. Our long-term goal is to establish an effective treatment for Wolfram syndrome and to test whether these strategies will address other diseases in which ER dysfunction is involved. We have recognized a new calcium signaling pathway that influences the progression of Wolfram syndrome. This pathway is known to be targeted by drugs and the effect of treatment can be assessed using non-invasive tests in mice (glucose tolerance test and hearing acuity). These tests, along with visual acuity assessment, can be translatable to human subjects.

Experimental data

The experimental data presented herein show that wo!framin and NCSl interact and that there are functional consequences of these interactions.

Wolframin/NCS 1 interaction

Recently, data showed that wolframin interacts directly with NCS 1 and it was suggested that this complex is necessary _' to promote calcium transfer between the ER and mitochondria (4). We tagged the cytoplasmic domain of wolframin to glutathione S- transferase (GST) and showed that NCS1 interacts directly with wolframin in a calcium dependent manner (FIGURE 2). From these pull-down experiments w'e localize binding of NCS1 to the cytoplasmic domain of w _' olframin (FIGURE 2), which is the N-temiinal region, residues 1-288. Our in-silico docking experiments using the crystal structure ofNCSl and the structure of wolframin deduced by available software (Robetta Server) are consistent with binding in tins region (FIGURE 2B). Note that the caipain cleavage site of NCSl (FIGURE 2B, red arrow) appears to be capped by wolframin. Using the Broad Institute cell line information for a separate project, we selected 4 cell lines with different endogenous levels of NCS 1. In these cell lines we found that NCS 1 and wolframin le vels are co-regulated, measured as both mRNA (FIGURE 3) and protein levels. In WFS1 patient derived fibroblasts, NCS1 was reduced and resting calcium was reduced (4). NCS1 overexpression in these patient derived cells restored calcium homeostasis and cell functions, measured as mitochondrial respiration (4). To compare with a mouse model of Wolfram syndrome, we examined the levels of wolframin and NCS1 in tissues from WFSl-KO mice. Wolframin was deleted in all tissues examined (brain tissue shown in FIGURE 4). As expected from the human cells, NCS1 levels were reduced to at least half of WT levels (FIGURE 4). These results provide support for the hypothesis that NCS i is a component of the pathogenic mechanism for Wolfram syndrome.

Functional effects of decreased wolframin

A. Decreased calcium signaling. The addition of glucose stimulates calcium oscillations in INS-1 cells, an insulin-secreting cell line derived from pancreatic tissue (45) This increase in calcium is needed for insulin secretion. Both intracellular calcium release channels and voltage gated calcium channels on the plasma membrane are regulated by NCS1 (7, 24 ), and both channels are needed to maintain glucose stimulated insulin release (45). INS-1 cells lacking wolframin had a decreased response to the addition of 30 mM glucose, when compared to WT cells. Both the amplitude of each calcium transient and the number of oscillations were diminished (FIGURE 5). This blunted response to glucose likely contributes to the decreased insulin secretion, a measure of the pathophysiology of Wolfram syndrome.

B. NCS1 levels change after glucose treatment. INS-1 cells w'ere incubated for 48 hours with 0, 2, or 30 mM glucose. The levels of NCS 1 after treatment with 0 or 2 mM glucose were the same in WT and WFSl-KO INS-1 cells. After prolonged exposure to 30 mM glucose, NCS1 was elevated in the WT cells, but decreased in the WFSl-KO cells (FIGURE 6). For both ceil lines the level of NCS1 was normalized to b-actin so that the percent change is assessed here, rather than the relative level of NCS 1 in the WT and NCS 1 -KO cells. The elevated NCS1 is consistent with our and others studies showing that NCS1 levels are increased with stress (8), presumably to promote cell survival. The lack of elevation of NCS1 in the WFSl-KO cells is consistent with decreased calcium homeostasis and poor survival of pancreatic b-cel!s.

C. Changes in pAKT phosphorylation. The level of phosphorylation of AKT was decreased in WFSl-KO INS-1 cells when compared to WT cells (FIGURE 7). The decreased pAKT in the WTSI-KO cells is consistent with poor cell survival in Wolfram syndrome.

Decreased celI survival. The colony formation assay showed that lack of wolframin reduced the number of colonies formed (FIGURE:, left panels), as shown using NCS1-KO cells (FIGURE, right panels). Similarly, cell survival is diminished in WFS1-KO cells when compared to WT cells after treatment with 30 raM glucose for 48 hours (FIGURE 9)

Decreased glucose tolerance in mice. After an injection of a bolus of glucose, we found that glucose tolerance was similar in mice lacking either wolframin or NCS1 (FIGURE 10). These results show that the two proteins regulate similar metabolic and cellular functions and that protecting NCS1 levels rnay compensate for the diminished functions associated with loss of wolframin activity in Wolfram syndrome.

Pharmacological interventions

The pathway leading to calpain activation provides potential therapeutic targets for Wolfram syndrome. To test this concept, my collaborator Dr. F. Urano (Washington

University, St. Louis) performed a small-scale screen to identify chemical compounds. The first compounds that my collaborator tested is dantrolene. This drag is a FDA -approved drag clinically used for malignant hyperthermia (54) and is an inhibitor of the ER-locaiized ryanodine receptors which will suppress leakage of calcium from the ER (55). Dantrolene restored cytosolic calcium levels in WFSl -deficient cells, suppressed apoptosis (56) and reduced calpain activity in INS-1 cells (FIGURE 1 1) and in brain lysates from WFS1-KO mice (57). These results support the hypothesis that inhibition of calpain activation would help slow the progression of Wolfram syndrome. However, dantrolene has side effects of prolonged use, primarily liver toxicity. Because of these long term effects, new, less toxic compounds are needed to treat Wolfram syndrome.

Calpain antagonists (eg, AK295, calpastatin) also have been tested as a treatment in several human diseases (79). However, direct calpain antagonists are not appropriate as a therapeutic for human subjects because calpains are ubiquitous and necessary for survival. That is, the side effects of inhibition of calpains are w'orse than the disease to be treated. The inventor tested drugs known to maintain NCS 1 -dependent expression and signaling (valproic acid, ibudilast, lithium, chlorpromazine) and calpain inhibitors (AK295). We found that valproic acid, lithium, and chlorpromazine had no effect on glucose induced cell death in WT (FIGURE 12) and WFS1 KO cells (FIGURE 13). In contrast, ibudilast and the calpain inhibitor rescued viability at high glucose concentration in WT (FIGURE 12) and WFS1 KO cells (FIGURE 13). Ibudilast had no effect on cell survival in 1 niM glucose, the normal level of glucose (FIGURE 14). The half max effect on survival occurred at approximately 300 nM, with nearly full recover _} ' at 1 uM ibudilast (FIGURE 15). The concentration range for protection from cell death by high glucose in WFS1 KO cells is similar.

Examples Second Set Materials and methods:

Reagents

All chemicals used were obtained from Sigma-A!drich unless stated otherwise. Calpain inhibitor XI was purchased from Calbiochem, ibudilast from Cayman Chemical Company, and ATP from AmericanBio. Stock of drugs was prepared in 100% DMSQ (AmericaBio), aliquoted, and stored at -20°C. For treatment, stock concentrations were diluted in cell medium and DMSO concentration was kept below 0.1% in all experiments.

Generation of stable cell lines and cell culturing

The WFSl-KO INS l 832/13 lines were generated by the Genome Engineering and iPSC Center (GEiC) at the Washington University in St. Louis. Briefly, gRNAs w'ere designed to target an early exon that is common to all transcription isoforms, cloned under a U6 promoter and validated for cleavage activity in K562 cells by cotransfecting with a plasmid expressing Cas9 under the CMV promoter (CMV-Cas9-NLS-HA). The most active gRNA (5’- construct was then cotransfected with the Cas9 plasmid to INS l cells using the nucleofection method in solution P3 and the program DS-150, following Lonza’s instructions. Transfected pool was genotyped to confirm presence of editing at the target site before it was single cell sorted into 96-well plates. Clones grown from the sorted plates w'ere genotyped at the target site to identify those carrying out-of-frame indeis in all alleles. Then positive clones were expanded. Genotype was confirmed when cells were transferred from a 6-well to a T75. A frozen vial was then test thawed to confirm good sundval, and the culture was tested for mycoplasma contamination before delivery . WFS1- OE cells were generated by transfecting WFS 1-KO clone #1 cells with a pcDNAS. l plasmid carrying the full-length WFSl sequence (Addgene, #13011} using Lipofectamine 2000 (Thermo Fisher), followed by 4 weeks of antibiotic selection with 2 mg/niL G418 (AmericanBio). WFS 1-WT and WFS 1-KO cells stably expressing mito-gCaMP6F (a gift from D. Stefam, University of Padua) were generated by transfection with mito-gCaMP6F using Lipofectamine 2000. GFP-positive cells were subsequently collected using fluorescence-activated cell sorting (FACS) NCS1 -OE and empty vector control cells were generated by transfecting WFS 1-KO clone #1 cells with a pIRES2-EGFP plasrnid (a gift from E. Gracheva, Yale University) with or without full-length NCS! sequence using Lipofectamine 2000, then collected using FACS. All INS1 cell hues were maintained at 37°C, 5% CO2, in RPMI 1640 supplemented with 10% FBS, 1% HEPES, 1% sodium pyruvate, 50mM b-mercaptoethanoi, and 1% penicillin/streptomycin (Gibco). Cell medium for WFS l-OE, mito-gCaMP6F, NCS1-OE, and the empty vector control was additionally supplemented with 1 mg/mL G418 for maintenance.

Calcium imaging

2x10 ⁵ ceils were plated on each coverslip 2 days before imaging in INS1 medium without G418. HEPES-buffered saline (140 mM NaCl, 1.13 mM MgCh, 4.7 mM KG, 2 niM CaCl ₂ , 10 mM D-glucose, and 10 mM HEPES, adjusted to pH 7.4 with NaOH) was used to prepare calcium dye solution and during imaging. In calcium-free HEPES buffer, CaCl2 was replaced with MgCl ₂ and 0.1 mM EGTA was added to chelate calcium. Fura-2-AM dye powder (Thermo Fisher) was dissolved to 4mM in calcium-containing HEPES buffer supplemented with 0.03% Pluronic acid (Thermo Fisher). On the day of imaging, each coverslip was incubated in dye solution for 45 min in the dark at room temperature. Each coverslip was then washed 3 times in HEPES-buffered saline solution before calcium imaging began. Calcium measurements were performed with a Hammamatsu Orca R2 camera attached to a Zeiss microscope with a Suter Lambda DG4 for excitation ratio imaging. Cells were imaged using sequential excitation at 340/380 nm (Fura-2-AM). Images were acquired with emission bandwidth of 501 to 550 nm every' second. The raw' 340 and 380 signals for each cell were subtracted by corresponding background signal before a 340/380 ratio was calculated. Max amplitude and area under the curve were calculated using PRISM Statistical Software 8. Rate of rise w¾s quantified as the gradient between 25% and 75% maximum amplitude. All experiments were conducted at room temperature. For calcium imaging at varying glucose concentrations, glucose concentration was maintained throughout the dye and imaging solutions.

For mitochondrial calcium imaging, cells stably expressing mito-gCaMP6F were prepared on coversiips as described above. 2 days after, cells were imaged using sequential excitation at 488 nm, and images were acquired with emission bandwidth of 501 to 555 nm. The experiment was earned out and analyzed similarly to cytosolic calcium recordings, except that after background subtraction, data were normalized to the first 10 s (baseline recording). All figures depicting calcium imaging traces show the average of 8-24 coversiips, each with 40-70 cells, from at least 3 independent recordings.

Western blot

Cultured cells were lysed in mammalian protein extraction reagent (MPER, Thermo Fisher) and mouse brains were lysed in radioimmunoprecipitation assay (RIPA) buffer containing SDS (Santa Cruz). Both MPER and RIP A were supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher). After spinning down at 13,000 rpm for 20 min at 4°C to clear cell lysate, the protein concentration was measured with the bicinchoninic acid assay (Thermo Fisher). Equal amounts of protein were loaded, and electrophoresis was performed in NuPAGE 4-12% gradient bis-tris polyacrylamide protein gels (Thermo Fisher). Proteins were transferred to a PVDF membrane and blocked with 5% milk in phosphate- buffered saline with 0.1% Tween-20 for 1 hour. Membranes were then incubated overnight with primary antibodies (see list of primary antibodies in SI Appendix, Table Si, below) at 4°C. Blots were washed and incubated with secondary' antibody for 2 h at room temperature. After washing, the secondary antibody was visualized by Pierce ECL chemiluminescence reagents (Thermo Fisher) or using a LI-COR Odyssey imaging system (Ll-CQR Biosciences).

Co-immunoprecipitation

500 pL of 1 mg/ mL mouse brain lysate (in RIPA with protease and phosphatase inhibitor) was incubated with 10 pL of NCS1 antibody (FL190, Santa Cruz) or 10 pL of rabbit IgG overnight at 4°C, followed by incubation with 30 pL of Pierce ^TM protein A/G magnetic beads (Thermo) for 2 h at 4°C. Incubated beads were washed 3 times with cold phosphate buffered saline (PBS) (American Bio), and then eluted by boiling with 20 pL of loading buffer. The eluted fractions were then analyzed via Western blot. Cell viability assay

CellTiter-Glo® (CTG) assay (Promega) was used to quantify ATP-dependent bioluminescence as an indicator of cell viability. To assess ceil viability, INS 1 cells were plated in white 96-well plates (Cat. 07-200-628, Fisher Scientific) at a density of 2x10 ³ cells per well and treated the following day with high glucose and/or the indicated drugs for 48 hours before imaging. After treatment was completed, 100 mL of CTG solution was added to each well and 20 minutes later, reading was performed using a Tecan Infinite M1000 Pro microplate reader using the following setting: 5 s orbital shaking (3 mm, 216 rpm), followed by imaging in luminescence mode with 500 ms integration time.

Calpain activity assay

Calpain-Glo™ protease assay (Promega) was used to quantify calpain activity. 1x10 ⁶ cells were plated per well on a 12-well plate. 24 h later, ceils were lysed in cytobuster (Novagen). Protein concentration was quantified using BCA assay. Calpain assay was carried out on a white 96-well plate in a 100 mI reaction set up. Per well, 25 mg protein were diluted in 50 mI of cytobuster. 50m1 of pure cytobuster was used as a negative control, and 50 mΐ of cytobuster with 2mM CaC12 and 1 mΐ pure calpain -2 was used as a positive control. Finally, 50mI of Calpain-Glo solution were added per well and 30 minutes later, reading was done using a Tecan Infinite M1000 Pro microplate reader with the same setting as described for the CTG assay.

Insulin secretion assay

For glucose-stimulated insulin secretion studies, INS1 cells were plated on 6-well plates at a density of 6x10 ⁵ ceils per well. After 24 hours, cells were incubated with drugs for another 24 hours. Two days after plating, the insulin ELISA assays were performed following a previously published protocol [79] In detail, preincubating cells in DMEM-base (Sigma) supplemented with 2.5 niM glucose for 1.5 h was followed by a 45 min incubation in DMEM-base with either 2.5 mM glucose for basal secretion or 9 mM glucose for stimulated secretion as indicated. 200 mI of supernatant were collected for analysis of insulin concentration using the Rat High Range ELISA kit (80-INSRTH-E01, ALPCO). Cells were washed with ice cold PBS and lysed in 1 mL 0.1% Triton X-100. Insulin levels were normalized to total protein measured by Micro BCA protein assay kit (23235, Thermo Fisher Scientific). Bata analysis

Data management and calculations were performed using PRISM Statistical Software 7. For comparison between two groups, unpaired, two-tailed student t-test was carried out. For comparison of more than two groups, one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, was performed. A p-value < 0.05 was considered to be statistically significant and the following notations were used in all figures: * for p<0.05, ** for pO.Ol, *** for p<0.001, and **** for p<0.0001. All error bars shown are standard deviation (SD). Detailed results of statistical analyses were included in the SI Appendix, Table S2.

Overveiw

Wolfram syndrome is an orphan, autosomal recessive genetic disorder that affects about 1 in 500,000 people worldwide and is characterized by diabetes insipidus, diabetes mellitus, optic nerve atrophy, and deafness (therefore also known by the acronym “DIDMOAD”) [1] Typically, a progressive childhood-onset of non-autoimmune, insulin-dependent diabetes mellitus is tire first diagnosed symptom at around age 6 [2] There is currently no disease- modifying treatment for Wolfram syndrome, and patients usually die in mid-adulthood [3] Up to 90% of cases can be attributed to pathogenic variants in the Wolfram syndrome 1 (WFSl) gene, which encodes for the protein wolframin (WFSl) [4] Tire remaining cases are due to mutations in the C1SD2 gene (a.k.a. WFSl) or oilier unknown genes [2] Heterozygous carriers of WFSl sequence variants make up around 1% of the world’s population and are at enhanced risk of psychiatric disorders and hearing loss [5-7] WFSl is a transmembrane protein and appears to localize to the endoplasmic reticulum (ER) [8] . It is expressed in most tissues, but at higher levels in the brain, heart, lung, and pancreas. Although the endogenous functions of WFSl remain unclear, several recent studies suggest that WFSl regulates ER stress [9, 10], mitochondrial health [11], and calcium homeostasis [12-14]

This study further investigates how WFSl regulates calcium homeostasis in the context of diabetes mellitus. Calcium is a universal second messenger and its concentration in the different cellular compartments has to be tightly regulated for proper cell functions [ 15 |. In particular, intact calcium homeostasis is integral to the survival of [16, 17] and insulin secretion from pancreatic b-cells [18-20] Additionally, dysregulation of calcium signaling has been proposed as a mechanism of many diseases such as Alzheimer’s disease [21], cancer progression [22], and diabetes mellitus [23, 24] Here, we showed that knocking -out (KO) WFS1 in rat insulinoma (INSi) cells led to elevated resting cytosolic calcium, reduced stimulus-evoked calcium signaling and consequently, to hypersusceptibility to hyperglycemia and decreased glucose-stimulated insulin secretion. Overexpressing WFS1 or WFSl’s interacting partner neuronal calcium sensor- 1 (NCS1) reversed the deficits observed in cells lacking WFS1. Moreover, caipam inhibitor XI and ibudilast rescued resting cytosolic calcium, cell viability, and insulin secretion in WFS1-KO cells. These findings further our understanding of Wolfram syndrome and other diseases caused by impaired calcium homeostasis.

Results:

Loss of WFS1 disrupts cellular calcium homeostasis

Several studies have implicated a role for WFS1 in regulating calcium homeostasis, including resting cytosolic calcium [11, 13], ER calcium storage [12], and agonist-induced ER calcium release [11, 14] To study the effects of WFS1 depletion on calcium homeostasis in pancreatic b-cells, we compared stable INSI 832/13 rat insulinoma cell lines with normal expression (WFS1 -WT) or loss of WFSi (WFSI-KO). Two WFSI-KO clones were created using clustered regularly interspaced short palindromic repeats (CRISPR) with a gRNA targeting an early, conserved exon (SI Appendix, FIGURE SI). All experimental results shown were obtained from WFSI-KO clone #1, and key findings were validated in WFSI- KO clone #2 (SI Appendix, FIGURE S3). WFS1-WT cell line was obtained from a clone with no CRISPR modification in the same preparation. We also generated stable WFS1- overexpressing (WFSl-OE) cells on the WFSI-KO background for validation experiments. Loss of WFSI in WFSI-KO cells as well as the successful re-expression of WFSI was verified using Western blot (FIGURE 16A). To confirm a previous observation that INSI ceils with reduced WFSI expression show higher resting cytosolic calcium [13], we measured resting cytosolic calcium in both cell lines using the ratiometric, cytosolic calcium dye Fura-2-AM. As expected, WFSI-KO cells showed an elevation in resting cytosolic calcium at baseline compared to WFS1-WT cells, which was normalized by re-expressing WFSI (FIGURE 16B). Similar results were obtained using the non-ratiometric, cytosolic calcium dye F!uo-4-AM (SI Appendix, FIGURE S2A). We also found that calpain activity was elevated in WFS1 -KO cells (FIGURE 16C), suggesting that our WFS1-KO cells recapitulate the deficits observed in an earlier WFS1 -knockdown cell model [13].

WFS1-KO cells show decreased ImP3R~dependent ER calcium release. Because the loss of WFS l expression has been linked to increased ER stress [9, 10] and reduced ER calcium release [11, 14], we next investigated agonist-induced calcium release from the ER in WFS i- WT and WFS1-KO cells. To measure ER calcium release via the inositol 1 ,4,5-trisphosphate- receptor (InsP3R), we used adenosine triphosphate (ATP) as the agonist for cells in calcium- free buffer. Measurements with Fura-2-AM dye showed that WFS 1-KO cells showed reduced ATP-induced InsP3R-dependent FIR calcium release into the cytosol (FIGURE 16D). Compared to the WFS1-WT cells, WFS 1 -KO cells exhibited a significant reduction in the max amplitude, area under the curve, and rate of rise (FIGURE 16E-G). Similar observations were made when cells were loaded with F!uo-4-AM dye (SI Appendix, FIGURE S2E-H). Reintroducing WFS l into WFS1-KO cells fully rescued the max amplitude and rate of rise (FIGURE 16E-G), and partially rescued the area under the curve (FIGURE 16F) Several effects of the loss of WFSl on ER calcium filling have been described in different cell lines [12, 14] . In INS 1 cells, ER calcium loading, as assessed by treatment with 1 mM thapsigargm, was not altered following the loss of WFSl (SI Appendix, FIGURE S2B-D). Furthermore, the protein expressions of InsP3Rl and InsP3R3 were not different between WFS 1 -WT and WFS 1-KO cells (SI Appendix, FIGURE S21-K).

WFS1-KO cells show decreased ER-mitochondrial calcium transfer. The ER releases calcium via the InsP3R not only into the cytosol but also into mitochondria at specialized interorganellar junctions called mitochondria-associated ER membranes (MAM) [25] Dysregulations of the MAM have been implicated in Alzheimer’s disease [26], diabetes mellitus [27, 28] and Wolfram syndrome [14] Therefore, we hypothesized that reduced cytosolic InsPBR-dependent calcium transients (FIGURE 16D-G) would be correlated with a reduction in mitochondrial calcium uptake in WFS1-KO cells. As expected, mitochondrial calcium uptake, as measured with the calcium sensor mito-gCaMP6F, was significantly smaller in WFS 1-KO cells after stimulation with ATP (FIGUE 16 H-K). Using two independent cellular fractionation protocols, we found that WFS 1 was present in the crude mitochondrial fraction, which contains MAM proteins (SI Appendix, Fig. S2L-M). This observation is consistent with several previously published proteomic analyses of the MAM structure [29-31], supporting that WFSl plays a role at the MAM. WFS1-KO cells show more severely impaired calcium signaling due to hyperglycemia Chronic hyperglycemia, or glucose toxicity, is a hallmark of diabetes mellitus and impairs b- cell physiology, particularly intracellular calcium signaling [18, 32-34] To mimic glucose toxicity in Wolfram syndrome, we treated both WFS1-WT and WFS1-KO cells with an additional 30 mM glucose for 24 h before calcium imaging w¾s performed. In the high glucose environment, resting cytosolic calcium in WFS1-WT cells rose significantly to a level comparable to WFSl-KO cells at baseline (FIGURE 17A). In contrast, resting cytosolic calcium in WFSl-KO cells treated with extra glucose remained at the same level as in untreated WFSl-KO cells, suggesting that untreated WFSl-KO cells already achieved a maximal resting cytosolic calcium. In response to increasing glucose concentrations (+ 0, 15, or 30 mM glucose for 24h), both WFS1-WT and WFSl-KO cells showed a concentration- dependent reduction in ATP-evoked ER-calcium release into the cytosol (FIGURE 17B). Nevertheless, WFSl-KO cells showed a lower calcium response at 0 mm and 15 mM additional glucose compared to WFSl-WT cells at the same concentrations. Tire response for both cell lines converged to a minimal level at the highest glucose concentration. Further analyses of max amplitude, area under the curve, and rate of rise suggested that WFSl-KO cells at baseline showed an ATP-response similar to WFSl-WT cells treated with 15 mM glucose, and WFSl-KO cells treated with 15 mM showed a similar response to WFSl-WT cells treated with 30 mM glucose (FIGURE 17C-E). These observations indicate that at baseline, WFSl-KO cells already show defects in calcium signaling comparable to WFSl- WT cells under diabetic hyperglycemia, which may result in an acceleration of functional impairments following hyperglycemia in WFSl-KO cells.

Overexpression of WFSl’s interacting partner NCS1 rescues calcium homeostasis in WFSl-KO cells

Neuronal calcium sensor-1 (NCS1) is a regulator of calcium-dependent signaling pathways

[35], such as survival [36] and insulin secretion [37], and was recently implicated in the disease mechanism of Wolfram syndrome in fibroblasts [14] WFSl and NCS1 were reported to interact, which we corroborated with co-mimunoprecipitation experiments (FIGURE 18A). Unlike the previous observation in fibroblasts, we saw no difference in NCSl protein expression between INS1 WFSl-WT and WFSl-KO cells, suggesting tissue-specific regulation (SI Appendix, FIGURE S4A-B). As several studies reported that NCSl protein expression was increased in response to cell stress [36, 38], we assessed NCS1 protein expression following hyperglycemia. We found that glucose toxicity led to an approximately 1.5-fold increase in NCS1 protein expression in WFSi-WT cells after 48 hours (FIGURE 18B-C). In contrast, in WFS1-KO cells, a significant decrease in NCS 1 protein expression was observed (FIGURE 18B-C). NCS1 rriRNA level was not changed between the different conditions (SI Appendix, FIGURE S4C), indicating that WFS1 likely regulates NCS1 protein levels post-transcriptionally Next, we overexpressed NCS1 in WFS 1-KO cells and showed that NCS 1 fully rescued both the ATP -evoked cytosolic calcium release (FIGURE 18E-H) and the resting cytosolic calcium (FIGURE 181). Overexpressing the empty vector with a green fluorescence protein (GFP) marker alone did not affect calcium response in WFS1-KO cells, suggesting that neither the transfection process nor the GFP signal interfered with our measurement. Consistent with a previous study [14], these results indicate that NCS1 plays a role m the disease mechanism of Wolfram syndrome and that NCS1 is a potential target for treatment, as previously described in other conditions [39, 40]

Calpain inhibitor XI and ibudilast rescue cell viability and resting cytosolic calcium in WFS1-KO cells

Because intracellular calcium is an important determinant of cell viability, we measured cell viability in WFSI-WT and WFS1-KO cells A luminescent, ATP-based assay was used. First, we established that knocking -out WFSI in IN SI cells did not reduce cell viability at baseline (FIGURE 19 A) or proliferation over one week (FIGURE 19B). Following hyperglycemia (additional 30mM glucose for 48b), we observed an approximately 40% reduction in cell viability in WFS1-KO cells, whereas cell viability in WFS I-WT cells was only 15% lower than under control conditions (FIGURE 19C). WFS 1-KO cells could be rescued by WFSI re expression (SI Appendix, FIGURE S5A). These findings are supported by previous reposts showing that WFS I deficiency causes progressive loss of pancreatic b-ceils [41 , 42]

Calpain inhibitor and ibndilast rescue cell viability in WFSl-KO cells. In order to reverse hyperglycemia-induced loss of cell viability pharmacologically, 6 different compounds previously shown to affect calcium homeostasis and WFSI- or NCS1 -dependent processes were tested in WFSI-WT, WFSl-KO and WFS1-OE cells (SI Appendix, FIGURE S5A). Two compounds, calpain inhibitor XI and ibndilast, fully rescued cell viability back to baseline in all three cell lines. Subsequently, we showed that calpain inhibitor XI and ibudilast did not significantly affect cell viability at baseline (FIGURE 19C) and reversed glucose toxicity-induced loss of cell viability in a dose-dependent manner in WFS1-KO cells (SI Appendix, FIGURE S5B-E). Calpain inhibitor XI is a potent, highly selective, reversible, and active site inhibitor of calpain- 1 and -2 [43] Ibudilast was developed as a phosphodiesterase 4 (PDE4) inhibitor and is approved for the treatment of patients with asthma and post-stroke dizziness in Japan [44] .

Calpain inhibitor and ibudilast rescue resting cytosolic calcium in WFS1-KO cells. To investigate a possible mechanism of drug action, we tested whether calpain inhibitor XI and ibudilast could rescue resting cytosolic calcium following the loss of WFS1. Sustained elevation in resting cytosolic calcium can lead to harmful cellular processes resulting in cell death [15] and impaired insulin secretion [18] . Both calpain inhibitor XI and ibudilast lowered the resting cytosolic calcium in WFS1-KO ceils to the level of WFS1-WT cells (FIGURE 19D-E), underscoring that disrupted calcium signaling is an important contributor to Wolfram syndrome pathology and can be targeted with calpain inhibitor XI and ibudilast.

WFS1-KO cells show' decreased insulin secretion, which can be reversed

by calpain inhibitor XI and ibudilast

In addition to reduced b-cell mass (FIGURE 19), decreased glucose-stimulated insulin secretion was observed in studies investigating animal models with WFS1 deficiency and corresponding pancreatic islets [42, 45, 46] When w'e measured glucose-stimulated insulin secretion, stimulation w'ith 9 mM glucose significantly increased insulin secretion in WFS1- WT cells, whereas WFS1-K0 cells failed to exhibit a significant increase (FIGURE 5A). This resulted in a significantly lower insulin secretion rate in WFS1-KO cells compared to WFS1-WT ceils at 9 mM glucose. Adding either calpain inhibitor XI or ibudilast reversed the impairment of glucose-stimulated insulin secretion in WFS1-KO cells. Treatment with calpain inhibitor XI did not affect insulin secretion in WFS1-WT cells and rescued secretion in WFS1-KO cells. Similar to another PDE4-inhibitor roflumilast [47], ibudilast enhanced insulin secretion at baseline in both cell lines. Following glucose stimulation, ibudilast ameliorated the difference between WFS1-WT and WFS1-KO cells

WFS1-KO cells show decreased insulin receptor and protein kinase B/Akt signaling. Studies performed in animal models lacking the insulin receptor (IR) and insulin-like growth factor I (TGFI) receptor indicate that insulin also exerts an important effect on b-cells, and that IR signaling regulates survival and insulin secretion in b cells [48-50] Therefore, w'e examined the expression levels of proteins involved in the insulin signaling network. Total IR and proteinkinase B (Akt) were similar between the WFS1-WT, WFSl-KO, and WFS1-OE cells (Si Appendix, FIGURE S6A-B). Phosphorylation of the insulin receptor (pIRb-Y1150/1151) and Akt (pS473 and pT308) was significantly reduced in WFS1-KO cells (FIGURE 5B-F). Reintroducing WFS1 in WFS1-KO cells significantly increased piRb-Y1150/1151 and rescued pAkt-S473. These data suggest that disruption of IR and Akt signaling plays a role in Wolfram syndrome pathology.

Discussion:

WFS1 regulates intracellular calcium homeostasis

Here, we describe how intracellular calcium is globally dysregulated in WFS1-KO b-ceIls. Consistent with previous studies in the field [11, 13, 14, 24], WFS1-KO cells showed elevated resting cytosolic calcium and reduced ATP-evoked calcium transients from the ER to both the cytosol and mitochondria. The exact mechanism of WFS1 -dependent InsP3R dysfunction is unclear. However, we were able to rule out reduced expression of InsPSRs or decreased ER-calcium loading as causes. There remain several possible, not mutually exclusive explanations. First, VVFSl may interact directly with InsP3R [14] and positively regulate InsP3R function similar to NCS1 [51, 52] Second, WFS1 may function as a calcium -permeable ion channel [53] Taken together, our data investigating calcium signaling in a cellular disease model of Wolfram syndrome emphasize that WFS1 is a versatile regulator of calcium homeostasis.

WFS1-KO cells are predisposed to hyperglycemia-induced impairments. When cells w'ere challenged with glucose toxicity, WFS1-KO ceils showed more severely impaired calcium signaling than WFS1-WT cells. Similar to wildtype rat islets that were cultured in high glucose over one week [32], WFS1 -KO cells showed no further increase of resting cytosolic calcium. Therefore, we propose that at baseline, WFS1-KO cells already show' signaling impairments like“prediabetic” cells. Such impairments predispose them to more severe hyperglycemia-induced defects, as supported by the lower cell viability we observed in WFSl-KO cells following hyperglycemia. This may explain w'hy Wolfram syndrome patients progressively develop more degenerative symptoms with age.

WFSl-KO cells show impaired IR and Akt signaling. Decreased IR and Akt signaling, likely linked through defects in PI3K and mTORC2 signaling [54], may contribute to impaired insulin secretion [55] and cell viability [56, 57] of WFS1-KO cells. Reduction of protein kinase B/Akt signaling may be due to the elevation of cytosolic calcium [58, 59] In addition, protein phosphatase 2A (PP2A) reduces the phosphorylation of IR, Akt, and other insulin signaling molecules and is known to be hyperactivated in diabetic states [60] Although we saw no changes in the protein expression of the catalytic subunit of PP2A (PP2Ac, SI Appendix, Fig. S6C-D), the activity of PP2a is regulated by multiple factors, including calcium [61] and post-translational modifications [60] Alternatively, the reduction in insulin secretion in WFS1-KO cells may downregulate the insulin signaling pathway. Akt signaling could be a new drug target for Wolfram syndrome as previously investigated in other conditions, including obesity and type 2 diabetes mellitus [62]

Restoring calcium homeostasis in WFS1-KO cells

Neuronal calcium sensor- 1 { NCSl) . We found that overexpressing NCS1 is a promising strategy to restore calcium homeostasis in INS1 cells. NCS1 may normalize calcium dysregulation through its enhancing effect on InsPSR activity [51, 52] and its function as a calcium sensor [35] Moreover, a recent study found that NCSl was mis-localized in adipocytes of a high-fat diet mouse model [63] Therefore, we speculate that localization or functions of NCS l are similarly altered in“prediabetic” WFS1-KO cells.

Calpain inhibitor XI and ibudilast. Pharmacological interventions with calpain inhibitor XI and ibudilast rescued resting cytosolic calcium as well as ceil viability and glucose-stimulated insulin secretion of WFS1-KO cells. The specific mechanism of action for both drugs in Wolfram syndrome has yet to be determined, but we provide evidence that they act through normalizing calcium homeostasis. Calpain, a calcium-dependent protease, is typically regulated by changes in cytosolic calcium [64] Our observation that calpain normalized resting cytosolic calcium in WFS1 -KO cells suggests feedback signaling between calpain activity and cytosolic calcium. The calpain pathway should be further investigated in b-cells in the context of hyperglycemia because calpain hyperactivity was observed in diabetic cardiomyocytes [65] and overexpression of calpastatin, the endogenous inhibitor of calpain, protected mice against diabetes [66] . We hypothesize that ibudilast normalizes calcium through its interaction with NCSl [39] . Furthermore, the effect of ibudilast on PDE4 - and hence cAMP levels - in WFS1-WT and WFS1-KO cells needs to he investigated because cAMP interacts with calcium signaling pathways and is similarly implicated in cell viability and insulin secretion of b-cells [67-69] Because it is already approved for use in humans [44], ibudilast appears to be a safe drug candidate for Wolfram syndrome. In addition to restoring b-cell function, ibudilast may also reduce neurodegenerative symptoms of Wolfram syndrome as it is known to reduce neurotoxic symptoms [70-72] and is currently in clinical trial for multiple sclerosis [73] and amyotrophic lateral sclerosis [74]

Proposed model and future directions

Dysregulations in calcium signaling have been implicated in the pathogenesis of diabetes mellitus [24, 75] and neurodegeneration [76, 77], the two hallmarks of Wolfram syndrome. Here, we propose a disease model for Wolfram syndrome where global dysregulation of intracellular calcium homeostasis disrupts associated pathways including calpain, NCS1, and Akt, and consequently causes reduced cell viability and insulin secretion (Fig. 5G). Calpain inhibitor XI and ibudilast reversed deficits caused by the loss of WFS1, which makes them promising drug candidates for the treatment of Wolfram syndrome. This effect should be recapitulated in cell lines expressing WFS1 -variants as seen in patients, and then further tested in an animal model of Wolfram syndrome [45, 46] To advance our understanding of the disease mechanism of Wolfram syndrome, the link between disrupted calcium and the IR/Akt pathway should be further investigated. Importantly, the IR signaling network is increasingly recognized as an essential and draggable pathway both in b-cells and the brain

[78] . Because Wolfram syndrome was proposed as a model system for diabetes mellitus and neurodegenerative diseases [2], w'e expect that the findings presented in this manuscript will be relevant to many fields of research.

Supporting Information (SI Appendix):

Supplementary methods:

Calcium imaging with Fluo-4-AM

Buffers were prepared following the same protocol as described for calcium imaging with Fura-2-AM. Fura-4-AM dye powder (Thermo Fisher) was dissolved to 4mM in calcium- containing HEPES buffer supplemented with 0.03% Pluronic acid (Thermo Fisher).

For measurements of ER-calcium release, cells were plated at a density of 2xl0 ⁵ cells per covers!ip. After 2 days, calcium imaging was carried out as described for Fura-2-AM. Following stimulation with 50nM ATP, cells were imaged using sequential excitation at 488 nm, and images were acquired with emission bandwidth of 501 to 555 nm. After background subtraction, data were normalized to the first 10 seconds of baseline recording. Subsequent data quantification was performed as described for Fura-2-AM. Ali figures depicting calcium imaging traces show the average of 8-12 coverslips, each with 40-70 cells, from at least 3 independent recordings.

For measurements of cytosolic calcium, 2.5xl0 ⁴ ceils were plated per well on a black, clear- bottom 96-well plate. After 2 days, wells were carefully washed two times with calcium- containing HEPES-buffered saline solution. Then, cells were incubated in Fluo-4-AM dye solution for 45 min in the dark at room temperature. After washing off the dye, cells were kept in calcium-containing HEPES-buffered saline solution and imaged a Tecan Infinite M1000 Pro microplate reader using the following setting: 2 s linear shaking (2mm, 654 rpm), followed by imaging in fluorescence mode with 40ms integration time. mRNA analysis

mRNA was isolated from TNS1 cells grown to confluency using the RNeasy Mini kit (Qiagen) and reverse-transcribed to complementary DNA (cDNA) using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For real-time reverse transcription PCR, 40 ng of cDNA was used as transcript in a reaction with POWER SYBR Green MasterMix (Life Technologies) in a 7500 Fast machine (Applied Biosystems). Each sample was run as three technical replicates on a 96-well plate. Fold change in mRNA transcript levels was determined by using the 2-AACt method [1 ] IBS was used as a control. The following primers were used: rat 18S (fwd, 5’ rev, 5’

Subcellular fractionation

Subcell ular fractionation to obtain homogenate, membrane, cytosolic, and mitochondrial fraction was carried out following the protocol provided by Abeam (R. Patten). Lysis buffer contained 250mM Sucrose, 20mM HEPES (pH 7.4), lOmM KC1, 1.5mM MgCb, ImM EDTA, and I mM EGTA. Cell lysate obtained from a 15cm dish of confluent INS 1 cells was passed through a 25G needle and centrifuged repeatedly resulting in the different fractions step by step. Additionally, we prepared crude mitochondrial extracts from HEK293 lysate using a mitochondrial extraction kit (Thermo Fisher, #89874). A cell suspension in HEK cell media (DMEM high glucose, 5% FBS, 1% PenStrep) was collected using TrypLE and pelleted at 850xg for 2 min. Between steps of centrifuging and vortexing, the reagents provided in the kit were added to the pellet step by step. Finally, mitochondria were lysed in 2% CHAPS with tris buffer. For further analysis, fractionation samples were prepared for Western Blot (see above).

Table SI: Inst of primary antibodies used

Table S2: Detailed statistical analysis for Figures S 1-S5. All data were included in statistical testing, relevant p-va!ues are shown below.

Reference for Supplemental Information SI

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