Cerebellar ataxias: Insights from recent preclinical and clinical studies Simon Kaja, Ph.D. Assistant Professor, Associate Director Preclinical Research University of Missouri – Kansas City, School of Medicine Dept. of Ophthalmology, Vision Research Center Chief Executive Officer and Co-Founder K&P Scientific LLC Kansas City, MO Director North American Operations Experimentica Ltd. Kuopio, Finland

Clinical PRESENTATION AND CURRENT TREATMENTS Cerebellar Ataxia Clinical PRESENTATION AND CURRENT TREATMENTS

Ataxia – a definition Ataxia – from Greek α-τάξις (= lack of order) Subtypes: - Cerebellar Ataxia Sensory Ataxia (loss of proprioception) Vestibular Ataxia (vertigo, nausea, vomiting) Cerebellar Ataxia: Incoordination of voluntary movements as a result of cerebellar disease, including: - a tendency for limb movements to overshoot or undershoot a target (dysmetria), a tremor that occurs during attempted movements (intention tremor), impaired force and rhythm of diadochokinesis (rapidly alternating movements), and gait ataxia. From: Kandel ER, Schwartz JH und Jessel TM: Principles of Neural Science. Norwalk: Appleton & Lange, 1991

Cerebellar ataxias - classification Cerebro-cerebellar dysfunction disturbances in carrying out voluntary movements intention tremor peculiar writing abnormalities, dysarthria (slurred speech, often characterized by explosive variations in voice intensity despite a regular rhythm) Spino-cerebellar dysfunction wide-based "drunken sailor" gait (characterized by uncertain start and stop, lateral deviations, and unequal steps) Vestibulo-cerebellar dysfunction postural instability (person tends to separate the feet on standing to gain a wider base, and avoid oscillations) Normal electropalatogram Electropalatogram of patient with dysarthria Main et al., 1997 Electropalatogram: ”smoke”

Causes of ataxia Non-hereditary ataxias Focal lesions Vitamin B12 deficiency Cerebellar degeneration due to chronic ethanol abuse Paraneoplastic cerebellar degeneration High altitude cerebral edema Coeliac disease Normal pressure hydrocephalus Hereditary ataxias (incomplete pentrance) Spinocerebellar ataxias Episodic ataxias Friedreich’s ataxia Ataxia-telangiectasia Abetalipoproteinaemia Fragile-X syndrome Cerebellar atrophy in chronic alcoholism Paraneoplastic cerebellar oedema

Prevalence of cerebellar ataxias Difficult to assess due to genetic heterogeneity, founder effects, etc.; Ataxia is commonly co-morbid with other neurological diseases, such as epilepsy, migraine, etc. existing medication for these illnesses do not improve ataxia; All figures are estimates, projected from regional studies. Autosomal-dominant: 4:100,000 Autosomal-recessive: 7:100,000 Fragile-X syndrome: 4:1,000

Current treatment options for cerebellar ataxia No confirmed pharmacological treatment Challenges: - Heterogeneous disease group Responders and non-responders Lack of well-designed clinical trials Difficult clinical assessment of ataxia Drugs: - Acetazolamide (for EA1 and EA2) Physostigmine – cholinesterase inhibitor alkaloid Choline (derivatives) Serotonin analogues Thyrotropin releasing hormone Sulfamethoxazole-trimethoprim – folate pathway inhibitors Cycloserine 4-Aminopyridine Ogawa, 2004

Acetazolamide (Diamox®) Accepted treatment for Episodic Ataxias 50% of EA1 (KCNA1) patients respond 75% of EA2 (CACNA1A) patients respond Other indications: - Glaucoma Altitude sickness Mode of action: - Unknown mode of action Carbonic anhydrase Inhibits conversion: CO2 + H2O  H+ + H2CO3 No direct effect of on Cav2.1 channels in vitro or ex vivo (shown). Activation of skeletal BKCa channels Tricario et al. 2000 Tricario et al. 2004 Acetazolamide Diuretic; Increased excretion of bicarbonate makes blood leves more acidic and enhances ventilation Kaja et al., 2008

Acetazolamide (Diamox®) Problems associated with long-term administration of Acetazolamide: - Loss of efficacy Increased incidence of side effects nephrolithiasis hyperhydrosis, paresthesia, muscle stiffness, gastrointestinal disturbance

MOLECUALR GENETICS AND GENETIC MODELS Cerebellar Ataxia MOLECUALR GENETICS AND GENETIC MODELS

Molecular Genetics of Autosomal-Dominant Ataxias CHRONIC PAROXYSMAL

Voltage-gated Calcium Channels Grouped into high- (HVA) and low- (LVA) voltage activated channels; Pore-forming α1 subunit; HVA are associated with auxiliary α2δ, β, and γ subunits Different degrees of homology; Extremely different biophysical properties, with an enormous repertoire of splice variants; Differential sensitivity to neurotoxins and organic peptides allows pharmacological dissection. High-voltage activated Low-voltage activated

Calcium channel mutations outside inside repeat I II III IV N Mouse mutations tottering leaner rolling Nagoya rocker knock-in R192Q knock-in S218L wobbly Tg-4J Tg-5J tg ln rn rk ki wb 4J 5J EA-2 Progressive ataxia SCA-6 Gln 11 2 3 1 5 6 4 7 12 9 13 8 10 14 15 16 17 18 4 6 17 7 10 12 13 14 15 1 3 11 5 8 2 9 FHM1 Human mutations 16 Interaction sites 4 subunit (specific)  subunit (unspecific) G-proteins synprint region: SNAP25, syntaxin, synaptotagmin structural presynaptic proteins: Mint-1, CASK, Velin calmodulin binding domain

Calcium Channel Mutant Mice

Variable mutation effects FHM1 S218L KI (—○—) Rolling Nagoya (—Δ—) Tg-5J (—■—) Current density 30% ↑ 70% ↓ 30% ↓ Activation voltage - 10 mV + 10 mV - 5 mV Ataxia  Reference Tottene et al 2005 Mori et al 2000 Miki et al 2008

A NEW APPROACH TO IDENTIFYING DRUG TARGETS Cerebellar Ataxia A NEW APPROACH TO IDENTIFYING DRUG TARGETS

Classical approach to therapeutic intervention Rectification of deficit Channel dysfunction (“loss of function”) Gene mutation Is this approach feasible for niche indications, such as cerebellar ataxia? Challenges:- Broad genetic heterogeneity of the group of disorders; Neglect of compensatory effects during development and adulthood. Ataxia score wild-type mutant + drug Disease phenotype (ataxia) Clinical improvement

A novel approach to treating ataxia Channel dysfunction (“loss of function”) Gene mutation Disease phenotype (ataxia) Ataxia score wild-type mutant Compensatory Mechanism(s) GABAA receptors SK channels Serotonergic system Glutamate receptors … + drug Clinical improvement Therapeutic intervention

Drug targets

Targeting Compensatory Mechanisms GABAA Receptors

GABAA receptors Heteropentamers 2x α, 2x β, γ or δ GABA Muscimol /

Tottering and Cerebellar GABAA Receptors GABAA receptor subunit expression is regulated by CaV channels, specifically CaV1.2; CaV1.2 channels are up-regulated in Tottering cerebellum, but not forebrain; The onset of ataxia temporally coincides with the maturation and migration of GABAA receptors in cerebellar granule cells. Kaja et al., 2007

Rolling Nagoya and GABAA Receptors * Yamaguchi et al., 1984; Kaja et al., 2014 Yamaguchi et al., 1984

Loss of GABAAR δ subunit in Rolling Nagoya

FHM1 Knock-In Mice R192Q KI mice S218L KI mice Gain-of-function No overt neurological phenotype S218L KI mice Cerebellar ataxia Absence epilepsy Episodic hemiplegia Increased mortality

Non-ataxic R192Q KI mice have normal GABAARs mRNA protein

Non-ataxic R192Q KI mice have normal GABAARs

Non-ataxic R192Q KI mice have normal GABAARs

Gain-of-function S218L KI mice

Summary of GABAAR changes Strain Synaptic phenotype Behavioral phenotype Change in GABAARs Affected subunits Tottering Loss of function Ataxia, epilepsy, dyskinesia, dystonia ↓ α6βxγ2 Rolling Nagoya Ataxia α6βxδ FHM1 R192Q Gain of function --- = - FHM1 S218L Ataxia, epilepsy ↑ ?

Mechanism of GABAAR

Novel Canine Model for Childhood-Onset Ataxia Kerry Blue Terrier Vermis Uvula

Haplotype Analysis

GABAA Receptor Abnormalities 6/8/2019

Reduced Ryanodine Type 1 Receptor Expression Skin Fibroblasts

Normal RyR2 and RyR Expression 6/8/2019

GABAA receptors as potential drug targets GABAA receptor expression is tightly coupled to Ca2+ influx Both increased and decreased Ca2+ influx can cause ataxia GABAA receptor abnormalities are present in both murine and canine models for cerebellar ataxia. Ligands targeting GABAARs may represent a feasible complementary strategy for treating cerebellar ataxias.

UMKC School of Medicine Vision Research Center Acknowledgements UMKC School of Medicine Vision Research Center Peter Koulen, Ph.D. Andrew Payne, Ph.D. Yuliya Naumchuk, B.S. Students: Vidhi V. Shah Alexandra N. Maynard Collaborators University of Missouri – Kansas City Nilofer Qureshi, Ph.D. Asaf Qureshi, Ph.D. Charles Van Way III, M.D. Ann Smith, Ph.D. University of British Columbia Terrance P. Snutch, Ph.D. NeuroSearch A/S Elsebet Østergaard-Nielsen University of Missouri – Columbia Dennis O’Brien, D.V.M., Ph.D. Gary S. Johnson, D.V.M., Ph.D.