1. 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

2. Clinical PRESENTATION AND CURRENT TREATMENTS
Cerebellar Ataxia
Clinical PRESENTATION AND CURRENT TREATMENTS

3. 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

4. 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”

5. 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

6. 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

7. 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

8. 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

9. 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

10. MOLECUALR GENETICS AND GENETIC MODELS
Cerebellar Ataxia
MOLECUALR GENETICS AND GENETIC MODELS

11. Molecular Genetics of Autosomal-Dominant Ataxias
CHRONIC
PAROXYSMAL

12. 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

13. 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

14. Calcium Channel Mutant Mice

15. 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

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

17. 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

18. 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

19. Drug targets

20. Targeting Compensatory Mechanisms
GABAA Receptors

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

22. 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

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

24. Loss of GABAAR δ subunit in Rolling Nagoya

25. 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

26. Non-ataxic R192Q KI mice have normal GABAARs
mRNA
protein

27. Non-ataxic R192Q KI mice have normal GABAARs

28. Non-ataxic R192Q KI mice have normal GABAARs

29. Gain-of-function S218L KI mice

30. 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 ↑ ?

31. Mechanism of GABAAR

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

33. Haplotype Analysis

34. GABAA Receptor Abnormalities
6/8/2019

35. Reduced Ryanodine Type 1 Receptor Expression
Skin Fibroblasts

36. Normal RyR2 and RyR Expression
6/8/2019

37. 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.

38. 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.