Side effects: the unexpected

1. Side effects: the unexpected Unreported findings in a mouse fALS model following gene therapy Alessandra Piersigilli DVM PhD DECVP InstitutfürTierpathologie – University of Bern/ Life Sciences Faculty - EcolePolitechnique Federal de Lausanne

2. ALS • Formerly called Charcot disease, Lou Gehring Disease in US • Third most common neurodegenerative cause of adult death, after Alzheimer and Parkinson’s disease • Sporadic (more common) and familial forms: no phenotypic signature, despite different gene and molecular profiles  phenotypic heterogeneous  syndrome • FALS: Most commonly associated with missense mutation of SOD1, 160 mutations • Autosomal dominant inheritance pattern • SALS: deposition of ubiquinated TDP-43  found also in most of non-SOD1 mutated fALS  proteinopathy • Subtle onset with minimal leg muscles (popliteus) fasciculations or muscle weakness, language disorders  neglected or underestimated  only advanced stage of disease well characterized  animal models  all stages. • 6.5 more common in people practicing sport (football players)  mechanic and metabolic causes or cofactors? • Adult onset, 10% diagnosed in under 40s • Fatal  Palliation

3. Animal model to study the human disease • Most of information from human patients refers to autopsy material  end stage lesions. • Animal models: access to neuronal tissue at all stages (especially subclinical) of development of the disease  gene expression and phenotype monitoring • Motor neuron disease caused/associated with gene mutation  2 possible models: 1) same genotype (mutation) with good recapitulation of phenotype (role and impact of additional factors); 2) unknown genotype but similar phenotype (identification of the gene/s mutations or altered pathway/s)

4. ALS mouse models • Transgenic mice with mutated SOD1 gene • Overexpression and knock outs  no ALS phenotype • Human patients: no correlation between clinical severity and enzyme activity levels • Mouse model: clinicopathological severity depends on transgenic copy numbers. • Hind limbs weakness/paralysis  muscle wasting (neurogenic amyotrophy)

5. Human mutated SOD1 mouse models of ALS • Developed more than 10 years ago • Commercially available • Different lines (exon-intorn mutation) • Morphologic phenotype influenced by: • Copy numbers of transgene • Gene mutation

6. Pathology of ALS like inG93A • Earliest change: Golgi apparatus fragmentation • Dilated mitochondria and endoplasmic reticulum intracytoplasmicvacuolation  onset clinical symptoms (high expressors only). • > inclusions < vacuolation • Atrophy of MNs, ubiquinated neuronal hyaline inclusions in cell body and processes (motorneurons in high expressors, astrocytes in low expressors)  seen in human patients

7. Mechanisms of neurotoxicity: cause or effect?

8. SOD1 • Superoxide dismutase 1 • Cytosolic • Binds Cu/Zn and catalyzes detoxification of superoxide radical (O.2)  H2O2 • Down or upregulation lead to oxidative stress

9. Pathogenesis of damage and disease • Peroxidation? Damage of mitochondria  > Ca and > ATP >>Ca  cell death • Phosphorylation of NFs  aggregation  impairment of transport • Glutamate excitotoxicity: loss of astrocytic glutamate transporters  glutamate inhibitors  life prolongation in G93A mice • Disrupted Calcium homeostasis: Glutamate binds to Ca permeable glutamate receptors  > intracellular Calcium  endonuclease activation • Apoptosis • SOD1+, TDP43- aggregation in fALS, Prion like propagation of misfolded proteins • RNA processing: TDP43 toxicity due to binding of RNAs  by removal of RNA binding toxicity is eliminated • Pathological templating 8misfolded SOD1 and TDP43)

10. Similarities/differences • Motoneurons loss + gliosis • Lewy bodies like and AST hyaline inclusions • Number of inclusions  clinical severity • Vacuolation of neurons and neuropil • In mice decrease of cell numbers start beyond 90 days, but vacuolation already apparent • Cortical neurons (V layer) • Lower motorneurons • Mild corticospinal tracts degneration

11. Gene therapy approach with AAV- miRNA • AAV  no tox in humans • Remain episomal  no insertional mutagenesis risk • Long term expression in stabile cells • PCR detection of human mSOD1 in puppies. Quantification of copies of TG and of vector • Compound muscular action potential measured in gastrocnemius + swimming test (time to reach 1 mt platform in a narrow plexiglas tank) • Endpoint: not able to right themselves over a 15’’ time when placed on their side (TG), discomfort (wound lesions, etc) in AAV6 mice

14. Brain mineralization • Described in vessels and/or neuroparenchymaof monkey, horses • Cellular and extracellular in brain nuclei in neurodegenerative diseases • In Purkinje cells of term neonates or newborn, associated with hypoxia/ischemia conditions • CC rat: cerebellar calcification (Purkinje cells) due to autosomal recessive spontaneous mutation  symmetric, glycoconiugates accumulation? • Never described in mouse Purkinje cells (w/o ALS)

15. Vet Pathol. 2002 Nov;39(6):732-6. Mitochondriopathy with regional encephalic mineralization in a Jack Russell Terrier. Gruber AD, Wessmann A, Vandevelde M, Summers BA, Tipold A. Mineralization of neurons, neuropil, smooth muscles cells of small arteries, capillaries in vestibulocochlear nerve, cerebellar nuclei, medulla oblongata, choroid plexus…. Mitochondrial abnormalities in liver, heart, brain  mitochondriopathy

16. ProcSocExpBiol Med. 1999 Sep;221(4):361-8 A new neurological mutant rat with symmetrical calcification of Purkinje cells in cerebellum. Ando Y, Ichihara N, Takeshita S, Nagata M, Kimura T, Tanase H, Kikuchi T. early change is accumulation of PAS+ material  storage disease ExpNeurol. 2003 Feb;179(2):127-38. Excitotoxiclesioningofthe rat basal forebrainwith S-AMPA: consequentmineralizationandassociatedglialresponse. Oliveira A, Hodges H, Rezaie P. Associated withglialresponse dystrophicmechanism

17. Hippocampal sclerosis • Associated in humans, domestic and lab animals (rodents) with seizures. Cause or effect? • Transient ischemia (primary or secondary?)  neuronal death and mineralization • Typical of mouse epilepsy models mimicking temporal lobe epilepsy (CA3)

18. What about translation into humans?

19. Acknowledments • CyliaRochat, Julianne Aebischer (Aebischer Group) • Gianni Mancini, AgnèsAutier, Jessica Dessimoz (Histology Core Facility) • Anna Oevermann (Neurocenter) • Nadine Regenscheit (itpa) • Manuela Bozzo, Eveline Rohrer, Erika Bürgi (itpa- histology lab)