Side effects: the unexpectedUnreported findings in a mouse fALS model following gene therapy

Alessandra Piersigilli DVM PhD DECVPInstitut für Tierpathologie – University of Bern/

Life Sciences Faculty - Ecole Politechnique Federal de Lausanne

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

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)

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)

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

Pathology of ALS like inG93A

• Earliest change: Golgi apparatus fragmentation• Dilated mitochondria and endoplasmic reticulum intracytoplasmic vacuolation 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

Mechanisms of neurotoxicity: cause or effect?

SOD1

• Superoxide dismutase 1• Cytosolic• Binds Cu/Zn and catalyzes detoxification of

superoxide radical (O.2) H2O2

• Down or upregulation lead to oxidative stress

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)

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

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

Brain mineralization

• Described in vessels and/or neuroparenchyma of 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)

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

Proc Soc Exp Biol 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

Exp Neurol. 2003 Feb;179(2):127-38.

Excitotoxic lesioning of the rat basal forebrain with S-AMPA: consequent mineralization and associated glial response.Oliveira A, Hodges H, Rezaie P.Associated with glial response dystrophic mechanism

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)

What about translation into humans?

Acknowledments

• Cylia Rochat, Julianne Aebischer (Aebischer Group)• Gianni Mancini, Agnès Autier, Jessica Dessimoz

(Histology Core Facility)

• Anna Oevermann (Neurocenter)• Nadine Regenscheit (itpa)• Manuela Bozzo, Eveline Rohrer, Erika Bürgi (itpa-

histology lab)