Transcript Piersigilli Alessandra (pptx)

Side effects: the unexpected
Unreported findings in a mouse fALS model following gene
therapy
Alessandra Piersigilli DVM PhD DECVP
Institut für Tierpathologie – University of Bern/
Life Sciences Faculty - Ecole Politechnique Federal de Lausanne
ALS
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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
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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
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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 AAVmiRNA
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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 (itpahistology lab)