Side effects: the unexpected

Side effects: the unexpected

Unreported findings in a mouse fALS model following gene therapyAlessandra 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 USThird most common neurodegenerative cause of adult death, after Alzheimer and Parkinson’s diseaseSporadic (more common) and familial forms: no phenotypic signature, despite different gene and molecular profiles  phenotypic heterogeneous  syndromeFALS: Most commonly associated with missense mutation of SOD1, 160 mutationsAutosomal dominant inheritance patternSALS: deposition of ubiquinated TDP-43  found also in most of non-SOD1 mutated fALS  proteinopathySubtle 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 40sFatal  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

)

 H

2

O

2

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

C

ellular 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.

C

ause 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)