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Research Models on alscience

ALS Research Models

Along with cellular models, animal models of a disease have become a critical tool for biomedical research and drug discovery. Given the inaccessibility of ALS affected patients tissues to monitor pre-symptomatic stages, these models provided an unprecedented tool to study disease pathogenesis and involved molecular pathways. They also provide a platform to identify putative neurotoxic agents as well as to test the potential of therapeutic compounds and approaches in preclinical settings. The aim of this section is to compile the list of published and commercially available research models that are relevant for ALS that will be updated in pace with upcoming reports.

Modeling ALS in rodents

In particular, identification of ALS-causative genes allowed genetic modeling of a disease in rodents as well as in easy-to-manipulate organisms including the fruit fly, worm and Zebra fish. Indeed, fALS gene knock-in mice recapitulate most of clinical symptoms and histopathological marks encountered in patients (Talbot and Turner 2008, Wergorzewska 2009). Nevertheless, it must be kept in mind that identical mutations result in surprisingly divergent phenotypes in different mouse strains and no model perfectly mimics the human disease.

Available genetic animal models comprise murine and rat strains bearing wt or mutated human gene, or gene knock-outs for SOD1, TDP-43, ALS2, ADAR2, Dynactine, while some other are being developed ahead with the discovery of novel ALS genetic determinants.

SOD1model

Since 1993, 139 mutations distributed throughout the SOD1 gene have been found in fALS patients. Mutant SOD1 transgenic mice have become widely accepted and, over last 15 years, an almost exclusive animal model of ALS. SOD1 is an enzyme that catalyses the conversion of superoxide anions into hydrogen peroxide that is further metabolized; as such it is a part of cellular defense mechanisms against oxidative stress. However, a strong body of evidence suggests that it is a toxic gain of function, rather than a loss of function of this enzyme, to cause selective motor neuropathy. Indeed wt SOD1 and  knock-out  mice do not develop ALS-like phenotype differently from mice bearing mutant SOD1 forms that often maintain their enzymatic activity. At present, transgenic expression of 12 human SOD1 mutations driven by the endogenous promoter is disease-causative and uniformly lethal in mice and rats, despite consistent biochemical and biophysical variation between different mutants (Turner and Talbot 2008). The model proved to be useful for identification of non neuronal cell targets of SOD1 toxicity, misregulated neurotrophic and cell death pathways, and survey for other genetic interactors that can modify the disease. Around 100 genetic cross-breeding experiments with transgenic mutant SOD1 mice have been performed to verify molecular mechanisms proposed to drive ALS pathogenesis in vivo (glutamate-induced excitotoxicity, axonal transport blockade, mitochondrial dysfunction, neuroinflammation protein misfolding and apoptosis). Furthermore, mounting evidence from mice with cell restrictive, repressible or chimeric expression of mutant SOD1 transgenes have evidenced the role of non-neural cells in disease pathogenesis and neuroprotection. Transgenic rodents have also provided the benchmark preclinical tool for evaluation of potential therapeutic pharmacological agents. Recent promising findings from gene and antisense therapies, cell replacement and combinatorial drug approaches in transgenic mutant SOD1 still await successful translation in patients. Finally, the mouse model is useful for the technical development that is also relevant to ALS diagnosis and therapeutics (e.g. spinal cord imaging, new methods for analysis of patient-derived tissues/cells).

The most prevalent and severe ALS related SOD1 mutations are A4V in the U.S.A. and H46R in Japan. The most commonly used ALS mouse model is G93A. Despite the relative rarity of this mutation it has been studied very intensely as it was the first mutation to be modeled in mice and due to the ready availability of the G93A mouse from The Jackson Lab. Established in 1995, the SOD1(G93A) mouse model is internationally accepted as a robust model for ALS research. In 2008, ALS TDI published guidelines for the model's use and interpretation of experimental results. Noteworthy, Jackson G93A mouse bears over 20 copies of the human gene. This mutation is a pseudo-WT mutation that leaves the enzyme activity intact. For a comprehensive overview of SOD1 murine models features, background and applications see also ALS mouse model guidelines published by ALS TDI and The Jackson Laboratory.

Failure of ALS clinical trials following successful preclinical testing in SOD1 mouse raised a question of how accurately does this model predict a human disease and can serve as gateway for novel therapeutic treatments. As the majority of familial cases are clinically and pathologically very similar to sporadic cases, it has been hypothesized that they share common pathogenic mechanisms. However, some pathological mechanisms that trigger sALS and fALS can be different and mutually exclusive. The striking example is the absence of pathological TDP-43 i.e., major component of ubiquitinated inclusions in 95% of sALS patients, in ALS with SOD1 mutation. The failure of  trials in the SOD1 mouse to translate into effective therapies for human ALS raised some questions about the validity of this model as a representation of sporadic disease. Recently some variables intrinsic to the model such as genetic background and vast phenotypic variability, as well as those associated with preclinical studies management have been revaluated. It has been thus suggested that the results of most of trials performed so far in SOD1 mouse are likely to reflect the noise in the distribution of survival means rather than actual drug effects. On the other side attempts to standardize preclinical testing in animal model have been made by publication of a set of guidelines by European ALS researchers consortium. These are aimed at addressing the compatibility between pre-symptomatic treatment in mice and the enrollment in the clinical trials of ALS patients with advanced disease progression, as well as still unclear differences in mouse and human pharmacokinetics. Currently, much attention is focused on clinical trials design that would also cope with inherent disease heterogeneity (genetic and phenotypic).

TDP-43 model

Identification of TDP-43 as a key pathological protein in a vast majority of sALS cases, as well as detection of mutations in a subset of fALS patients raised hopes of development of a new research and preclinical model that would mimic a disease and be representative of both genetic and sporadic forms. First published TDP43 transgenic mouse that was engineered to express human ALS related mutant A315T recapitulated well both histopathological features and clinical symptoms of a disease. Surprisingly, this mutant lacked TDP-4 inclusions that are major hallmark of high majority of both TDP-43 linked and sporadic disease. This aberrant inclusions are instead present in another mouse model overexpressing wt human protein that as well displays degeneration of specific neurons in the central nervous system, including spinal and cortical motor neurons and non-motor cortical neurons, and causes spastic quadriplegia in a dose-dependent manner, reassuming thus features of both ALS and FTLD (Wils H 2010). Wide range of new transgenic murine lines are currently under development carrying other human pathogenic mutations such as G348C, QQ31K or M337V). This is also true for the mouse lines being made overexpressing WT and mutant versions of FUS (e.g.R521C or R514G).

Rat transgenic model  constitutively and conditionally overexpressing human M337V TDP-43 mutant has a prominent motor deficit phenotype with widespread and progressive degeneration of motor neurons and denervation and atrophy of skeletal muscles and formation of TDP-43 inclusions at cellular level (Zhou 2010). Rat model for FTLD and ALS was reported in which the human TDP-43 overexpression was targeted to substantia nigra via adeno associated virus vector mediated transduction (Tatom 2009 http://www.ncbi.nlm.nih.gov/pubmed/19223871 This model recapitulated some ALS features such as TDP-433  cytoplasmic expression, ubiquitination, gliosis, neuronal loss, and motor impairment. .

Non genetic rodent ALS models

Generation of two immune-mediated animal models have been reported for the loss of motor neurons (ref). Experimental autoimmune motor neuron disease with lower motor syndromes has been induced in guinea pigs by the repeated injection of bovine spinal motor neuron antigen. Affected animals demonstrated extremity weakness associated with electromyographic and morphologic evidence of denervation, a loss of spinal cord motor neurons and localization of IgG immunoreactivity to the neuromuscular junction and motor neuron cytoplasm. Experimental autoimmune grey matter disease is a more acute and severe disorder involving both upper and lower motor neurons, induced in guinea pigs by inoculation of bovine ventral spinal cord homogenate. Both models closely resembled clinical, electrophysiological and morphological features of human ALS. Engelhardt 1986, 1989, Smith 1993.

Other experimental models of motor neuron disease has been described where the motor system damage, muscle wasting and subsequent limb paralysis were caused by toxins (Coutts 2007).

Alternative models that were indeed used as the motor neuron disease animal model before the G93A SOD1 transgenic mouse was established are murine models of spontaneous motor neuron disease like the wobbler and pmn mouse.  This mouse does not replicate a cause of ALS in that it actually represent a defect in  Golgi-associated retrograde protein (GARP) complex, impairing the cell's ability to cleanup misfolding proteins. However, it replicates common features of ALS and MND in general: primary motor neuronopathy ,Axonal pathology predominated at the level of the ventral root a substantial decline in motor neurons. Noteworthy, it has been demonstrated recently that wobbler mouse undergos TDP-43 and ubiquitin changes characteristic of sporadic ALS. (Mitsumoto and Bradley 1982, Dennis  and Citron 2009).  


Non murine models for motor neuron disease

Canine Model of ALS:  researchers have found that certain dog breeds prone to degenerative myelopathy which is accompanied by lower motor neuron involvement and motor neuron loss in the ventral horns and eventually cause paraplegia, have a mutation in the SOD1 gene that is similar to ALS linked human mutation (Awano 2009, Coates 2010, Green 2002)

In order to understand the proteins role in the disease and perform genetic screening by taking advantage of genetically well defined and easy-studied organisms ALS models have been developed using Drosophila melanogaster (Fruit fly), Caenorhabditis elegans, Danio rerio (Zebrafish) and Chick embryo.

Zebrafish is a convenient organism for modeling human motor neuron disease as it  shares the same basic organ and tissue layout with humans, have simple neural physiology, embryos develop quickly, it is relatively easy to spot motor defects and  motor neuron pathology,  and exhibits high homology and amino acid identity with human proteins. Fish ALS models were created that knock down or express mutations in zebrafish homologs of human genes or directly express human genes bearing ALS related mutations (Kabashi 2010  and Best and Alderton 2009). 

Transgenics and KO for ALS-linked genes have some motor and axon defects that recapitulate some features of human ALS. motoneuron loss, muscle atrophy, paralysis and premature death. SOD1 transgenic fish was developed overexpressing human WT and G93R mutant (Ramesh 2010) TDP-43 Zebrafish transgenics are developed bearing WT human protein and A315T, G348C, and A382T mutants, while FUS transgenics were created using WT as well as disease-associated mutations: R521C, R521H and a deletion after S57. Als2 knock-down zebrafish had severe developmental abnormalities, swimming deficits and motor neuron perturbation (Gros Louis 2008). Finally, combining the knockdowns of crucial ALS linked genes such as SOD1, TDP-4 and FUS with overexpression of both wt and mutant proteins in fish embryos provides an excellent tool for studying genetic interactions.

Several Drosophila models for ALS have so far been characterized. Noteworthy modeling disease in fly allows easy genetic manipulations and transgenic expression in selected cell populations. Expression of wild type or disease-linked (A4V, G85R) mutants of human SOD1 selectively in motor neurons induced progressive climbing deficits accompanied by defective neural circuit electrophysiology, focal accumulation of human SOD1 protein in motor neurons, and a stress response in surrounding glia but no signs of SOD1 oligomerization and no evident neuronal loss (Watson 2008). Expression of wild-type human TDP-43 protein in Drosophila motor neurons led to motor neuron loss , motor dysfunction and dramatic reduction of life span (Hanson 2010 and Li 2010). This model indicated that simply increasing human TDP-43 expression is sufficient to cause neurotoxicity in different fly cell populations. TDP-43 expression in flies recapitulates several biochemical key features of human TDP-43 proteinopathies, including abnormal phosphorylation on a disease-specific site and processing of the protein. Moreover, our TDP-43 Drosophila models indicate distinct pathways of TDP-43 toxicity might operate depending on the cell type (Miguel 2010). Instead, flies lacking Drosophila TDP-43 appeared externally normal but presented deficient locomotive behaviors, reduced life span and anatomical defects at the neuromuscular junctions. These phenotypes were rescued by expression of the human protein in a restricted group of neurons including motoneurons (Feguin 2009). ALS8 fly model was generated to express VAPB P58S mutant. Robust pathological phenotypes produced by neuronal expression of VAP(P58S) resemble VAP loss of function mutants and are opposite those of VAP overexpression, suggesting that VAP(P58S) may function as a dominant negative (Ratnaparkhi 2008).

Caenorhabditis elegans provides a simple model to study basic disease biology, protein folding and genetic interactions. It was used for instance  to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of diverse SOD1 mutants in C. elegans resulted in severe locomotor defects and paralysis, partly due to aberrant protein misfolding and aggregation of SOD1 (Wang 2009). Transgenic worms with the neuronal expression of human TDP-43 exhibit an 'uncoordinated' phenotype and have abnormal motorneuron synapses (Ash 2010). Neuronal overexpression of the worm homologue TDP-1 also results in an uncoordinated phenotype, while genetic deletion of the tdp-1 gene does not affect movement or alter motorneuron synapses.

Reported rodent ALS mod

GENE

GENETIC BACK

GROUND

REGOLATORY ELEMENTS

  PHENOTYPE

AVAILIBILITY

REFERENCES

SOD1

SOD1 WT

B6SJL or

C57BL/6

Carries the normal allele of the human SOD1 gene, insertion site is maps to chromosome 3

Mice exhibit correlates of Down Syndrome, hind limb neuromuscular pathology, subclinical motor neuron degeneration

The Jackson Lab 

Epstain 1987  Shi 1994 Avraham 1988 Jaarsma 2006

SOD1 KO

C57BL/6JEi

Targeted gene deletion by replacement of exons 1 and 2 with a PGK-hprt expression cassette

Homozygous null mice are viable, with no motor abnormalities but hypersensible to toxic or ischemic injury. They age prematurely, exhibit age related peripheral axonopathy and muscle denervation, develop macular degeneration and liver tumors

The Jackson Lab

Reaume 1996, Ho 1998, Imamura 2006, Muller 2006, Turner&Talbot 2008

SOD1 G93A

C57BL/6J

mutant human SOD1 gene expression is driven by its endogenous human SOD1 promoter

Hemizygotes exhibit a phenotype similar to ALS in humans; paralysis in one or more limbs due to loss of motor neurons from the spinal cord and abbreviated life span

The Jackson Lab 

Gurney 1994

SOD1 G37R

C57BL/6J x C3H/HeJ

Mutant human SOD1 gene expression is driven by its endogenous human promoter

Hemizygous mice are viable and fertile, develop symptoms and pathology resembling human ALS, with paralysis of one or more limbs attributable to the loss of motor neurons from the spinal cord and brainstem. Also report widespread degenerative changes in other neuronal populations, and mild-to-moderate vacuolar changes in kidney.

The Jackson Lab 

Wong 1995

SOD1 G85R

C57BL/6

Mutant human SOD1 gene expression is driven by its endogenous human promoter

Hemizygous mice are viable and fertile, exhibit unaltered endogenous SOD1 activity; develop symptoms and pathology resembling human ALS; becoming paralyzed in one or more limbs due to loss of motor neurons from the spinal cord, with rapid progression to death.

The Jackson Lab 

Bruijn 1997

SOD1 G93A

Rat Sprague-Dawley (SD)


Typically develop motor neuron disease presenting as hind limb abnormal gait and quickly progressing to overt hind limb paralysis. The rapid decline coincides with substantial loss of spinal cord motor neurons as well as marked increases in gliosis and degeneration of muscle integrity and function.

See reference

Howland 2002

TDP-43

TDP-43 WT

C57BL/6;SJL

Human TDP-43 cDNA driven by the mouse Thy1.2 promotor

Male mice develop severe tremor, abnormal reflex of hindlimbs and gait abnormalities. Females develop fine tremor only after three months of age.

See reference

Shan 2010

TDP-43 WT


full-length human TDP-43 driven by the mouse prion promoter

 Expression of human gene causes a dose-dependent downregulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, and intranuclear and cytoplasmic aggregates, TDP-43 phosphorilation. Also present reactive gliosis, axonal and myelin degeneration, gait abnormalities, and early lethality

See reference

Xu 2009

TDP-43 KO

C57BL/6

Conditional deletion of exon 3 of Tardbp

Homozygous mice fertile. Postnatal deletion of Tardbp caused dramatic loss of body fat followed by rapid death. Neurological phenotype N/A

See reference

Chiang 2010

TDP-43 +/-

C57Bl/6;

Genetrap/β-geo fusion protein/loss of functional domains of native protein

Mice are viable, fertile, normal in size and display no overt phenotype.

See reference

Sephton 2010

TDP-43 A315T

C57BL/6J x CBA.

Full-length human TARDBP/A315T inserted between exon 2-3 under mouse PrP promotor

Mice are iable, fertile, develop a progressive and fatal neurodegenerative disease reminiscent of both ALS and FTLD. Present weight loss and an abnormal gait. The TDP43 proteinforms clumps in the main part of the cell and the mouse experiences motor neuron (nerve cell) loss, muscle atrophy and shortened life span

The Jackson Lab

Wegorzewska 2009

TDP -43 M337V

Rat Sprague-Dawley (SD)

TDP-43M337V transgene under

the control of the TRE promoter

TRE-miniCMV

Constitutive expression of a transgene causes early death Conditinal ecpression causes widespread neurodegeneration that predominantly affects the motor system, progressive degeneration of motor neurons and denervation atrophy of skeletal muscles. formation of TDP-43 inclusions, cytoplasmic localization of phosphorylated TDP-43, and fragmentation of TDP-43 protein.

See the reference

Zhou 2010

TDP-43 WT

Rat Sprague-Dawley (SD)


N/A

See the reference

Zhou 2010

ALS2

ALS2 -/-

129/SV and C57BL/6

ALS2 deletion lacking exon 2 and part of exon 3

Mice developed mild signs of neurodegeneration compatible with axonal transport deficiency

See the reference

Gros Louis 2008

ALS2 KO



Mice demonstrated progressive axonal degeneration in the lateral spinal cord slowed movement without muscle weakness, consistent with upper motor neuron defects that typically lead to spasticity in humans.

See the reference

Yamanaka 2006

ALS2 KO

C57BL/6

Exons 3 and 4 of the Als2 gene replaiced by β-galactosidase-neomycin cassette

Mice have subtle motor neuron pathology and motor behavior abnormalities; neurons show marked defects in specific endosomal trafficking pathways with a severe deficit in early endosome fusion stimulating activity in vitro

See the reference

Devon 2006

ALS2 KO

129/SvJ and C57BL/6J

Exon 3 replaiced by β-galactosidase-neomycin cassette

Mice exhibited elevated anxiety responses and were impaired in motor coordination and motor learning with no evidence consistent with classic motor neuron disease

See the reference

Cai 2005

ALS2 KO

129Ola/C57BL6J

Als2 gene was disrupted by inserting a stop codon, followed by the neomycin resistance gene transcribed under the control of the Pkg1 promoter

Both the Als2-null mice and the Als2 heterozygotes are viable and fertile with no evidences for motor abnormality, with normal growth, reproductivity, survival and motor performance

See the reference

Hadano 2006

VEGF

VEGF  -/-

Swiss/129

Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF

Results in adult-onset motor neuron degeneration that resembles ALS, show signs of denervation and compensatory reinnervation, muscle histology shows neurogenic atrophy, and peripheral nerves show loss of large myelinated motor axons in the spinal cord and brainstem, reduction in motor neurone numbers, with a reactive astrocytosis, and neurofilament inclusions in surviving neurones

See the reference

Brockington 2010


Vinores 2006

Dynein/Dynactin

BICD2-N

FVB

Thy1.2-GFP-BICD2-N mice as a dynein/dynactin loss-of-function

Mice has impaired dynein/dynactin function; motor neurons show accumulation of dynein and dynactin in the cell body, Golgi fragmentation and several signs of impaired retrograde trafficking; do not develop signs of motor neuron degeneration and neurofilament and motor abnormalities, show increased lifespan

See the reference

Teuling 2008

Cai 2009

Dynamitin

C57BL/6

Human dynamitin cDNA under Thy1.2 promotor


Targeted disruption of the dynein-dynactin complex causes a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, aberrant accumulation of neurofilament proteins and denervation of muscle

See the reference

LaMonte 2002









 

In vitro models currently used in ALS research comprise both transgenic and wild-type motor neuron-enriched and astrocyte cultures, as well as ALS patients derived lymphocytes and fibroblasts. Motoneuron cultures include either primary cultures of fetal motoneurons (Gingras 2007), organotypic cultures of spinal cord sections from postnatal rodents (Drachman 2000), motoneuron like hybridoma cell line NSC-34.  In particular, NSC-34 cell lines stably expressing WT and mutant ALS related genes have been employed, as for SOD1 and TDP-43, or are in development (Gomes 2008, Ferri 2006, Colombrita 2009, Duan 2010).

Also microglial cell lines such as N9 and astrocytes transfected or not with mutant SOD1 have been used to study cell non autonomous mechanisms of ALS pathogenesis and identify neuroprotective pathways (Bendotti and Carri 2004). Such ALS model glia can mediate toxicity to motor neurons in a coculture system (Hedlund 2009). Recently, neuronal cultures are obtained from both embryonic cells and  induced pluripotent stem cells (iPS). The advantage of the latter is  the ability to create patient specific lines that carry human ALS linked mutations. Also glial restricted precursor cells derived from fibroblasts of SOD1 mice were validated for cell therapy in animal models and as a tool to sudy a response  to environmental toxins such as organophosphates (Lepore 2008).

 

 

 

 

 

 

 




     
     
     
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