**2. In vivo models to study ALS**

The exact mechanisms by which all the above-described gene products are involved in ALS pathogenesis are the subject of many ongoing researches. Current thinking about ALS pathogenesis revolves around the interplay between pre-existing genetic susceptibility and environmental factors that may trigger disease. The search for putative environmental factors has remained elusive however; transgenic animal studies have yielded the greatest wealth of information to date. Through these studies, multiple cellular pathways have been identified including, protein misfolding, RNA processing, oxidative stress, excitotoxicity, axonal transport, mitochondrial dysfunction and abnormal secretion of proteins. Nevertheless, how accurately these animal models replicate all ALS clinical symptoms of the human illness remains an unanswered and troublesome question. This section will include the complete description of all published invertebrate and vertebrate models of ALS. Other considerations such as advantages, disadvantages, cost and availability of each model will also be discussed. Each model organism has its own advantages and disadvantages. Choosing an appropriate model depends on the question being asked. Many laboratories find it useful to perform parallel experiments in two or more model systems to understand different aspects of a biochemical process.

### **2.1 Invertebrate models**

84 Amyotrophic Lateral Sclerosis

degeneration (FTLD) (Strong and Yang, 2011). Cognitive and behavioral impairment is well described in ALS with a significant proportion of patients meeting strict diagnostic criteria for FTLD. Neuroimaging and pathological studies have shown clear involvement of nonmotor areas of the brain. More recently, distinct subtypes of frontotemporal lobar dysfunction have been identified in ALS and further work to distinguish these phenotypes and their correlate neuropathology is under way. Attention has been devoted lately to a gene coding for a DNA/RNA binding protein, which have been implicated in the pathogenesis of ALS with cognitive impairments. Indeed, dominant mutations in the *TARDBP* gene, encoding for TDP-43, were reported by several groups as a primary cause of ALS in about 3% familial cases and 1.5% sporadic cases (Chio, et al., 2011; Corrado, et al., 2009; Daoud, et al., 2009; Gitcho, et al., 2008; Kabashi, et al., 2008; Millecamps, et al., 2010; Sreedharan, et al., 2008; Van Deerlin, et al., 2008). The discovery of gene mutations linked to human ALS has provided plethora opportunities to develop model systems for

Motor neuron degeneration can also occasionally occurs in patients with Parkinson's disease and frontotemporal dementia (FTD). This disease is also called Disinhibition-dementiaparkinsonism-amyotrophy complex (DDPAC) or FTDP17 (Lynch, et al., 1994). The pathologic features distinguish this disease from the ALS-parkinsonism-dementia complex of Guam seen in the peninsula of Japan and from ALS-FTD linked to chromosome 9. Mutations in the *microtubule-associated protein tau* gene (*MAPT*) have been shown to be associated with FTD and Parkinsonism (Hutton, et al., 1998). Tau is a member of the microtubule-associated protein family, which have the principal function stabilizing microtubules and promoting their assembly by binding to tubulin. As a number of reviews (Gros-Louis, et al., 2006; Julien and Kriz, 2006) have already described the relationship between MAPT and ALS, the following sections will not discuss in details about this gene and FTDP17 models that have been

The exact mechanisms by which all the above-described gene products are involved in ALS pathogenesis are the subject of many ongoing researches. Current thinking about ALS pathogenesis revolves around the interplay between pre-existing genetic susceptibility and environmental factors that may trigger disease. The search for putative environmental factors has remained elusive however; transgenic animal studies have yielded the greatest wealth of information to date. Through these studies, multiple cellular pathways have been identified including, protein misfolding, RNA processing, oxidative stress, excitotoxicity, axonal transport, mitochondrial dysfunction and abnormal secretion of proteins. Nevertheless, how accurately these animal models replicate all ALS clinical symptoms of the human illness remains an unanswered and troublesome question. This section will include the complete description of all published invertebrate and vertebrate models of ALS. Other considerations such as advantages, disadvantages, cost and availability of each model will also be discussed. Each model organism has its own advantages and disadvantages. Choosing an appropriate model depends on the question being asked. Many laboratories find it useful to perform parallel experiments in two or more model systems to understand different aspects of a

investigating mechanisms of TDP-43 associated disease.

generated.

biochemical process.

**2. In vivo models to study ALS** 

Invertebrate model organisms include systems such as the simple yeast (fungi), fly, and nematode. The study of these experimental systems began with genetics and development, moved into molecular and cellular biology prior to most recently propelled into functional genomics and proteomics. These model organisms have highly manipulable genomes allowing for rapid generation of transgenic lines to provide insight on gene functions and protein network interactions. In 2002, Sydner Brenner, Robert Horvitz and John Sulston received the Nobel Prize in Physiology and Medicine for their establishment of the nematode *Caenorhadditis elegans* as a novel model organism to explore the molecular bases of organ development and cell death. Their discoveries identified key genes involved in cell division, differentiation and apoptosis. They determined that these genes were in fact highly conserved both structurally and functionally with higher organisms, including humans. Their studies provided the framework in which simpler organisms can be used to define key pathways and processes of relevance with the important benefit that the results are often directly applicable to understand human diseases.

The key points into generating models for human disease using smaller invertebrate organisms should encompass high degree of conservation with mammals in order for the model to be useful for the identification of the molecular components implicated in disease pathogenesis. A fully sequenced genome of the studied organism should ideally be also easily accessible for genome comparison with higher vertebrates and mammals in order to facilitate evolutionary genomics studies and to quickly generate transgenic animals through DNA transformation. The model organism should also provide significant experimental advantages over their mammalian counterparts, including a short generation time, small size, ease and reasonable cost of maintenance. It should be also amenable to both forward (phenotype to gene) and reverse (gene to phenotype) genetic approaches, which are essential molecular tools to dissect out and understand gene function. Classic forward genetic characteristics allow for the identification of novel molecules or pathways involved in a particular cellular process. This can be one of the most powerful attributes of invertebrate models. Forward-genetic screens using chemical mutagens are one of the most universal ways to generate mutants to elucidate gene function. On the other side, reverse genetics allows the quick identification of pathways on which a particular gene acts. Genetic knockdown mutants, using RNA interference (RNAi) technology in which a gene product is dramatically reduced by introducing double-stranded RNA (dsRNA) into the organism, can also provide invaluable information on the role a gene plays in a biological process.

The obvious disadvantage of using invertebrate model organisms such as fly or worm models is that there are evolutionarily far from mammals and that many physiological functions are not conserved, e.g. the immune system. Furthermore, their organs are extremely undeveloped and simple compared to other animals. The limited cellular diversity also represents a major disadvantage.

#### **2.1.1 Caenorhabditis elegans**

Yet, for the vast majority of neurodegenerative disorders including ALS, the mechanisms underlying neuronal dysfunction and death remain poorly understood despite the identification of relevant disease genes. Given the fact that molecular conservation in neuronal signaling pathways across vertebrate and invertebrate is relatively high and since the *Caenorhabditis elegans* nervous system contains almost all of the known signaling and

*In Vivo* and *In Vitro* Models to Study Amyotrophic Lateral Sclerosis 87

formation to the terminal steps of differentiation and morphogenesis (Sulston, et al., 1983). Additionally, *C. elegans* have a simple nervous system of 302 neurons out of 959 cells in the adult hermaphrodite, in which each neuron has a unique position (Sulston, 1983). Twenty of these neurons are located inside the pharynx, which has its own nervous system. The remaining 282 neurons are located in various ganglia in the head and tail and also along the ventral cord, the main longitudinal axon tract (Fig. 1). The majority of the neurons develop during embryogenesis. However, 80 of them, mainly motor neurons, develop postembryonically. The structure of the nervous system is highly reproducible from animal to animal and has been described in detail by electron microscopic reconstruction (White, et al., 1986) This technique allowed White and colleague to obtain high-resolution images to identify all the synapses formed in *C. elegans* (about 5000 chemical synapses, 2000 neuromuscular junctions and some 500 gap junctions) and to map all the connections in

In addition, gene knockdown by RNA interference can be relatively easily achieved in most cell types *in vivo* or *in vitro* by injecting the double stranded RNA for a specific gene of interest, by simply soaking the animals in dsRNA, or by feeding the animals with bacteria expressing the desired dsRNA (Fire, et al., 1998; Maeda, et al., 2001). Primary neuronal and muscular cell cultures obtained by dissecting this animal have also been optimized to allow

For all these reasons, *C. elegans* has emerged as an attractive and powerful *in vivo* model system for studying pathological mechanisms in several major neurodegenerative disorders, including ALS, providing clear leads towards the identification of potential targets for the

A number of *C. elegans* models have been developed that recapitulate many aspects of ALS pathogenesis. These transgenic models mainly expressed either the SOD1 or the TDP-43 protein under the control of various gene promoters. The first *C. elegans* ALS model was first generated in 2001 (Oeda, et al., 2001). This transgenic *C. elagans* model has been generated by introducing human wild type and various human FALS *SOD1*-linked mutations (A4V, G37R and G93A) under the control of hsp16-2 heat shock and myo-3 muscle-specific promoters. The heat inducible hsp16-2 promoter allows expression of mutant SOD1 in almost all tissues, including neurons, while the myo-3 muscle-specific promoter allows high level of protein expression in all of the muscle tissues except for the pharynx. Unfortunately, no morphological abnormalities and no discernable changes in survival or behavior were observed. However, the authors reported some interesting findings. The mutant SOD1 expressing nematodes showed a reduced resistance to paraquat-induced oxidative stress. Furthermore, oxidative stress significantly reduced the degradation rate of mutant SOD1 protein, and finally aberrant accumulation of mutant human SOD1 proteins was also observed when expressed in muscle cells. Interestingly, this later pathological phenotype is

In a subsequent study, pan-neuronal expression of the G85R ALS-linked mutant form of human SOD1, using the *C. elegans* syntobrevin gene promoter (*snb-1*), coupled to a yellow fluorescent protein (YFP) produces strong locomotor defects and paralysis in this transgenic snb-1/G85RSOD1-YFP *C. elegans* model (Wang, et al., 2009a). Interestingly, the observed phenotype correlated with intra-neuronal SOD1 aggregation. Another SOD1 mutant *C. elegans* model in which aggregation, toxicity, and cellular interactions can be directly

order to work out the entire neuronal circuit of this model organism.

for stable growth of embryonic cells (Christensen, et al., 2002).

**2.1.1.1 C. Elegans SOD1 models** 

development of new therapeutic interventions against human diseases.

in line with the pathology observed in human post-mortem ALS tissues.

neurotransmitter systems found in mammals (Bargmann, 1998), many researchers have turned to this model organism to identify the mechanisms underlying neurodegenerative disease pathology.

Research in *C. elegans* has been instrumental, especially over the last decade, for the elucidation of molecular pathways implicated in many human diseases. This is highly related to the fact that the *C. elegans* genome was the first multicellular organism to be completely sequenced in 1998 (Anonymous, 1998). The worm genome contains approximately 20,000 genes, compared with 25,000 in the human genome highlighting that *C. elegans* is a powerful model system for genetic analysis and for exploring the molecular mechanisms of neuron development, function, and disease. Furthermore, at least 42% of human disease related genes have a *C. elegans* ortholog, suggesting that most biochemical pathways are conserved across evolution (Culetto and Sattelle, 2000).

### Fig. 1. Transgenic UNC-47:GFP *C. elegans*

In *C. elegans*, 26 neurons of 5 different classes express the neurotransmitter Gamma-Aminobutyric Acid (GABA) (McIntire, et al., 1993). Nineteen of these GABAergic neurons, known as the type D neurons, are required for normal locomotion by providing dorsoventral cross-inhibition to body wall muscles (McIntire, et al., 1993; White, et al., 1986). Therefore, this transgenic worm would be a good model to depict pathological changes associated with locomotor dysfunction. Arrowheads point to type D neurons (only some of the D neurons are indicated). *Courtesy of Dr Alex J. Parker from the Centre of Excellence in Neuromics, University of Montreal, Quebec, Canada.*

*C. elegans* is a free-living nematode of about 1 millimeter in length with a short generation cycle (3 days) and lifespan (around 3 weeks), large brood size (approximately 300 progeny from a single hermaphrodite), and a transparent anatomically simple body that allows for the visualization of all cell types at all stages of development (Brenner, 1974). This last feature allows researchers to easily detect and quantify neuronal cell death and protein inclusions using optical techniques. The complete *C. elegans* embryonic cell-lineage is also known, making it possible to follow organogenesis from the earliest stages of embryos

neurotransmitter systems found in mammals (Bargmann, 1998), many researchers have turned to this model organism to identify the mechanisms underlying neurodegenerative

Research in *C. elegans* has been instrumental, especially over the last decade, for the elucidation of molecular pathways implicated in many human diseases. This is highly related to the fact that the *C. elegans* genome was the first multicellular organism to be completely sequenced in 1998 (Anonymous, 1998). The worm genome contains approximately 20,000 genes, compared with 25,000 in the human genome highlighting that *C. elegans* is a powerful model system for genetic analysis and for exploring the molecular mechanisms of neuron development, function, and disease. Furthermore, at least 42% of human disease related genes have a *C. elegans* ortholog, suggesting that most biochemical

pathways are conserved across evolution (Culetto and Sattelle, 2000).

In *C. elegans*, 26 neurons of 5 different classes express the neurotransmitter Gamma-

known as the type D neurons, are required for normal locomotion by providing

Aminobutyric Acid (GABA) (McIntire, et al., 1993). Nineteen of these GABAergic neurons,

dorsoventral cross-inhibition to body wall muscles (McIntire, et al., 1993; White, et al., 1986). Therefore, this transgenic worm would be a good model to depict pathological changes associated with locomotor dysfunction. Arrowheads point to type D neurons (only some of the D neurons are indicated). *Courtesy of Dr Alex J. Parker from the Centre of Excellence in* 

*C. elegans* is a free-living nematode of about 1 millimeter in length with a short generation cycle (3 days) and lifespan (around 3 weeks), large brood size (approximately 300 progeny from a single hermaphrodite), and a transparent anatomically simple body that allows for the visualization of all cell types at all stages of development (Brenner, 1974). This last feature allows researchers to easily detect and quantify neuronal cell death and protein inclusions using optical techniques. The complete *C. elegans* embryonic cell-lineage is also known, making it possible to follow organogenesis from the earliest stages of embryos

Fig. 1. Transgenic UNC-47:GFP *C. elegans*

*Neuromics, University of Montreal, Quebec, Canada.*

disease pathology.

formation to the terminal steps of differentiation and morphogenesis (Sulston, et al., 1983). Additionally, *C. elegans* have a simple nervous system of 302 neurons out of 959 cells in the adult hermaphrodite, in which each neuron has a unique position (Sulston, 1983). Twenty of these neurons are located inside the pharynx, which has its own nervous system. The remaining 282 neurons are located in various ganglia in the head and tail and also along the ventral cord, the main longitudinal axon tract (Fig. 1). The majority of the neurons develop during embryogenesis. However, 80 of them, mainly motor neurons, develop postembryonically. The structure of the nervous system is highly reproducible from animal to animal and has been described in detail by electron microscopic reconstruction (White, et al., 1986) This technique allowed White and colleague to obtain high-resolution images to identify all the synapses formed in *C. elegans* (about 5000 chemical synapses, 2000 neuromuscular junctions and some 500 gap junctions) and to map all the connections in order to work out the entire neuronal circuit of this model organism.

In addition, gene knockdown by RNA interference can be relatively easily achieved in most cell types *in vivo* or *in vitro* by injecting the double stranded RNA for a specific gene of interest, by simply soaking the animals in dsRNA, or by feeding the animals with bacteria expressing the desired dsRNA (Fire, et al., 1998; Maeda, et al., 2001). Primary neuronal and muscular cell cultures obtained by dissecting this animal have also been optimized to allow for stable growth of embryonic cells (Christensen, et al., 2002).

For all these reasons, *C. elegans* has emerged as an attractive and powerful *in vivo* model system for studying pathological mechanisms in several major neurodegenerative disorders, including ALS, providing clear leads towards the identification of potential targets for the development of new therapeutic interventions against human diseases.

#### **2.1.1.1 C. Elegans SOD1 models**

A number of *C. elegans* models have been developed that recapitulate many aspects of ALS pathogenesis. These transgenic models mainly expressed either the SOD1 or the TDP-43 protein under the control of various gene promoters. The first *C. elegans* ALS model was first generated in 2001 (Oeda, et al., 2001). This transgenic *C. elagans* model has been generated by introducing human wild type and various human FALS *SOD1*-linked mutations (A4V, G37R and G93A) under the control of hsp16-2 heat shock and myo-3 muscle-specific promoters. The heat inducible hsp16-2 promoter allows expression of mutant SOD1 in almost all tissues, including neurons, while the myo-3 muscle-specific promoter allows high level of protein expression in all of the muscle tissues except for the pharynx. Unfortunately, no morphological abnormalities and no discernable changes in survival or behavior were observed. However, the authors reported some interesting findings. The mutant SOD1 expressing nematodes showed a reduced resistance to paraquat-induced oxidative stress. Furthermore, oxidative stress significantly reduced the degradation rate of mutant SOD1 protein, and finally aberrant accumulation of mutant human SOD1 proteins was also observed when expressed in muscle cells. Interestingly, this later pathological phenotype is in line with the pathology observed in human post-mortem ALS tissues.

In a subsequent study, pan-neuronal expression of the G85R ALS-linked mutant form of human SOD1, using the *C. elegans* syntobrevin gene promoter (*snb-1*), coupled to a yellow fluorescent protein (YFP) produces strong locomotor defects and paralysis in this transgenic snb-1/G85RSOD1-YFP *C. elegans* model (Wang, et al., 2009a). Interestingly, the observed phenotype correlated with intra-neuronal SOD1 aggregation. Another SOD1 mutant *C. elegans* model in which aggregation, toxicity, and cellular interactions can be directly

*In Vivo* and *In Vitro* Models to Study Amyotrophic Lateral Sclerosis 89

as a functional unit. Its importance for human health was recognized by the award of the Nobel Prize in Physiology and Medicine to Ed Lewis, Christiane Nusslein-Volhard and Eric Wieschaus in 1995 for their discoveries concerning the genetic control of early embryonic

Fig. 2. Expression of human *Ataxin3* gene in the compound eye of transgenic flies using the

Transgenic *drosophila* expressing human full-length WT or mutated Ataxin 3 (ATXN3) gene

*Drosophila* is cheap, of small size (approximately 2.5mm) and easy to maintain in the laboratory. One of the most attractive aspects of *Drosophila* for use as a model organism is its short reproductive cycle and large number of genetically identical progeny. Adult females can lay, following a circadian pattern, over 400 eggs within 10 days and their short 2-week generation time allows for quick analysis of mutant animals. The fly has only four pairs of chromosomes, including three autosomal chromosomes and one set of sexual X/Y chromosomes which can be directly visualized in the giant polytene chromosome of the larval salivary gland. The *Drosophila* genome has been completely sequenced (Adams, et al., 2000). Its entire genome is encoded by roughly 13,600 genes as compared to 25,000 human genes. Another interesting feature of the fly is that it is relatively easy to drive time- and tissue-specific expression of any gene of interest using the yeast transcription activator protein Gal4 in combination with the Upstream Activation Sequence (UAS) to which Gal4 specifically binds to activate gene transcription (Brand and Perrimon, 1993). To study neurodegenerative diseases, the compound eye is predominantly used because it allows the

in the compound eye. Only mutant ATAXN3 flies showed an external and internal degeneration, which is characterized by cell death and irregular ommatidia and photoreceptor distribution. *Courtesy of Dr Guy A. Rouleau from the Centre of Excellence in* 

development using *D. melanogaster* as a model.

GAL4/UAS system.

*Neuromics, University of Montreal, Quebec, Canada.*

compared between different SOD1 mutants was also reported. This mutant SOD1-YFP model, expressing various SOD1 mutants (G85R, G93A, 127X) in *C. elegans* muscle cells by the use of the muscle specific *unc-54* gene promoter resulted in mild cellular dysfunction (Gidalevitz, et al., 2009). However, when mutant SOD1 was introduced into genetic backgrounds harboring destabilizing temperature-sensitive mutations, the toxicity was enhanced significantly and a variety of toxic phenotypes were observed. Based on theses results, the authors concluded that the specific toxic phenotypes may not be simply due to aggregation toxicity of the causative mutant proteins, but may be modulated by the genetic interactions with cellular pathways harboring mildly destabilizing polymorphisms in the genetic background.

### **2.1.1.2 C. Elegans TDP-43 models**

In order to study TDP-43 function and neurotoxicity, transgenic *C. elegans* model was generated allowing pan-neuronal expression of the wild-type TDP-43 human protein (Ash, et al., 2010). This was done using the *snb-1* gene promoter driving the expression of human TDP-43 cDNA in all neurons. The transgenic snb-1/hTDP-43 worms displayed a distinctive uncoordinated phenotype characterized by non-sinusoidal, slow movement and inappropriate responses to stimulus. Transgenic worms first display this phenotype during larval stages, and the phenotype remains constant throughout the adulthood. The authors have also found that this uncoordinated phenotype correlated with abnormal motor neuron synapses. The mechanism by which nuclear TDP-43 activity leads to abnormal synapses is still unknown, but the authors hypothesized that excessive TDP-43 activity may alters some component of RNA metabolism (e.g. alternative splicing), subsequently leading to altered production of specific proteins required for proper synaptic function. In another study, also describing a *C. elegans* model of TDP-43 proteinopathy, Liachko and colleagues similarly found that overexpression of normal human TDP-43 in all *C. elegans* neurons causes motor defects (Liachko, et al., 2010). However, they also found that overexpression of various human ALS-associated TDP-43 mutants (G290A, A315T, or M337V) cause a more severe motor dysfunction phenotype. Interestingly, the authors also demonstrated that the observed motor phenotype worsen over time and that the described mutant TDP-43 *C. elegans* model recapitulates some characteristic features seen in ALS and FTLD-U patients including progressive paralysis, reduced lifespan, and degeneration of motor neurons accompanied by hyperphosphorylation, truncation, and ubiquitination of the TDP-43 protein that accumulates in detergent insoluble protein deposits.

All the above described *C. elegans* models provide a good *in vivo* system to further dissect cellular and molecular mechanisms underlying disease. Further investigations using these models may reveal insights into SOD1 and TDP-43 functions, potentially reveal neurotoxic mechanisms relevant to ALS and other neurodegenerative diseases, and ultimately lead to the development of novel therapeutic targets.

#### **2.1.2 Drosophila melanogaster**

The fruit fly *Drosophila melanogaster* is a powerful genetic tool to study neurodegenerative diseases. *Drosophila* is a complex organism, with a functioning brain and nervous system, capable of many behaviors like learning, motility, and visual acuity. This model organism is one of the oldest multi cellular eukaryotic genetic models and has been used for almost a century to examine a variety of basic biological principles, including genetic inheritance, behavioral and developmental processes, and the first experimental description of the gene

compared between different SOD1 mutants was also reported. This mutant SOD1-YFP model, expressing various SOD1 mutants (G85R, G93A, 127X) in *C. elegans* muscle cells by the use of the muscle specific *unc-54* gene promoter resulted in mild cellular dysfunction (Gidalevitz, et al., 2009). However, when mutant SOD1 was introduced into genetic backgrounds harboring destabilizing temperature-sensitive mutations, the toxicity was enhanced significantly and a variety of toxic phenotypes were observed. Based on theses results, the authors concluded that the specific toxic phenotypes may not be simply due to aggregation toxicity of the causative mutant proteins, but may be modulated by the genetic interactions with cellular pathways harboring mildly destabilizing polymorphisms in the

In order to study TDP-43 function and neurotoxicity, transgenic *C. elegans* model was generated allowing pan-neuronal expression of the wild-type TDP-43 human protein (Ash, et al., 2010). This was done using the *snb-1* gene promoter driving the expression of human TDP-43 cDNA in all neurons. The transgenic snb-1/hTDP-43 worms displayed a distinctive uncoordinated phenotype characterized by non-sinusoidal, slow movement and inappropriate responses to stimulus. Transgenic worms first display this phenotype during larval stages, and the phenotype remains constant throughout the adulthood. The authors have also found that this uncoordinated phenotype correlated with abnormal motor neuron synapses. The mechanism by which nuclear TDP-43 activity leads to abnormal synapses is still unknown, but the authors hypothesized that excessive TDP-43 activity may alters some component of RNA metabolism (e.g. alternative splicing), subsequently leading to altered production of specific proteins required for proper synaptic function. In another study, also describing a *C. elegans* model of TDP-43 proteinopathy, Liachko and colleagues similarly found that overexpression of normal human TDP-43 in all *C. elegans* neurons causes motor defects (Liachko, et al., 2010). However, they also found that overexpression of various human ALS-associated TDP-43 mutants (G290A, A315T, or M337V) cause a more severe motor dysfunction phenotype. Interestingly, the authors also demonstrated that the observed motor phenotype worsen over time and that the described mutant TDP-43 *C. elegans* model recapitulates some characteristic features seen in ALS and FTLD-U patients including progressive paralysis, reduced lifespan, and degeneration of motor neurons accompanied by hyperphosphorylation, truncation, and ubiquitination of the TDP-43

All the above described *C. elegans* models provide a good *in vivo* system to further dissect cellular and molecular mechanisms underlying disease. Further investigations using these models may reveal insights into SOD1 and TDP-43 functions, potentially reveal neurotoxic mechanisms relevant to ALS and other neurodegenerative diseases, and ultimately lead to

The fruit fly *Drosophila melanogaster* is a powerful genetic tool to study neurodegenerative diseases. *Drosophila* is a complex organism, with a functioning brain and nervous system, capable of many behaviors like learning, motility, and visual acuity. This model organism is one of the oldest multi cellular eukaryotic genetic models and has been used for almost a century to examine a variety of basic biological principles, including genetic inheritance, behavioral and developmental processes, and the first experimental description of the gene

protein that accumulates in detergent insoluble protein deposits.

the development of novel therapeutic targets.

**2.1.2 Drosophila melanogaster** 

genetic background.

**2.1.1.2 C. Elegans TDP-43 models** 

as a functional unit. Its importance for human health was recognized by the award of the Nobel Prize in Physiology and Medicine to Ed Lewis, Christiane Nusslein-Volhard and Eric Wieschaus in 1995 for their discoveries concerning the genetic control of early embryonic development using *D. melanogaster* as a model.

Fig. 2. Expression of human *Ataxin3* gene in the compound eye of transgenic flies using the GAL4/UAS system.

Transgenic *drosophila* expressing human full-length WT or mutated Ataxin 3 (ATXN3) gene in the compound eye. Only mutant ATAXN3 flies showed an external and internal degeneration, which is characterized by cell death and irregular ommatidia and photoreceptor distribution. *Courtesy of Dr Guy A. Rouleau from the Centre of Excellence in Neuromics, University of Montreal, Quebec, Canada.*

*Drosophila* is cheap, of small size (approximately 2.5mm) and easy to maintain in the laboratory. One of the most attractive aspects of *Drosophila* for use as a model organism is its short reproductive cycle and large number of genetically identical progeny. Adult females can lay, following a circadian pattern, over 400 eggs within 10 days and their short 2-week generation time allows for quick analysis of mutant animals. The fly has only four pairs of chromosomes, including three autosomal chromosomes and one set of sexual X/Y chromosomes which can be directly visualized in the giant polytene chromosome of the larval salivary gland. The *Drosophila* genome has been completely sequenced (Adams, et al., 2000). Its entire genome is encoded by roughly 13,600 genes as compared to 25,000 human genes. Another interesting feature of the fly is that it is relatively easy to drive time- and tissue-specific expression of any gene of interest using the yeast transcription activator protein Gal4 in combination with the Upstream Activation Sequence (UAS) to which Gal4 specifically binds to activate gene transcription (Brand and Perrimon, 1993). To study neurodegenerative diseases, the compound eye is predominantly used because it allows the

*In Vivo* and *In Vitro* Models to Study Amyotrophic Lateral Sclerosis 91

(Feiguin, et al., 2009; Lin, et al., 2011; Lu, et al., 2009). These phenotypes were rescued by expression of the human TDP-43 protein in a restricted group of neurons including motor neurons (Feiguin, et al., 2009). These findings support the notion that loss of normal TDP-43 function may contribute to the pathogenesis of ALS and FTLD. On the other hand, ubiquitous or tissue-specific overexpression of either dTDP or human TDP-43 also recapitulated key hallmark features of ALS pathology including premature lethality, neuronal loss, neuromuscular junctions architecture defects and locomotor deficits (Elden, et al., 2010; Estes, et al., 2011; Hanson, et al., 2010; Li, et al., 2010; Lu, et al., 2009; Miguel, et al., 2011; Ritson, et al., 2010; Voigt, et al., 2010). Furthermore, TDP-43 expression appears to be independent of ALS/FTLD-linked mutations (Elden, et al., 2010; Estes, et al., 2011; Ritson, et al., 2010; Voigt, et al., 2010). These results also support the view of a toxic dominant gain-of-

To further explore the role of TDP-43 pathogenesis and identify pathogenic mechanisms, several independent research groups have used different genetic approaches to identify modifier genes that could suppress or enhance TDP-43 toxicity (Elden, et al., 2010; Hanson, et al., 2010; Ritson, et al., 2010). Interestingly, upregulation of Pab1-binding protein 1 (Pbp1), an ortholog of the human *ATXN2* gene, in a transgenic model of TDP-43 enhanced TDP-43 toxicity and led to a more severe TDP-43 associated phenotypes (Elden, et al., 2010). Similarly, overexpression of ubiquilin 1, a previously identified TDP- 43 interacting partner (Kim, et al., 2009), reduced steady-state TDP-43 expression but enhanced the severity of TDP-43 phenotypes (Hanson, et al., 2010). Another study also showed that the TDP-43 associated phenotypes observed in a transgenic *Drosophila* TDP-43 model was modulated by coexpression of valosin-containing protein (VCP), a member of the ATPases associated with multiple cellular activities (AAA+) family of proteins regulating a wide array of cellular processes (Ritson, et al., 2010). Further investigations on these interesting findings may enable the development of novel therapeutics targets that can regulate TDP-43 expression in

The pathogenic mechanisms underlying FUS proteinopathy remain largely unknown, although it is clear that *FUS* mutations affects motor neurons and other neuronal populations such as cortical neurons. In order to study FUS related function *in vivo*, a FUS/TLS Drosophila model was generated in which targeted expression of mutant human FUS/TLS (R518K, R521C and R521H) caused severe neurodegeneration in Drosophila eyes, whereas expression of WT human FUS/TLS resulted in very mild eye degeneration (Lanson, et al., 2011). Locomotor dysfunction and premature lethality was also observed in the mutant FUS/TLS transgenic flies. In addition, overexpression of mutant FUS/TLS caused an accumulation of ubiquitinated proteins, a pathological hallmark feature of ALS. Similarly, a pathogenic role of human ALS-associated FUS/TLS mutations (R524S and P525L) using Drosiphila has been described (Chen, et al., 2011). In this model overexpression of either Wt or ALS-mutant in different neuronal subpopulations, including photoreceptors, mushroom bodies and motor neurons led to an age-dependent progressive neuronal degeneration, including axonal loss, morphological changes and functional impairment in motor neurons. The human FUS/TLS drosophila ortholog (*caz*) has been also disrupted in order to study FUS function. The *caz* deficient fly exhibited reduced life span and locomotor deficits as compared with controls. Interestingly, these phenotypes were fully rescued by WT human FUS, but not ALS-associated mutant FUS proteins suggesting

function mechanism(s) associated with TDP-43 proteinopathies.

patients and hopefully delay or cure TDP-43-linked patients.

**2.1.2.3 Drosophila FUS models** 

generation of a neurodegenerative phenotype (rough eye phenotype) that can be easily appreciated under a standard light microscope (Fig. 2). In turn, the Gal4/UAS system can be also used to screen for genetic modifier (enhancer or suppressor) genes.

Based on these observations, it is obvious that *Drosophila melanogaster* can offer unique opportunities in the study of human neurodegeneration. Nonetheless, the major disadvantage of using *D. melanogaster* as a model is that forward genetic screen using RNA interference technology is more tedious than in the *C.elegans* model. For instance, at the moment, knockdown of genes by RNAi cannot be carried out by simply feeding flies with double-stranded RNA (as in the worm model); it has to be injected into the embryo. Although very laborious, this problem could be overcome by generating transgenic constructs expressing dsRNA for each transcript.

### **2.1.2.1 Drosophila SOD1 models**

Initial ALS studies in *Drosophila* showed either reduced longevity and fertility, increased susceptibility to oxidative stress, motor deficits and/or necrotic cell death in the fly eye of *Sod* null flies (Phillips, et al., 1989). Surprisingly, subsequent studies in transgenic fly model, overexpressing human WT SOD1 only in motor neurons, showed an extension of lifespan, without affecting locomotion or motor neuron survival (Parkes, et al., 1998). In contrast, another study showed that selective expression of WT or human SOD1 (hSOD1) diseaselinked (A4V, G85R) mutants in motor neurons induced progressive motor dysfunctions, coupled with electrophysiological defects and abnormal accumulation of the protein and a stress response in surrounding glial cells (Watson, et al., 2008). These effects were accompanied by synaptic transmission deficits, focal accumulation of hSOD1 in motor neurons, and up-regulation of heat shock protein in glia. This work suggests that SOD can cause cell-autonomous damage to motor neurons, and highlights that motor neuron selective expression of hSOD1 can induces change in glial cells. These results are in line with findings that disease is not strictly autonomous to motor neurons and that toxicity can propagate from one cell to another (Boillee, et al., 2006; Clement, et al., 2003). Expression of a human *SOD1* transgene in *Drosophila* motor neurons was achieved by using the yeast GAL4/UAS system. Interestingly, Mocket and colleagues showed that human WT SOD1 expression, placed in a *Drosophila* Cu-Zn Sod null background, at very low levels was sufficient to rescue the life-span reduction, increased oxidative stress, and impaired physiological function associated with the *Drosophila* Sod null model described above (Mockett, et al., 2003). However, the introduction of FALS-linked SOD1 alleles (A4V, G37R, G41D, G93C, and I113T) only partly reversed these effects. These results, in conjunction with earlier findings from FALS SOD1 expression restricted to motor neurons, suggest that the introduction of FALS mutant SOD1 alleles in *Drosophila* does not result in a dominant gainof-function, as it is observed in human patients.

### **2.1.2.2 Drosophila TDP-43 models**

A number of models for TDP-43 toxicity in flies have also been generated, revealing that the protein shows toxicity *in vivo*. Mainly, these independent studies have examined phenotypes associated with manipulation of both endogenous *Drosophila* TDP-43 ortholog (dTDP) and transgenic expression of human TDP-43. The major findings of these complementary studies were largely consistent with one other. Flies lacking dTDP appeared externally normal but presented deficient locomotive behaviors, reduced life span, anatomical defects at the neuromuscular junctions and decreased dendritic branching

generation of a neurodegenerative phenotype (rough eye phenotype) that can be easily appreciated under a standard light microscope (Fig. 2). In turn, the Gal4/UAS system can be

Based on these observations, it is obvious that *Drosophila melanogaster* can offer unique opportunities in the study of human neurodegeneration. Nonetheless, the major disadvantage of using *D. melanogaster* as a model is that forward genetic screen using RNA interference technology is more tedious than in the *C.elegans* model. For instance, at the moment, knockdown of genes by RNAi cannot be carried out by simply feeding flies with double-stranded RNA (as in the worm model); it has to be injected into the embryo. Although very laborious, this problem could be overcome by generating transgenic

Initial ALS studies in *Drosophila* showed either reduced longevity and fertility, increased susceptibility to oxidative stress, motor deficits and/or necrotic cell death in the fly eye of *Sod* null flies (Phillips, et al., 1989). Surprisingly, subsequent studies in transgenic fly model, overexpressing human WT SOD1 only in motor neurons, showed an extension of lifespan, without affecting locomotion or motor neuron survival (Parkes, et al., 1998). In contrast, another study showed that selective expression of WT or human SOD1 (hSOD1) diseaselinked (A4V, G85R) mutants in motor neurons induced progressive motor dysfunctions, coupled with electrophysiological defects and abnormal accumulation of the protein and a stress response in surrounding glial cells (Watson, et al., 2008). These effects were accompanied by synaptic transmission deficits, focal accumulation of hSOD1 in motor neurons, and up-regulation of heat shock protein in glia. This work suggests that SOD can cause cell-autonomous damage to motor neurons, and highlights that motor neuron selective expression of hSOD1 can induces change in glial cells. These results are in line with findings that disease is not strictly autonomous to motor neurons and that toxicity can propagate from one cell to another (Boillee, et al., 2006; Clement, et al., 2003). Expression of a human *SOD1* transgene in *Drosophila* motor neurons was achieved by using the yeast GAL4/UAS system. Interestingly, Mocket and colleagues showed that human WT SOD1 expression, placed in a *Drosophila* Cu-Zn Sod null background, at very low levels was sufficient to rescue the life-span reduction, increased oxidative stress, and impaired physiological function associated with the *Drosophila* Sod null model described above (Mockett, et al., 2003). However, the introduction of FALS-linked SOD1 alleles (A4V, G37R, G41D, G93C, and I113T) only partly reversed these effects. These results, in conjunction with earlier findings from FALS SOD1 expression restricted to motor neurons, suggest that the introduction of FALS mutant SOD1 alleles in *Drosophila* does not result in a dominant gain-

A number of models for TDP-43 toxicity in flies have also been generated, revealing that the protein shows toxicity *in vivo*. Mainly, these independent studies have examined phenotypes associated with manipulation of both endogenous *Drosophila* TDP-43 ortholog (dTDP) and transgenic expression of human TDP-43. The major findings of these complementary studies were largely consistent with one other. Flies lacking dTDP appeared externally normal but presented deficient locomotive behaviors, reduced life span, anatomical defects at the neuromuscular junctions and decreased dendritic branching

also used to screen for genetic modifier (enhancer or suppressor) genes.

constructs expressing dsRNA for each transcript.

of-function, as it is observed in human patients.

**2.1.2.2 Drosophila TDP-43 models** 

**2.1.2.1 Drosophila SOD1 models** 

(Feiguin, et al., 2009; Lin, et al., 2011; Lu, et al., 2009). These phenotypes were rescued by expression of the human TDP-43 protein in a restricted group of neurons including motor neurons (Feiguin, et al., 2009). These findings support the notion that loss of normal TDP-43 function may contribute to the pathogenesis of ALS and FTLD. On the other hand, ubiquitous or tissue-specific overexpression of either dTDP or human TDP-43 also recapitulated key hallmark features of ALS pathology including premature lethality, neuronal loss, neuromuscular junctions architecture defects and locomotor deficits (Elden, et al., 2010; Estes, et al., 2011; Hanson, et al., 2010; Li, et al., 2010; Lu, et al., 2009; Miguel, et al., 2011; Ritson, et al., 2010; Voigt, et al., 2010). Furthermore, TDP-43 expression appears to be independent of ALS/FTLD-linked mutations (Elden, et al., 2010; Estes, et al., 2011; Ritson, et al., 2010; Voigt, et al., 2010). These results also support the view of a toxic dominant gain-offunction mechanism(s) associated with TDP-43 proteinopathies.

To further explore the role of TDP-43 pathogenesis and identify pathogenic mechanisms, several independent research groups have used different genetic approaches to identify modifier genes that could suppress or enhance TDP-43 toxicity (Elden, et al., 2010; Hanson, et al., 2010; Ritson, et al., 2010). Interestingly, upregulation of Pab1-binding protein 1 (Pbp1), an ortholog of the human *ATXN2* gene, in a transgenic model of TDP-43 enhanced TDP-43 toxicity and led to a more severe TDP-43 associated phenotypes (Elden, et al., 2010). Similarly, overexpression of ubiquilin 1, a previously identified TDP- 43 interacting partner (Kim, et al., 2009), reduced steady-state TDP-43 expression but enhanced the severity of TDP-43 phenotypes (Hanson, et al., 2010). Another study also showed that the TDP-43 associated phenotypes observed in a transgenic *Drosophila* TDP-43 model was modulated by coexpression of valosin-containing protein (VCP), a member of the ATPases associated with multiple cellular activities (AAA+) family of proteins regulating a wide array of cellular processes (Ritson, et al., 2010). Further investigations on these interesting findings may enable the development of novel therapeutics targets that can regulate TDP-43 expression in patients and hopefully delay or cure TDP-43-linked patients.

### **2.1.2.3 Drosophila FUS models**

The pathogenic mechanisms underlying FUS proteinopathy remain largely unknown, although it is clear that *FUS* mutations affects motor neurons and other neuronal populations such as cortical neurons. In order to study FUS related function *in vivo*, a FUS/TLS Drosophila model was generated in which targeted expression of mutant human FUS/TLS (R518K, R521C and R521H) caused severe neurodegeneration in Drosophila eyes, whereas expression of WT human FUS/TLS resulted in very mild eye degeneration (Lanson, et al., 2011). Locomotor dysfunction and premature lethality was also observed in the mutant FUS/TLS transgenic flies. In addition, overexpression of mutant FUS/TLS caused an accumulation of ubiquitinated proteins, a pathological hallmark feature of ALS. Similarly, a pathogenic role of human ALS-associated FUS/TLS mutations (R524S and P525L) using Drosiphila has been described (Chen, et al., 2011). In this model overexpression of either Wt or ALS-mutant in different neuronal subpopulations, including photoreceptors, mushroom bodies and motor neurons led to an age-dependent progressive neuronal degeneration, including axonal loss, morphological changes and functional impairment in motor neurons. The human FUS/TLS drosophila ortholog (*caz*) has been also disrupted in order to study FUS function. The *caz* deficient fly exhibited reduced life span and locomotor deficits as compared with controls. Interestingly, these phenotypes were fully rescued by WT human FUS, but not ALS-associated mutant FUS proteins suggesting

*In Vivo* and *In Vitro* Models to Study Amyotrophic Lateral Sclerosis 93

afflict humans but normally do not strike mice, such as cystic fibrosis, obesity, blindness, anxiety, aggressive behavior, alcoholism, drug addiction and neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and ALS. Immunodeficient mice can also be used as hosts to facilitate cancer and AIDS research. Researchers have used a collection of innovative genetic technologies to produce custommade mouse models for a broad array of specific diseases, as well as to study the function of targeted genes. One of the most important technological advances has been the ability to produce transgenic mice, in which a new gene (cloned from human or other various species, wild-type or mutated) is inserted into the mouse's germline. Indeed, the 2007 Nobel Prize in Physiology and Medicine was awarded to Drs Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells. Even more potent approaches, it is now possible to knockout or to artificially drive the expression of an inserted gene in specific tissue or at various time during development or adulthood using the Cre/Loxp system (Sauer, 1998). There are many mouse models commercially available for genetic research including

Researches using the laboratory mouse led to major advances in our ability to treat a number of serious diseases and conditions. Genetically, mice are more closely related to humans than invertebrates in the sense that most human genes have functional mouse counterparts and the mouse genome is organized in a very similar manner to the human genome. The mouse and human genomes are approximately the same size. They most likely contain the same number of genes and show extensive synteny and conserved gene order. Importantly, mice have also genes that are often not represented in other models, such as *C. elegans* and *Drosophila*. All these advantages make the laboratory mouse the model organism of choice to study human diseases. Although mice are widely used in research, questions remain however about their reliability as a model for human diseases. In occurrence, how to explain that many drugs worked well during preclinical trials in mice, but turned out to be ineffective when used in clinical trials on humans? Here, we will review the mouse studies that contributed toward understanding the pathogenic pathways of motor neuron disease

The use of mouse models has been of particular importance in studying the pathogenesis of Amyotrophic Lateral Sclerosis. The initial description of *SOD1* gene mutations in familial ALS patients, in 1993, first led to the hypothesis that the disease resulted from compromised enzymatic activity due to the loss of the enzyme function (Rosen, et al., 1993). However, this loss of function hypothesis was rapidly refuted, as *Sod1* KO mice in which the murine *Sod1* gene was disrupted, do not develop disease (Reaume, et al., 1996). To date, over 150 different *SOD1* missense mutations have been reported. In contrast, transgenic mice ubiquitously overexpressing various *SOD1* gene mutations with different biochemical properties, even in the presence of endogenous mouse *Sod1* gene, develop a neurodegenerative disease that is quite similar to the human illness. Of particular interest, transgenic mice overexpressing WT human SOD1 or specifically expressing mutant SOD1 only in neurons or only in glial cells do not develop disease (Bruijn, et al., 1998; Gong, et al., 2000; Pramatarova, et al., 2001). A toxic gain-of-function rather than a loss-of-function of mutant *SOD1* gene is therefore believe to be involved in ALS-linked SOD1 patients. All published mouse models have used the endogenous murine *Sod1* promoter resulting in high

thousands of unique inbred strains and genetically engineered mutants.

and the testing of therapeutic approaches.

**2.2.1.1 Mice SOD1 models** 

that ALS-associated FUS/TLS mutations are toxics (Walker, et al., 2011). Through crossrescue analysis, it has been demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, the authors found that these proteins associated with each other in an RNA-dependent complex. These results establish that FUS and TDP-43 function together *in vivo* and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.
