C81 MICE: HOW THEY HELP US STUDY MOTOR NEURON DEGENERATION
Fisher EMC
Institute of Neurology, London, UK
E‐mail address for correspondence: [email protected]
Mice have long been used in the study of basic biology and as models for understanding the pathology of human disease, and in first trials for treatments. However, there are many different types of mouse model, each relevant to different types of study, each with specific pros and cons. In ALS research the SOD1 transgenics are of paramount importance, and they are now joined by several recent models that have modifications of other genes, all of which are adding in to the big picture of what causes motor neuron death. New data are also coming from mice in which we do not yet know the causal genetic mutation.
As with all animal studies we have to carefully evaluate biomedical research needs and what society finds acceptable in the level of distress to individual mice. We also need to be clear about what our phenotype testing is really telling us, as while mice share almost the same biochemistry and cell biological pathways as humans, our physiology can be different.
Overall many mouse models, especially the SOD1 transgenics, are providing key information in the study of motor neurons in health and disease, and in the search for effective therapies.
C82 IDENTIFICATION OF MODIFIER GENES THAT CAN DELAY DISEASE ONSET IN A MOUSE MODEL FOR ALS USING GENETIC MAPPING AND GENE EXPRESSION PROFILING
van Vught PWJ1, Veldink JH1, Groeneveld GJ1, Vliem MJ1, Kruitwagen CLJJ1, Kunst CB2 & van den Berg LH1
1Rudolf Magnus Institute of Neuroscience, Department of Neurology, University Medical Centre Utrecht, Utrecht, The Netherlands, and 2Eleanor Roosevelt Institute, University of Denver, Denver, USA
E‐mail address for correspondence: [email protected]
Background: Mice carrying mutated SOD1 in a FVB genetic background are widely used as a model to study ALS. They normally develop symptoms resembling ALS and die between 90 and 120 days of age. When these mice are crossed with mice from the C57B16/129Sv background, the offspring develop a delayed ALS onset, ranging from 140 days to 2 years, despite the presence of mutated SOD1 (1). This indicates that the expression of the ALS phenotype depends upon the genetic background and suggests that certain genes can delay the toxic effects of mutated SOD1.
Objective: To identify modifier genes that can delay disease onset in a mouse model for ALS using genetic mapping and gene expression profiling.
Methods: For the identification of the genes that can delay disease onset as a result of the mixed genetic background, we use a combination of gene expression analysis, genotype differences and clinical data (the ‘genetical genomics’ approach). Here, mRNA transcripts are treated as quantitative traits and correlated with DNA marker information. This hopefully points toward specific regions on the genome responsible for differential gene expression (expression QTL) which may lead to the identification of candidate genes responsible for delayed disease onset.
Results: Mice with mixed background and delayed onset were genotyped previously using nearly 200 genomewide polymorphic markers, which linked several loci to the delayed disease phenotype (1). So far we have isolated and amplified RNA from spinal cord tissue. Samples were labeled with either Cy3 or Cy5 and hybridized to oligo microarrays containing over 32,000 mouse‐specific genes and splice variants using an extended loop design. Linking transcript abundance to the genetic markers is in progress.
Discussion: Results will be presented at the meeting. It is hoped that this study identifies genes involved in delayed disease onset that may be targets for future treatment of ALS.
Reference
- Kunst CB, Messer L, Gordon J, et al. Genomics 2000;70:181–9.
C83 THE ROLE OF ALSIN IN JUVENILE ONSET ALS
Devon RS1, Orban P2, Schwab C2, Helm JR2, Davidson T‐L2, Rogers DA2, Simpson EM2, Leavitt BR2 & Hayden MR2
1University of Edinburgh Molecular Medicine Centre, Edinburgh, UK, and 2Centre for Molecular Medicine and Therapeutics, Vancouver, Canada
E‐mail address for correspondence: [email protected]
Background: Mutations in the ALS2 gene, encoding alsin, cause autosomal recessive, juvenile onset ALS (ALS2) and related conditions. The nine mutations that have been detected thus far are all predicted to lead to premature truncation of the alsin protein and a complete loss of function. The intact alsin protein has been shown to act as a guanine nucleotide exchange factor (GEF), or activator, of the GTPases Rab5 and Rac1. This suggests a role for alsin in endocytosis and/or vesicle trafficking.
Objectives: We are seeking an understanding of the function of alsin and its role in ALS2 pathogenesis by studying its detailed expression pattern, and by the generation and analysis of a mouse model of the human disease.
Methods: To study the expression of alsin we generated two new reagents: 1) a highly specific monoclonal antibody (N‐alsin 24), directed towards the N‐terminus of alsin; and 2) transgenic mice expressing the β‐gal gene under the control of the ALS2 promoter, enabling in‐vivo visualization by lacZ staining of the cell types in which ALS2 is expressed. The β‐gal gene in these mice replaced exons 3 and 4 of the normal gene, resulting in a null allele, thereby concurrently generating a model of ALS2.
Results and conclusions: In the adult mouse, alsin is predominantly expressed in the CNS, with high levels in the cerebellum, choroid plexus and alpha motor neurons of the spinal cord. Alsin expression colocalized with the neuronal marker NeuN but not the glial marker GFAP. In the cerebellum, the alsin protein appears to reside in the axons (but not the cell bodies) of the granular cells, which make up the bulk of the molecular layer. Expression was not observed in the Purkinje cells. Other areas of the brain showed moderate expression (including hypothalamus, amygdala and hippocampus) (1). In the periphery, expression was detected in testis and kidney, and weakly in heart and liver. Alsin was expressed throughout mouse development, although primarily in the periphery. CNS expression became predominant in neonates, largely reflecting the postnatal development of the cerebellum (1). ALS2 null mice are viable and are born at the expected Mendelian frequency. They are fertile and exhibit no gross abnormalities. In later life, they exhibit mild behavioural deficits and neuropathological changes. These mice, and cells derived from them, will prove useful in future studies of ALS2 pathogenesis.
Reference
- Devon RS, Schwab C, Topp J, et al. Neurobiol Dis. 2005;18:243–57.
C84 DEVELOPMENT OF AN ALSIN KNOCKOUT MOUSE MODEL
Deng HX, Fu R, Zhai H & Siddique T
Northwestern University, Chicago, USA
E‐mail address for correspondence: h‐[email protected]
Background: ALS2 is characterized by bilateral pyramidal syndrome, weakness with atrophy and fasciculation of the hands and/or legs without sensory disturbance. ALS2 has an early onset at an infantile and a juvenile age with very slow progression. We have previously identified a novel gene named alsin, mutations in which cause juvenile ALS type 3 (ALS2) or juvenile primary lateral sclerosis (JPLS) depending on the location of the mutations. All the mutations identified in alsin so far lead to truncated alsin protein. Thus it is postulated that loss of normal function of alsin leads to motor neuron degeneration in ALS2. Alsin is relatively a large gene with 83kb genomic DNA in size. It has 34 exons with the first exon being non‐coding. Alsin has two transcriptional forms with two distinct poly(A) signals and encodes one short and one long form of protein product, alsin. The short form of alsin gene has four exons, encoding 396 amino acids (aa). The long form of alsin has 34 exons, encoding 1657aa. Both forms share the first four exons. The physiological function and the pathogenic mechanism underlying ALS2 are not known.
Objective: To investigate the physiological function of alsin and the pathogenesis of ALS2.
Methods: We constructed a targeting vector designed to replace exon 4 and a part of exon 3 with neo‐cassette. This vector was designed to target both short and long forms of alsin, leading to a very short, truncated polypeptide consisting of only nine amino acids. We selected two positive ES cells. We developed alsin knockout mice from these ES cells.
Results: The alsin knockout mice show normal lifespan with mild deficit in motor function on RotaRod test. Degenerative pathology is mainly observed in the corticospinal tracts in the dorsal column of the spinal cord. No apparent motor neuron pathology was found in the cortex, brain stem and spinal cord. We also crossbred the alsin knockout mice with SOD1G93A transgenic mice to generate SOD1G93A transgenic mice on the null alsin background. We found that the disease course of the SOD1G93A mice without alsin is not changed.
Conclusion: Our findings from alsin knockout mouse model suggest that ALS2 in mice is predominantly an axonopathy of corticospinal tracts; loss of alsin is not the pathogenic mechanism underlying ALS1; although motor neuron degeneration is a shared late consequence in both ALS1 and ALS2, the upstream signaling pathways triggering motor neuron degeneration in ALS1 and ALS2 are independent.
C85 MOTOR CO‐ORDINATION AND LEARNING DEFICITS, INCREASED ANXIETY, AND SUSCEPTIBILITY TO OXIDATIVE STRESS IN MICE LACKING ALS2
Cai H1, Lin X1, Xie C1, Laird F2, Lai C1, Wen H2, Chiang H2, Shim H1, Hoke A2, Price D2 & Wong P2
1National Institute on Aging, Bethesda, and 2The Johns Hopkins University School of Medicine, Baltimore, USA
E‐mail address for correspondence: [email protected]
Background: Mutations in ALS2 have been linked to autosomal recessive juvenile onset amyotrophic lateral sclerosis (ALS2). With the exception of the Tunisian mutation in ALS2 suggested to be associated with both upper and lower motor neuron defects, at least eight other mutations in ALS2 have been identified and all cause the juvenile or infantile‐onset motor neuron disease, particularly affecting upper motor neurons. This Tunisian mutation, a single nucleotide deletion in exon 3 resulting in a premature stop codon, probably abrogates all the potential functions of alsin (the protein encoded by ALS2), including activities from its guanine‐nucleotide‐exchange factor (GEF) domains.
Objective: To study the physiological role of ALS2 and to clarify the pathogenic mechanisms of ALS2‐linked disease.
Methods: To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knockout (ALS2−/−) mice. A series of mouse behavioural tests, particularly on its motor functions, was applied. Electrophysiological studies were used to study the action potential transduction in both of periphery motor and sensory nerves. Histological and immunohistological studies were employed to study the neuropathological abnormalities of these animals. Primary cortical cultures were used to study the viability of neurons under oxidative stress.
Results: While ALS2−/− mice develop normally, they exhibit age‐dependent deficits in motor coordination and motor learning. Moreover, ALS2−/− mice show a higher level of anxiety as judged by either the open field or elevated plus maze tasks. Although they have not yet developed clinical or neuropathological abnormalities consistent with lower motor neuron disease by 20 months of age, ALS2−/− mice or primary cultured neurons derived from these mice were more susceptible to oxidative stress compared to wild‐type controls.
Discussion and conclusion: Taken together with findings that the majority of mutations in ALS2 are linked to upper motor neuron diseases, such as primary lateral sclerosis (PLS) and infantile onset ascending hereditary spastic paralysis (HSP), our observations are consistent with the view that loss of ALS2 is not sufficient to cause lower motor neuron disease and raise the possibility as to whether ALS2‐linked mutation found in the Tunisian family should be classified as part of a spectrum of HSP.
C86 ZEBRAFISH AS A MODEL FOR MOTOR NEURON DISEASES
Beattie CE
The Ohio State University, Columbus, USA
E‐mail address for correspondence: [email protected]
Animal models are a vital resource for studying human disease with each model organism offering different strengths. The zebrafish is both a genetic model system and is superbly suited for developmental biology studies. These attributes have resulted in it increasingly being used to model human diseases. Models generated by both forward genetics; isolating mutations that affect development of a particular organ or tissue, and by reverse genetics; using targeted gene knockdown or scanning genes in mutagenized fish to find gene‐specific mutations, have been successful at uncovering novel aspects of human diseases. For diseases not caused by gene deletion but by expression of a mutant form of a gene, transgenic fish can be generated. Once the disease model is established, a number of powerful techniques can be used to analyze the biological basis of the disease. In the context of motor neuron diseases, transgenic fish with GFP‐expressing motor neurons can be used to visualize motor axon outgrowth, genetic mosaics can be generated to determine the contribution of specific cell types to the disease process, and electrophysiological and behavioral analysis can be applied to examine the consequences of the lesions. Furthermore, it is also possible to perform suppressor screens to reveal genetic pathways or drug screens to identify therapeutic agents.
The biological basis of two of the most common motor neuron diseases, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are not well understood thus hindering development of therapeutic strategies. Due to their external development and stereotyped neuromuscular system, zebrafish offer an excellent opportunity to address the biological basis of these diseases. SMA is an autosomal recessive disease and the number one genetic cause of infant and toddler mortality. Low levels of the ubiquitous Survival Motor Neuron (SMN) protein cause SMA; it is unclear, however, how decreased SMN protein causes motor neuron cell death. Because low levels of SMN cause SMA, we modeled this disease in zebrafish using morpholino (MO) mediated protein knock down. The earliest phenotype associated with low SMN levels in zebrafish is aberrant motor axon outgrowth. Motor axons innervating the fin and axial muscle stall inappropriately and are excessively branched in animals with decreased SMN. By injecting morpholino into single motor neurons, we can recapitulate these defects indicating that SMN is needed cell‐autonomously for correct motor axon outgrowth. By following smn MO fish over time, we find a strong correlation between the severity of the motor nerve defects and decreased longevity. This, and data from others showing that SMN is transported down axons to growth cones, indicate an important role for SMN in axon development and suggest that SMA is a motor axon disease. We have more recently begun to model ALS in zebrafish by generating transgenic lines carrying SOD1 mutations. By taking advantage of the strengths of the zebrafish; genetics, accessible development, embryonic manipulations, and trangenesis, we hope to contribute to the understanding of the biological basis of both of these motor neuron diseases.