C85 OPTIMISING STEM-CELL DERIVED MODELS FOR ALS RESEARCH
EGGAN K
Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA
Email address for correspondence: [email protected]
A number of devastating diseases, including amyotrophic lateral sclerosis (ALS), specifically affect the neuromuscular system. Progress in understanding the molecular pathology underlying these conditions has been slow, partly because it has been impossible to access significant quantities of the disease-affected cell, the spinal motor neuron. With recent advances in stem cell and reprogramming biology, we can now produce millions of spinal motor neurons with control and diseased genotypes. Motor neurons made by stem cell and reprogramming approaches possess a molecular signature similar to embryonic motor neurons and are functional both in vivo and in vitro. These new cellular resources can now be used to design in vitro disease models for both mechanistic studies and for the discovery of novel small-molecule therapeutics.
C86 ARE MICE A GOOD MODEL FOR HUMAN ALS?
COX G
The Jackson Laboratory, Bar Harbor, Maine, USA
Email address for correspondence: [email protected]
Keywords: mouse models
My research program at The Jackson Laboratory focuses on the power of mouse genetics to understand the cellular and molecular mechanisms of neuromuscular disease. In recent years, however, significant questions about the utility of individual genetic models for predicting the efficacy of therapeutic interventions for amyotrophic lateral sclerosis (ALS) have been advanced by both investigators and funding agencies. Some concern is legitimate and limitations need to be understood and addressed so that investigators can properly plan and interpret their results. For example, the high levels of transgenic over-expression required for initiation of disease symptoms (in the case of SOD1) or the sensitivity of mice to TDP43 or FUS manipulations need to be considered when making conclusions. I would argue, however, that some of these limitations are self-inflicted due to attempts at initiating a disease in mice in less than 4-6 months when the human disease can take longer than 50 years to manifest itself in patients. This is compounded by poorly planned and under-powered mouse genetics experiments that often do not consider the effects that genetic background can have on both disease onset and progression. In my own research, we have taken advantage of these differences in genetic background effects to map modifiers of disease. The genetic heterogeneity observed in familial forms of ALS (FALS) and the high proportion of sporadic cases suggest that several genes are likely to be necessary for the survival of motor neurons. The identification of genetic modifiers in a tractable model system like the mouse offers tantalizing hope that specific proteins and biochemical pathways can be identified to serve as targets for rational therapy development. In addition, ES cell-derived motor neurons from these mouse models along with patient-derived iPS cells will allow direct comparisons of cellular pathway defects, allow modifier gene candidates to be efficiently interrogated and can provide a platform for high-throughput screening of compounds that can then be tested for efficacy in an appropriate animal model.
C87 NEURONAL EXPRESSION OF ALS-LINKED TDP-43 IN MICE IS SUFFICIENT TO TRIGGER ADULT-ONSET MOTOR NEURON DISEASE
TSAO W, SHAN X, PRICE D, WONG P
The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
Email address for correspondence: [email protected]
Keywords: TDP-43, mouse model, cell-autonomous
Background: Mutations in TDP-43, a DNA/RNA binding protein involved in RNA transcription and splicing cause familial and sporadic ALS. Accompanied with the clearance of nuclear TDP-43, cytoplasmic and intranuclear aggregates containing TDP-43 have been observed in both neurons and glia in cases of ALS. Whether disease arises from a loss of nuclear TDP-43 function and/or gain of a toxic property by cytoplasmic aggregates remains to be established. It is likewise not clear as to whether both cell-autonomous and non-cell autonomous mechanisms contribute to disease linked to TDP-43. To begin to clarify these issues, we employed both gain- and loss-of-function approaches in mouse model systems and showed that TDP-43 participates in pathways critical for motor neuron physiology, including those that regulate the normal distributions of SMN-associated GEMs in the nucleus and mitochondria in the cytoplasm.
Objectives: The fact that neuronal over-expression of wild type or ALS-linked mutant TDP-43 showed early post-natal lethality in mice precludes the opportunity to assess whether accumulation of wild type or mutant TDP-43 in neurons is sufficient to trigger adult-onset motor neuron disease. To test this hypothesis, we focused on developing transgenic mice expressing lower levels of TDP-43 in efforts to obtain a mouse model that exhibits an adult-onset motor neuron disease.
Methods: In order to drive TDP-43 expression in a neuronal specific manner, we used the Thy 1.2 promoter to develop lines of mice expressing either wild type or ALS-linked mutant TDP-43.Lines of mice expressing low levels of TDP-43 were identified by protein blot analysis. Standard behavioral and pathological analysis of TDP-43 transgenic mice and littermate controls were performed.
Results: We have identified mice expressing a lower level of either wild type or mutant TDP-43. These mice are characterized clinically by weakness and showed evidence of hindlimb paralysis near end stage disease; pathologically, there is significant loss of ventral horn motor neurons. In contrast to high expressing lines of TDP-43 mice that exhibit a developmental phenotype, we have developed TDP-43 mouse models that faithfully recapitulate both clinical and pathological features of motor neuron disease.
Discussion and conclusions: The fact that expression of wild type TDP-43 causes adult-onset motor neuron disease would suggest the possibility that increase in TDP-43 gene dosage or mechanism whereby TDP-43 is upregulated may contribute to disease in ALS. Neuronal expression of TDP-43 is sufficient to trigger adult-onset motor neuron disease indicating that cell-autonomous mechanisms are a major driving force in initiating disease in TDP-43 linked ALS. How non-cell autonomous mechanisms contribute to disease pathogenesis remains to be explored. Availability of these new TDP-43 mouse models will be useful for further clarifying disease mechanism and testing therapeutic strategies in the future.
C88 OVEREXPRESSION OF HUMAN WILD-TYPE FUS RESULTS IN MOTOR DYSFUNCTION AND DEATH IN HOMOZYGOUS MICE
MITCHELL J1, LIU L1, ROGELJ B1, KLASEN C2, SHAW CE1
1Institute of Psychiatry, Kings College, London, London, UK, 2European Molecular Biology Laboratory, Heidelberg, Germany
Email address for correspondence: [email protected]
Keywords: FUS, transgenic mouse, frontotemporal dementia
Background: Mutations in the gene encoding the RNA binding protein, Fused in Sarcoma (FUS), have been identified as causing 3-5% of familial ALS cases. FUS containing inclusions have been identified in a subset of both ALS and frontotemporal dementia cases without mutations.
Objectives: To examine the effect of wild-type FUS over expression on motor function in the mouse.
Methods: Mice over-expressing HA tagged human wild-type FUS under the control of the mouse PrP promoter were generated. General health status and motor function was assessed longitudinally using a battery of tests including the rotarod and balance beam. Immunohistochemical analysis of the brain and spinal cord was undertaken using an anti-HA antibody to assess FUS expression and inclusion formation. Antibodies to ubiquitin, TDP-43 and GFAP to assess the extent of any pathology.
Results: Homozygous over-expression of FUS resulted in progressive motor dysfunction, with overt symptom onset at approximately 4 weeks of age. Mice developed a severe tremor and limb paralysis, with an average survival time of 10-12 weeks. Compared to both non-transgenic, and heterozygous littermates, animals displayed a significant reduction in weight, along with a significantly impaired performance on the rotarod and balance beam. Because of the early and severe motor dysfunction it was not possible to reliably assess cognitive function. Immunohistochemical comparison of heterozygous and homozygous FUS over-expressing mice, demonstrated a shift in the cellular distribution of the protein from the nucleus to the cytoplasm in the spinal cord of the homozygous animal.
Discussion and conclusions: The onset of progressive, severe motor dysfunction in homozygous mice over-expressing the wild-type human FUS protein suggests that FUS pathology in ALS and FTD may act via a gain of function mechanism. In addition, the correlation between motor dysfunction and the shift in cellular localisation from the nucleus to the cytoplasm in the spinal cord of the homozygous mouse supports the hypothesis that FUS mislocalisation is linked to pathogenesis.
C89 ALS-LIKE SPINAL CORD PATHOLOGY IN TRANSGENIC MICE WITH A MUTATION IN THE VALOSIN-CONTAINING PROTEIN GENE
MOZAFFAR T, YIN H, KIMONIS V, WEISS J
University of California, Irvine, Irvine, CA, USA
Email address for correspondence: [email protected]
Keywords: transgenic, valosin containing protein, TDP-43
Kimonis et al. identified a human genetic syndrome, Inclusion Body Myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD), and subsequently found it to be associated with mutations in the valosin-containing protein (VCP) gene (Watts, Nature Genetics 2004). A knock-in VCP mouse model of IBMPFD (R155H) developed by this group exhibited muscle, bone and brain pathology characteristic of the human disease, including TDP-43 positive inclusions (Badadani, PLoS One. 2010). Recent studies have extended the list of diseases associated with VCP mutations to include ALS (Johnson, Neuron 2010). We have thus undertaken studies of spinal cord pathology in heterozygous R155H mice. Preliminary examinations of 18-24 month old R155H mice show degenerative changes in ventral horn motor neurons (MNs), and increased astrocyte activation. In addition, we find evidence for TDP-43 positive cytosolic inclusions in many damaged MNs. These studies suggest that the R155H VCP mouse may provide a valuable new animal model for ALS, which reproduces key aspects of human disease, including the presence of MN cytosolic aggregates, and pronounced astrocytic as well as MN pathology.
C90 DEVELOPMENT OF C. ELEGANS MODELS FOR AMYOTROPHIC LATERAL SCLEROSIS
VACCARO A1,2, TAUFFENBERGER A1,2, PARKER A1,2
1Université de Montréal, Montreal, QC, Canada, 2CRCHUM, CENUM, Montreal, QC, Canada
Email address for correspondence: [email protected]
Keywords: C. elegans, TDP-43, FUS/TLS
Background: Two recently discovered causative genes for ALS, TDP-43 (TAR DNA Binding Protein 43) and FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma), are under further investigation regarding their biological roles in neuropathies. Since TDP-43 and FUS are evolutionarily conserved we turned to the model organism C. elegans to learn more about their biological functions.
Objectives: The following objectives are being carried out: investigate the biological role of tdp-1/TDP-43 and fust-1/FUS in order to better understand where those genes function; generate transgenic C. elegans models to investigate the consequences of TDP-43 and FUS/TLS mutations in worms by expressing the 2 genes in the motor neurons, and obtain strains useful for screening.
Methods: The worm orthologues of TDP-43 and FUS are tdp-1 and fust-1 and we have obtained deletion mutants for each gene. These mutants are being characterized for their contribution to cellular stress resistance and longevity. We also have taken a transgenic approach to study the in vivo consequences of TDP-43 and FUS mutations. We engineered strains to express wild type and mutant human TDP-43 or FUS in worm motor neurons.
Results: We performed lifespan and stress response assays and observed that tdp-1 and fust-1 have roles in the response to oxidative and osmotic stress. tdp-1 regulated lifespan and may be linked to the Insulin/IGF pathway.
For our transgenic experiments, the expression of mutant TDP-43 or FUS in worm motor neurons produces robust, adult onset, age-dependent motility defects ultimately leading to paralysis. These phenotypes are useful for genetic and pharmacological suppressor screening. We have conducted a genetic screen and isolated a number of suppressors of mutant TDP-43 toxicity.
Discussion: Under normal conditions tdp-1/TDP-43 seems to have a role in the regulation of specific aspects of cellular stress response. Furthermore, in terms of tdp-1's biological role we have identified tdp-1 as a new downstream regulator of a major and central pathway, the Insulin/IGF signaling path. The transgenic toxic gain of function models allowed us to isolate genetic suppressors of motor neuron cell death and will be useful to find compounds that can suppress cell death.
Conclusion: Together these data provide clues to help unravel the mechanism for TDP-43 and FUS toxicity that should also provide leads for early drug discovery.
C91 ZEBRAFISH MODELS OF ALS/MND: DELINEATING THE ROLE OF MUTANT FUS AND TDP-43
WHITEMAN I1,2, GOH D1,2, WINNICK C1,2, DON E1, BANDARA U1, SOLSKI J2, HALL T3, BLAIR I2, NICHOLSON G2, COLE N1
1Discipline of Anatomy, University of Sydney, Sydney, NSW, Australia, 2ANZAC Research Institute, University of Sydney, Sydney, NSW, Australia, 3Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC, Australia
Email address for correspondence: [email protected]
Keywords: zebrafish, FUS, TDP-43
Background: Motor neuronal cytoplasmic aggregations containing Fused in Sarcoma (FUS) or TDP-43 proteins are a pathological hallmark of ALS. FUS and TDP-43 are highly related, predominantly nuclear proteins that share a similar structure and common role of RNA-binding. Interestingly, mutations in each of the genes encoding these proteins, TARDBP and FUS respectively, have been identified in ALS patients, providing compelling evidence of a putative role for these aberrant proteins in the pathogenesis of ALS.
Objective and method: We have created in vivo models of ALS in zebrafish, an animal model that is becoming widely regarded for its advantages in both developmental biology and human disease research. By generating transgenic zebrafish lines expressing specific human FUS or TARDBP mutations identified in ALS families, we are able to investigate in vivo the role of mutant FUS and TDP-43 in the pathogenesis of ALS at high temporal and spatial resolution.
Results: Specifically, we have examined the development and morphology of primary motor neurons and neuromuscular junctions, patterns of redistribution and aggregation of FUS and TDP-43, formation of associated stress granules and neuronal degeneration profiles. Functionally, we have also characterised the effects of these mutations on motor activity and general survival.
Discussion and conclusion: Indeed, our transgenic zebrafish models recapitulate several key aspects of human ALS and as such, provide crucial insight into the pathogenic mechanisms involved in this largely enigmatic disease. Moreover, these models are a valuable tool that will ultimately be used for high throughput drug screening and development of more effective therapeutics.