C37 THE SOD1 MOUSE AS A MODEL OF SPORADIC ALS
Figlewicz DA1, Blankenhorn EP2, Heiman‐Patterson TD2
1University of Michigan, Michigan, USA, 2Drexel University College of Medicine, Philadelphia, USA
E‐mail address for correspondence: [email protected]
Amyotrophic lateral sclerosis (ALS) is a progressive disorder of the motor system leading to death within two to five years. Although most cases of ALS are sporadic in origin, 10–15% are inherited. Among the population with familial ALS, 15–20% possess a mutation in the SOD1 gene that codes for the enzyme Cu/Zn superoxide dismutase. The phenotypic variability observed between affected FALS individuals from the same family, in particular in onset and severity, lends strong support to the likelihood that there are important additional genetic modifiers of the disease.
Transgenic (Tg+) mice expressing a mutated human SOD1 gene (G93A) demonstrate clinical symptoms and neuropathological findings similar to human ALS. We have observed that the genetic background of these hSOD1(G93A) Tg+ mice can affect disease phenotype (onset and survival). Similar observations in related mouse models of motor neuron degeneration have been reported by others. Hence, we proposed that these differences could be used to identify genes that modify severity in this species and that potentially contribute to susceptibility or neuroprotection in human motor neuron degenerative disease.
Our collaborative work is based on these hypotheses: 1) There are variants of genes between mouse strains that alter phenotype; 2) These genes are likely to alter phenotype by influences on one of several pathways important to the mechanism of neuronal death in the G93A SOD Tg+ mouse including changes related to intracellular processing of mutated SOD1, altered inflammatory responses, altered oxidative stress, altered glutamate clearance, or altered cell death.
Thus far, we have bred the Tg on five genetic backgrounds, C57Bl/6J (abbreviated B6), SJL, C3H, B10, and DBA. Compared to the mixed background (B6 x SJL)Tg+, mean survival on the SJL and C3H backgrounds are significantly shorter while the survival on the B6 background is longer. In addition, gender‐based influences on phenotype include the fact that Tg+ females live longer in some congenic lines (SJL, C3H) but not others (B6); thus the congenic lines may also provide insight into gender‐based differences in human disease. The B6 and SJL congenic lines were used to create an F2 intercross, and a genome scan revealed significant linkage with lifespan to chromosome 2, 5, 10, and 15, and to disease latency on chromosome 2.
Because the human FALS and SALS clinical phenotypes are indistinguishable, the two phenotypes are likely to share important disease mechanisms; thus, identification of the gene(s) and related pathways based on these studies with the G93A Tg+ mice should lead to an increased understanding of the disease mechanisms and design of new treatment strategies pertinent to all ALS/MND patients.
Acknowledgement: DAF is supported by MDA‐USA and NIEHS. EPB and THP are supported by ALS Hope Foundation and MDA‐USA.
C38 DESIGN, POWER AND INTERPRETATION OF STUDIES IN THE STANDARD MURINE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Scott S, Kelly N, Kranz J, Lincecum J, Ramasubbu J, Vieira F, Heywood J
ALS Therapy Development Foundation, Cambridge, MA, USA
E‐mail address for correspondence: [email protected]
Identification of SOD1 as the mutated protein in a significant subset of familial amyotrophic lateral sclerosis (FALS) cases has led to the generation of transgenic rodent models of autosomal dominant SOD1 FALS. Mice carrying 23 copies of the human SOD1G93A SOD1G93A transgene are considered the standard model for FALS and ALS therapeutic studies. To date, there have been at least 50 publications describing therapeutic agents that extend the lifespan of this mouse. However, no therapeutic agent has shown corresponding, substantial, clinical efficacy. We used computer modelling and statistical analysis of 5429 SOD1G93A mice from our efficacy studies to identify several critical confounding biological variables that must be appreciated and should be controlled for when designing and interpreting efficacy studies. Having identified these biological variables we subsequently instituted parameters for optimal study design in the SOD1G93A mouse model. We repeated several of the major animal studies (minocycline, creatine, ritonavir, celecoxib, sodium phenyl butyrate, ceftriaxone, WHI‐P131, thalidomide, and riluzole) using an optimal study design and found no survival benefit in the SOD1G93A mouse for any compounds administered by their previously reported routes and doses. The presence of these uncontrolled confounding variables in the screening system, and the failure of these drugs to demonstrate efficacy in adequately designed and powered repeat studies, leads us to conclude that the majority of published effects are most likely measurements of noise in the distribution of survival means as opposed to actual drug effect.
C39 AN ARRAY‐BASED APPROACH TO IDENTIFY BIOMARKERS FOR ALS
De Zutter J, Lincecum J, Vieira F, Wang M, Scott S
ALS Therapy Development Foundation, Cambridge, MA, USA
E‐mail address for correspondence: [email protected]
Background: The SOD1 G93A transgenic mouse model is the standard tool for the investigation of new therapeutic candidates for ALS. These transgenic mice are asymptomatic at 60 days of age, and display progressively worsening symptoms from approximately 90 days until death, which occurs on average at 136±10 days. Our studies suggest that a minimum of 24 animals are required per group because of variability in the survival of the animal. As such, it would be highly desirable to have a surrogate endpoint that could facilitate drug screening using fewer animals per group.
Objective: The primary goal of this study is to establish a panel of biomarkers for disease progression in the SOD1 G93A mouse. In achieving this goal we will further our understanding of the molecular mechanisms in ALS and also generate a diagnostic tool for evaluating therapeutic candidates. In addition, if a disease progression‐modifying compound also induces altered expression levels of a particular gene, then a target validation approach will be initiated to determine if that gene and/or gene product is a therapeutic target.
Method: We analysed spinal cord tissue from SOD1 G93A mice at various time points during the mouse lifespan. We used a real time‐quantitative PCR based, targeted array approach to systematically examine expression levels of functionally categorized genes. More focused RT‐qPCR assays were performed to confirm expression changes observed by the array approach.
Results: Over the time course examined, we detected increasing RNA levels of: the glial marker Gfap (4.1‐fold), proteasome subunits Psmb8 and Psmb9 (5.8‐ and 3.8‐fold, respectively), apoptosis associated cyclinD1 (3.7‐fold), and inflammatory mediators Tnf alpha (9.5‐fold), Trem2 (11.6‐fold) and Tyrobp (12.6‐fold), among others. Decreasing RNA levels were measured for, among others: the neuronal markers NSE/Eno2 and NF‐M/Nef3 (2.0‐ and 3.1‐fold, respectively), the molecular motor subunits Kif3 alpha and Dnchc1 (1.7‐fold and 2.3‐fold, respectively). The genes, whose RNA reproducibly changed over the SOD1 G93A mouse lifespan, were categorized as progressively changing (over the course of disease) or late stage (changed at or near end stage of disease). To assemble the biomarker panel we included or excluded genes based on their temporal expression. To follow up results that suggest a gene's product is a target for therapeutic intervention, immunoblotting, ELISA and in situ hybridization experiments were performed to examine protein levels of genes of interest.
Conclusion: The end product of this effort is a panel of genes which may be used as a diagnostic tool to monitor disease progression in the SOD1 G93A mouse and to examine the effects of therapeutic candidates on that progression. By‐products of this work are potential targets for therapeutic intervention in ALS.
C40 GUIDELINES FOR THE PRECLINICAL EVALUATION OF PHARMACOLOGICALLY ACTIVE DRUGS FOR ALS/MND: REPORT ON THE 142nd ENMC INTERNATIONAL WORKSHOP
Ludolph AC1, Bendotti C2
1University of Ulm, Ulm, Germany, 2Mario Negri Institute for Pharmacological Research, Milan, Italy
E‐mail address for correspondence: [email protected]
A transgenic animal model for anterior horn cell loss was established in 1994. This model is based on the insertion of a high copy number of disease‐causing human Cu/Zn SOD mutations into the intact mouse genome. It serves to establish hypotheses for the pathogenesis of anterior horn cell death, but also to test potential pharmacological approaches to therapy in human ALS. To date, more than 100 (published and unpublished) compounds have been tested in this animal model, with a large proportion of them being successful. However, it has proved to be difficult to translate these therapeutic successes in the animal model into human trials. In addition, a number of disease‐modifying strategies have been difficult to reproduce, even by the same group. On the other hand, the step from mice to men means a huge investment for the sponsors of clinical trials and the scientific community. Therefore, establishment of standard methods for drug testing in ALS models is mandatory. In this workshop, clinical and preclinical researchers established in the field of ALS/MND met in Holland in March 2006 in order to establish guidelines for the community for drug testing in mouse models.