The Role of ALS2/Alsin in ALS and Motor Neuron Diseases

National Institute On Aging

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Abstract

Background and Specific Aims: Recently, a gene termed ALS2 was linked to a recessive form of juvenile ALS in several families. ALS2 encodes a protein termed alsin, which shares homology to GTPase regulatory proteins (guanine-nucleotide exchange factors) that participate in critical cellular functions including signal transduction, regulation of the cytoskeleton and intracellular trafficking. Mutations in ALS2 lead to a premature truncation of encoded protein, suggesting that the disease is associated with a loss of ALS2 function. Thus, we opted to use a genetic approach to ablate the gene encoding ALS2 to model this rare form of ALS. Our specific aims for project 1 are: [unreadable] Aim 1: To generate and characterize ALS2 knockout mice; [unreadable] Aim 2: To define the biochemical pathways and intracellular processes in which ALS2/alsin participates[unreadable] [unreadable] Progress Report:[unreadable] For Aim 1: we have generated ALS2 knockout (ALS2-/-) mice by disrupting the second coding exon of the ALS2 allele. While ALS2-/- mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2-/-mice showed a higher anxiety response in the open field and elevated plus maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypes consistent with motor neuron disease up to 20 months of age, ALS2-/- mice and primary cultured neurons derived from these mice were more susceptible to oxidative stress compared to wild type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model, but predisposes neurons to oxidative stress. Some of this work has been published in the Journal of Neuroscience (Cai et al., 2005).[unreadable] For Aim2: to identify the intracellular molecular pathways in which ALS2/alsin is involved, we screened proteins interacting with ALS2/alsin. We found that ALS2 is associated with the glutamate receptor interacting protein (GRIP1) as evidenced by co-immunoprecipitation of ALS2 and GRIP1 in extracts from heterologous expression systems and from mouse brain. ALS2 also co-localizes with GRIP1 in primary cultured neurons. GRIP1 is involved in the regulation of transportation and stabilization of AMPA type glutamate receptor type 2 in the synapses (Song and Huganir, 2002). The enrichment of ALS2 in the synaptic membrane fraction of the brain lysate in conjunction with altered distribution of GRIP1 in ALS2-/- spinal motor neurons implies that ALS2 is involved in the regulation of AMPA receptor trafficking. In support of this hypothesis, we found that loss of ALS2 leads to increased reverse rectification of AMPA receptor-mediated synaptic response in ALS2-/- neurons as compared to wild type controls (Fig. 3), indicating a lack of calcium-impermeable AMPA receptors in the synapses. Furthermore, we found that cell surface presentation of calcium-impermeable AMPA receptors was significantly decreased in ALS2-/- neurons as compared to wild type neurons following AMPA treatment. These observations are correlated with our finding of increased susceptibility of ALS2-/- spinal motor neurons to AMPA or kainic acid-induced neurotoxicity in organotypic spinal cord slice culture and in animals. Therefore, we propose that loss of ALS2 increases the vulnerability of motor neurons to glutamate receptor-mediated excitotoxicity due to a lack of calcium-impermeable AMPA receptors at the cell/synaptic surface: a potential pathogenic mechanism of ALS resulting from mutant ALS2. This work has been published in the Journal of Neuroscience (Lai et al., 2006).[unreadable] [unreadable] Future Directions:[unreadable] ALS2 KO mice have been generated by 7 different laboratories around the globe. None of them display any obvious spinal motor neuron degeneration. However, a recent work indicated the existence of an alternative translational initiation site at the 4th coding exon of ALS2, which remains intact in many of these ALS2 KO mouse lines. To address whether the alternative translational variants of alsin compensate for the loss of full-length protein, we will generate a new line of ALS2 KO mice that disrupts the transcription of exon 4 and other down-stream exons by the gene-trapping technique. We will closely examine the motor behavioral of neuropathological phenotypes of these newly developed ALS2 KO mice and to further investigate whether the loss of alsin causes motor neuron diseases.