The Section of Transgenesis (TS) in Laboratory of Neurogenetics (LNG) was created on May 2003. We devote our efforts to study the pathogenic mechanisms and experimental therapeutics of neurodegenerative diseases by a combination of in vivo mouse modeling and in vitro neurobiology approaches. Our research covers three major neurological diseases: Alzheimer?s disease (AD), Parkinson?s disease (PD), and Amyotrophic lateral sclerosis (ALS). Part 1. Alzheimer?s Disease (AD) A. BACE1 is Critical for Cognition A transmembrane aspartyl protease termed beta-site APP cleavage enzyme1 (BACE1) that cleaves the amyloid precursor protein (APP) is required for the generation of amyloid-beta (A-beta) peptides implicated in the pathogenesis of AD. Significantly, deletion of BACE1 in a mouse model of AD, the APPswe;PS1delE9 double transgenic mice prevents both A-beta deposition and age-associated cognitive abnormalities that occur in this model of A-beta amyloidosis. Moreover, the A-beta burden is sensitive to BACE1 dosage in young but not in aged APPswe;PS1delE9 mice, suggesting that A-beta clearance mechanisms in aged animals may be compromised. Although BACE1 null mice do not exhibit overt developmental abnormalities or adult-onset neuropathology, however, these animals do manifest alterations in performance on tests of cognition and emotion. Importantly, the memory deficits occurring in BACE1 knockout mice are prevented in APPswe;PS1delE9;BACE1knockout mice. Our results establish that BACE1-dependent APP processing is critical for cognitive and emotional behaviors, suggesting that future studies should be alert to potential mechanism-based toxicities associated with BACE1 inhibitors designed to ameliorate A-beta amyloidosis in AD. In response to the initial review of Neuron, we are gathering the complementary electrophysilogy data supporting the notion of cognitive deficits in BACE1 knockout mice for a second review. B. A Regulated APP Transgenic Mouse Model: How the A-Beta Aggregates Cause the Dysfunction and Death of Neurons How the A-beta aggregates cause the dysfunction and death of neurons is not clear. We plan to address this issue by expressing mutant APP transgene in selected neurons at a chosen time, so that we are able to determine where the A-beta peptides are initially deposited: synapses or non-synapses; whether intracellular Ab acts in a cell autonomous or in a heterologous fashion to cause neuronal damage. To generate this conditional APP transgenic mouse, we have built the transgenic construct in which a loxP-flanked reporter gene (lacZ) is inserted before the APP and EGFP transgenes. Once we have generated this regulated APP transgenic mice, we will cross them with the CRE-estrogen receptor or other cell-type specific CRE transgenic mice, which will initiate the expression of APP and EGFP by removing their fronting lacZ gene. Through these genetic maneuvers, we are able to control where or when the neurons express APP and EGFP, which will provide us a valuable tool to dissect the mutant APP-initiated neuropathogenic cascades. We have constructed the transgenic vectors and are ready to introduce them into mouse genome with help from Dr. Bob Nussbaum?s group. Part 2. Amyotrophic Lateral Sclerosis (ALS) and Other Motor Neuron Diseases A. Loss Function of Alsin Predispose Neurons to Glutamate-Mediated Excitotoxicity through Up-regulation of GRIP1 A subtype of inherited juvenile onset amyotrophic lateral sclerosis (ALS) is caused by the loss function of ALS2. ALS2 knockout mice have been generated to reveal the pathogenic mechanisms of ALS2 and the physiological functions of its encoded protein, alsin. Unlike ALS2 affected patients who showed movement disability at their early ages, ALS2 null mice developed a slowly progressive muscle weakness and motor deficits only after their midlife. Protein co-immunoprecipitation experiments revealed that alsin specificalllly interact with the N-terminal three PDZ domains of GRIP1, an adaptor protein involved in the trafficking and targeting of GluR2/3 receptors in neurons, though its N-terminal RCC1-like domain. Surprisingly, loss of alsin, particularly its C-terminal DH/PH and VPS9 domains, dramatically increased the expression level of GRIP1 protein. This increase of GRIP1 protein resulted in up-regulation of AMPA receptors and their cell surface expression, which rendered the ALS2 knockout neurons more susceptible to AMPA-mediated excitotoxicity. We are preparing for publication and will present these findings in the coming annual Neuroscience Meeting on October. B. A New Mouse Model of Motor Neuron Disease: Testing Intracellular Retro-transport in the Pathogenesis and Therapeutics of Neurodegenerative Diseases Deficiency in axonal transport has been reported in many neurodegenerative diseases, including AD, Huntington?s disease, and ALS. Recently, a single-base-pair misense mutation has been identified in the dynactin (DCTN1) gene that causes a slowly progressive, autosomal dominant form of lower motor neuron disease. This misense mutation in dynactin results in one amino acid substitution of serine for glycine in the mutant protein, which might impair its microtubule binding ability and cause the disease by reduction of axonal transport, or by destabilization of the nerve-muscle junctions. To distinguish these two major hypotheses, we have developed a mouse model that harbors the Dynactin mutation by gene targeting techniques. We have obtained heterozygous dynactin mutant mice; meanwhile, we will examine axonal trafficking in primary cultured neurons derived from these mutant mice. 3.Parkinson?s disease (PD) A. A PD-Related Gene DJ-1: Involvement in Oxidative Stress and Mitochondrial Function? Recently, genetic analyses have suggested that loss of DJ-1 functions cause a rare form of early onset PD. In vitro biomedical and cell biological studies have implied that DJ-1 serves as a multi-functional protein, including molecular chaperon and oxidative sensor. To investigate DJ-1?s physiological functions in vivo, we have generated a DJ-1 knockout mouse model by deleting its second coding exon. The DJ-1 knockout mice are viable and fertile, but yet to display any obvious motor deficits. Meanwhile, we have conducted research on whether DJ-1 knockout mice are more susceptible to MTPT-induced neuronal loss by injecting MPTP into age-matched DJ-1 wild type and knockout animals. We haven?t detected any significant decrease of motor activities in the knockout mice after the injection. We are now working on quantification of neuronal loss in these mice to examine whether absence of DJ-1 increase the vulnerability of dopaminergic neurons to MPTP-mediated cellular stress. We are going to present some of these data in the coming Neuroscience Society Meeting on October. B. The Role of Pink1, A Mitochondria-Bond Protein Kinase in PD In consistence with the notion that malfunction of mitochondria contributes to the pathogenesis of PD, two homozygous mutations affecting the mitochondria-located PINK1 (PTEN-induced kinase 1) at its predicted kinase domain have been identified from PD patients in three consanguineous families. Cultured cell lines overexpressing mutant Pink1 were more susceptible to protease inhibitor induced cellular stress. However, the function of PINK1 and its related pathogenic pathway are not clear. So, we decided to generate PINK1 knock mice to model for this type of PD. We have constructed the PINK1 targeting vector, which deleted the kinase domain of PINK1 and are ready to introduce them into mouse genome with help from Dr. Bob Nussbaum?s group. We also have conducted yeast-two hybrid screen to identify proteins interacting with PINK1. We have found 7 candidate proteins after initial screening and ready for verification.
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