The Section of Transgenesis (TS) in Laboratory of Neurogenetics (LNG) was devoted 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). Project 1. 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 Journal of Neuroscience, we are gathering the complementary electrophysilogy data supporting the notion of cognitive deficits in BACE1 knockout mice for a second review. Project 2. A Conditional 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 obtained several lines of chimeric mice with help from Dr. Bob Nussbaum?s group. Project 3. Loss Function of Alsin is Not Sufficient to Trigger Major Motor Neuron Degeneration but Predispose Neurons to Oxidative Stress ALS, the most common motor neuron disease, is caused by a selective loss of motor neurons in the central nervous system. Mutations in the ALS2 gene have been linked to one form of autosomal recessive juvenile onset ALS (ALS2). To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knockout (ALS2-/-) mice. 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 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. 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. These data were published in Journal of Neuroscience this August. Project 4. A New Mouse Model of Motor Neuron Disease: Testing Intracellular Retrograde-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 missense 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. Project 5. Mice Deficient in DJ-1 Exhibited Decreased Locomotor Activities But Maintained Normal Dopaminergic Transmission in the Midbrain PD is a progressive neuropathy characterized by the loss of dopaminergic neurons in the substantia nigra that underlies clinical symptoms of muscle rigidity, resting tremor and bradykinesia. While the etiology of sporadic PD remains unknown, several genes linked to rare familial forms of the disease have been identified. The same pathway is hypothesized (based on clinical symptoms and disease pathology) to underlie both genetic and environmental forms of PD. DJ-1 is a gene recently identified (Bonifati et al, 2003) to have loss-of-function mutations that can cause familial PD. To better understand DJ-1 and its role in PD, we developed a DJ-1 gene knockout mouse. Mice at 2, 5, and 9 months of age exhibited significant (male; n=10; p<0.005) deficits in locomotor activity over a 30 minute interval measured using an automated open field (flexfield) monitor (San Diego Instruments) that is equipped with photocells. Previously, mice deficient in dopamine metabolism and synaptic transmission all had deficits in locomotor activity compared to wild-type controls. We analyzed dopamine production and transmission in DJ-1 knockout mice. However, our data showed no significant difference on basal dopamine dynamics in dorsal striatum; and, no significant alteration in KCl-evoked dopamine release or dopamine receptor 2-mediated inhibition of dopamine release in DJ-1 knockout animals. The physiological roles of DJ-1 still need to be addressed. Project 6. The Role of PINK1, A Mitochondria-Bond Protein Kinase in PD In line 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 obtained several lines of PINK1 chimeric mice for generating PINK1 heterozygous knockout animals.
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