The Establishment And Phenotypic Analysis Of WT HTDP-43Transgenic Mice And Wt HTDP-43/MUT HSYN Bi-transgenic Mice

Background:Frontotemporal lobar degeneration (FTLD) is a relatively common form of dementia manifesting with behavioral and language dysfunction. Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder characterized by degeneration of motor neurons in the brain and spinal cord. Both FTLD and ALS patients are characterized by ubiquitinated neuronal cytoplasmic and intranuclear inclusions (NCIs and NIIs) mainly composed of TAR DNA binding protein (TDP-43) in affected regions. TDP-43is a ubiquitously expressed nuclear protein of43kd encoded by TARDBP gene (TARDBP) on chromosome1. Mutations in the TDP-43gene have been identified in a subset of both familial and sporadic forms ALS of and ALS with FTLD cases. However, the majority of ALS and FTLD-TDP cases with TDP-43pathology are not associated with TARDBP mutations and that TDP-43pathology is indistinguishable between sporadic and familial ALS cases, suggesting a possible mechanism by which the wild-type TDP-43(WT TDP-43) could lead to neurotoxicity in these neurodegenerative diseases. Its physiological functions remain to be elucidated.TDP-43proteinopathy, a hallmark of sporadic ALS and FTLD, is also observed in the other neurodegenerative diseases such as Parkinson's disease (PD), which is a devastating neurological condition typified by loss of dopaminergic (DA) neurons in midbrain, and related diseases. Mutation of the human alpha-synuclein (hSYN) gene is associated with PD. Over-expression of pathogenically mutated a-synuclein can lead to dopaminergic neuron death in transgenic mice at advanced ages. Given that TDP-43inclusion was found in the pathology of PD, and neurodegenerative diseases are commonly thought of multifactorial pathogenesis, we propose that TDP-43may play a role in the pathogenesis of DA neuron loss.Perspectives:1. To determine if WT hTDP-43can directly cause neurodegeneration in a genetically tractable model;2. To test the hypothesis that TDP-43potentiates SYN toxicity to DA neurons in transgenic mice.Methods:Part1We generated WT hTDP-43transgenic mouse lines, and identified the WT hTDP-43transgenic mice by PCR amplification of the hTDP-43gene as well as immunoblotting. Routine observation and Rotarod test were performed to analyze disease phenotypes. After the age of400days, all transgenic mice and nontransgenic mice (NT) were sacrificed and analyzed for neuropathological abnormalities in the brain, spinal cord and skeletal muscle by performing immunohistochemistry and histochemistry staining, double immunofluorescence staining, Toluidine blue staining, cresyl violet staining as well as stereological neuron counting in the prefrontal cortex and dentate gyrus with fractionator-based stereology software (Stereologer). Part2Dual-mutant hSYN transgenic mice were received from Dr. Richfield. We generated the WT hTDP-43/MUT hSYN bi-transgenic mice model by crossing WT hTDP-43mice with dual-mutant hSYN mice. These mice were genotyped by PCR analysis. Routine observation and behavior test were performed and analyze the phenotypes. At the age of more than200days, WT hTDP-43/MUT hSYN bi-transgenic mice and MUT hSYN transgenic mice were sacrificed and analyzed for neuropathological abnormalities in the midbrain by performing immunohistochemistry staining, immunofluorescence staining as well as stereological estimates of DA neurons in the mid-brain substantia nigra with stereology software.Results:Part1(1) PCR and western bolt showed TDP-43expression in the WT hTDP-43mice, but not in the NT.(2) A robust expression of human TDP-43that primarily localized to the nucleus was detected in the brain, spinal cord, and skeletal muscles of WT hTDP-43transgenic mice but not in the tissues of nontransgenic controls. Cytoplasmic distribution of hTDP-43was occasionally detected within the anterior horn of the spinal cord, but no TDP-43-positive inclusion was found.(3) No detectable phenotype was observed in WT hTDP-43transgenic mice.(4) WT hTDP-43mice displayed no neuropathology of ALS:neither detectable pathological impairment in the motor system, nor ubiquitin aggregation or activation of glial cells in these mice even at advanced ages.(5) Overexpression of the normal TDP-43causes cortical neuron degeneration in the prefrontal cortex but not in the dentate gyrus in these transgenic mice. Part2(1) Expression of both hTDP-43and hSYN in the DA neurons of WT hTDP-43/MUT hSYN bi-transgenic mice, but not in the NT.(2) Concomitant over-expression of WT hTDP-43and MUT hSYN caused a more severe loss of DA neurons in the bi-transgenic mice as compared to MUT hSYN mice.Conclusions:(1)Our study demonstrates that overexpression of WT TDP-43led to moderate loss of prefrontal cortical neurons characteristically affected in FTLD-TDP, and the neurodegeneration occurred in the absence of ubiquitin aggregation or TDP-43inclusion in cytoplasm. In vivo TDP-43neurotoxicity can result from overexpressed full-length TDP-43in nucleus;(2)Overexpression of TDP-43potentiates SYN toxicity to dopaminergic neurons in living animals. Our finding provides in vivo evidence suggesting that disease proteins such as TDP-43and SYN may play a synergistic role in disease induction in neurodegenerative diseases. Therefore this model will provide a useful tool to explore the common pathogenic mechanisms in these neurodegenerative diseases.