Chenjian Li - US grants

The long term goal is to understand estrogen's role in learning and memory, in hippocampal neuron function, in synaptogenesis, and how the malfunction of these is relevant to human diseases. Clinical study reveals that estrogen treatment affects women~s spatial tasks, verbal memory, and fine motor skills, and improves the mental status of female Alzheimer~s patients. Parallel study in rat also demonstrated estrogens impact on cognitive functions. Furthermore, at the cellular level, it has been shown that estrogen induces dendritic spines and new synapses in the hippocampal neurons. Hence, it is imperative to understand the molecular mechanisms of the estrogen induced synaptogenesis in hippocampal neurons, because: a) synaptogenesis is one of the most fundamental neuronal activities and a critical cellular basis for learning and memory; and b) hippocampus is an essential brain region that accounts for many learning and memory tasks. The specific aim of this proposed study is to identify molecular components and mechanisms of this process. A hippocampal cell culture system has been developed, in which synaptogenesis can be induced by estrogen treatment. Utilizing this system, a)molecules known to be important in synaptogenesis, such as cAMP responsive element binding protein (CREB), protein phosphotase I (PPI) and NMDA receptor will be examined for their changes in transcription, translation and phosphorylation, with or without estrogen induction; b) a hypothesis of the pathway will be tested; c)mRNA differential display technique will be used to identify new molecules that are induced or suppressed in estrogen induced synaptogenesis.

[unreadable] DESCRIPTION (provided by applicant): The newly identified LRRK2, a disease gene of familial dominant late-onset ParkS, encodes a kinase. No mouse models are available for ParkS; no LRRK2 kinase substrates are known; and little is known about LRRK2 normal functions and pathogenic roles in Parkinson's disease. In this early exploratory phase of research which is most suitable for R21 mechanism, we have established LRRK2 mouse models and now propose to characterize them, and utilize these new mouse models to identify LRRK2 substrates. Mutations in LRRK2 have been identified as the most prevalent ones in familial Parkinson's disease, as well as in sporadic PD unexpectedly. All ParkS mutations are missense mutations that cause a dominant phenotype, indicating a "gain of function" or "hyperactivity" mechanism of mutant proteins for pathogenesis. Therefore, transgenic (not knockout) mouse models are appropriate for modeling ParkS. Our CENTRAL HYPOTHESIS is that ParkS pathology is caused by hyperactivity of the mutant LRRK2 to hyper-phosphorylate its substrates. To test this hypothesis, we have generated transgenic LRRK2 mice that carry wild type sequence or ParkS mutations in HUMAN bacterial artificial chromosome (BAG). SPECIFIC AIM 1: to analyze the phenotypes of the LRRK2 transgenic mice for progressive behavioral deficits in motor function and in niagrostriatal pathways, neuronal pathology in substantia nigra, and dopamine production and release. SPECIFIC AIM 2: to use our new mouse models to identify substrates of LRRK2 kinase with both candidate and non-biased approaches. SIGNIFICANCE: Mouse models for ParkS will be critical tools for mechanistic studies and future therapeutic drug testing. The identification of LRRK2 substrates is important because kinase pathways and components are highly "drug-able" targets. The study of LRRK2 is also very likely to impact sporadic PD research, because ParkS mutation G2019S was discovered to be also highly prevalent in sporadic PD. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Mitochondria dysfunction and oxidative stress have been suggested to be associated with many neurodegenerative diseases, including Parkinson's Disease (PD). The most direct CAUSALITY evidence of mitochondria dysfunction to PD pathogenesis arises from the newly identified PINK1, a disease gene of familial, recessive early-onset PD. PINK1 protein is a nuclear genome encoded protein with a mitochondria targeting signal peptide and a conserved kinase domain, however, its normal functions and roles in pathogenesis remain to be elucidated. The mutations in PINK1-PD patients are causing the PINK1 proteins to be truncated or most likely functionally inactive. HYPOTHESIS: Our overall hypothesis is that PINK1-PD pathology is caused by disruption of mitochondria functions because the mutant PINK1 protein fails to phosphorylate its normal substrates. To test this hypothesis, we have generated genetic PINK1-/- knockout mice and other valuable reagents, and we propose the following specific aims. SPECIFIC AIM 1: we will analyze whether PINK1-/- mice develop progressive motor deficits and neuronal pathology in substantia nigra and other brain regions. SPECIFIC AIM 2: Subsequently, but not dependent on the Aim 1 outcome, we will investigate mitochondria dysfunction in PINK1 -/- mice and in PINK1-/- dopaminergic cell lines. SPECIFIC AIM 3: Subsequently, but not dependent on the Aim1 outcome, we will identify and confirm the substrates of PINK1 kinase. SIGNIFICANCE: Collectively, the generation and characterization of the PINK1 knockout mice as well as the dopaminergic PINK1-/- cell lines, the investigation of the mitochondria dysfunction caused by PINK1 mutations, and the identification of PINK1 kinase substrates are critical for understanding of the molecular mechanisms of PINK1-PD, for identifying potential therapeutic targets, and for drug screening and testing. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the second most common neurodegenerative disease. Mutations in Leucine-rich-repeat-kinase 2 (LRRK2), the newly identified causative gene for PARK8 type PD with autosomal dominant inheritance, are the most prevalent genetic causes in both familial and sporadic PD. Therefore it is important to understand the mechanisms of LRRK2 mediated pathogenesis. We have generated the first transgenic mouse model for LRRK2 that recapitulated robust motor behavioral, neurochemical and pathological features of PD. These mice develop an age-dependent progressive decrease in motor activity that is responsive to treatment with levodopa. At the level of pathology, the early and most robust phenotype is the axonopathy of the nigrostriatal dopaminergic projection, accompanied by hyperphosphorylated tau. Therefore, among all the aspects of LRRK2 induced pathogenesis, the tau-mediated axonal degeneration is particularly important and worth a major effort of investigation. We will address important questions at molecular, cellular and animal model levels. Hypothesis 1: At the molecular level, LRRK2 induces tau hyperphosphorylation that mediates the pathogenic effects. Specific Aim 1: To confirm that tau is an integral component of mutant LRRK2 kinase signaling. Hypothesis 2: At the cellular level, tau mediated axonal degeneration is a critical aspect of LRRK2 PD. Specific Aim 2: To investigate whether LRRK2-induced tau hyperphosphorylation causes axonal pathology in primary neuronal cell cultures. Hypothesis 3. At the organismal level, tau-mediated axonopathy leads to dopaminergic neuronal degeneration and progressive motor deficits. Specifc Aim 3: To test the interaction of tau and LRRK2 in genetic mouse models. This set of studies will identify the molecules and the pathways in LRRK2 pathogenesis. This is not only important from a scientific point, but also critical for the therapeutic development that directly addresses a major public health issue. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is the second most common neurodegenerative disease. Mutations in Leucine-rich-repeat-kinase 2 (LRRK2) are the most important genetic causes in PD. Therefore the understanding of the LRRK2 pathogenic mechanisms is not only important from a scientific point, but also critical for the therapeutic development that directly addresses a major public health issue.