Sangram Sign Sisodia, PhD - US grants

DESCRIPTION (provided by applicant): Autosomal dominant inheritance of mutations in PS1 and PS2 cause early-onset familial forms of AD. A series of neuropathological studies of humans and transgenic mice expressing FAD-linked PS1 have revealed that these mutant polypeptides likely cause AD by elevating production of amyloidogenic AB42 (43) species, thus fostering AB deposition. Despite the strengths of these conclusions, it is not clear that FAD-linked mutant PS1 exerts pathophysiological effects solely by promoting deposition of AB peptides. We propose investigations that address the function of PS1 and FAD-linked PS1 variants in neuronal physiology that are independent of AB42 production and deposition. In Specific Aim 1, we propose a series of electrophysiological, pharmacological and imaging studies in cultured neurons from wt and PS1-deficient mice to identify the mechanism(s) that underlie our observations that PS1 influences glutamatergic neurotransmission at both pre-and post-synaptic sites. In Specific Aim 2, we propose electrophysiological studies in hippocampal slices from transgenic mice expressing wt or FAD-linked mutant PS1 to assess the mechanism(s) underlying our demonstration that expression of mutant PS1 potentiates long-term potentiation (LTP) at Schaffer collateral CA1 synapses. In addition, we will examine LTP in hippocampal slices from mice with selective forebrain ablation of PS1, using a conditional gene knockout strategy. In Specific Aim 3, we propose to test the hypothesis that expression of mutant PS1 alters the threshold for vulnerability to lesion-induced excitotoxicity, or other insults. For these studies, we will exploit a perforant path lesion paradigm to examine neuronal vulnerability in the entorhinal cortex of mice expressing wt or FAD-linked mutant PS1. In addition, we will extend our intriguing observation that dentate neurogenesis is markedly elevated following perforant path lesions and examine the influence of PS1 and mutant PS1 in these processes.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is manifested by progressive memory loss and cognitive deterioration. The pathological hallmarks of the disease include neuronal loss and the deposition of p-amyloid peptides (AP) in specific brain areas, including the cortex and hippocampus. Compelling evidence has accrued to suggest that the cortex and the hippocampus exhibit environment-induced plasticity throughout adult life. Exposure of rodents to an "enriched environment" has been shown to have numerous beneficial effects, including: enhancement of learning and memory;protection from age-associated cognitive decline;increased neurogenesis;and upregulation of neurotrophic factors and immediate-early genes (IEG) associated with learning and memory. We have tested the hypothesis that environmental conditions might play a role in modulating hippocampal plasticity in a manner that impact on amyloid deposition. We reported that amyloid deposition and steady-state levels of AP peptides are markedly reduced in brains of transgenic mice exposed to enriched environment conditions, compared to mice that were maintained in standard housing conditions (Lazarov et al., 2005). Moreover, we observed an inverse correlation between the level of physical activity and the extent of amyloid deposition. We established that the activity of neprilysin, a zinc metallopeptidase known to be involved in AP degradation in vivo, is elevated in the brains of mice exposed to the enriched environment. Finally, high density oligonucleotide arrays revealed upregulation of genes associated with vasculogenesis, neurogenesis, AP sequestration and learning and memory processes. Encouraged by these provocative findings, we now propose a series of experiments to further explore the effect of environmental conditions on AD-related processes in the brains of transgenic mice. In Specific Aim 1 we will isolate the stimulus involved in enriched- mediated modulation of AP peptide accumulation and deposition and assess the effects on the transcriptome and APR processing in vivo. In Specific Aim 2 we will ask whether reduced expression of selected genes identified in our microarray analyses will affect histological and biochemical endpoints in the brains of mice following enrichment. In Specific Aim 3 we will examine the role of enrichment on the cerebrovasculature and hippocampal neurogenesis of these animals. In summary, our research program is designed to provide a molecular, cellular and physiological framework for understanding the mechanism(s) by which environmental enrichment modulates amyloid deposition in transgenic mice. These efforts will provide valuable new information relevant to defining new preventative measures and therapeutic strategies for Alzheimer's disease.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by impairments in memory and cognition, neuronal loss and deposition of beta-amyloid (Abeta) peptides that are derived from larger amyloid precursor proteins (APP). Inheritance of mutated PSEN1 and PSEN2 genes, encoding presenilin 1 and presenilin 2 (PS1 and PS2) variants, respectively, cause early-onset, autosomal dominant forms of familial AD (FAD), an aggressive form of disease that affects patients in the second to fifth decades of life (Price and Sisodia, 1998). We have demonstrated that expression of human FAD-linked PS1 variants in all CNS cell types in transgenic mice impairs environmental enrichment (EE)-induced division (termed proliferation) and production of new neurons (termed neurogenesis) of precursor cells (termed NPCs) in the hippocampus (Choi et al., 2008), and the experiments proposed in this application are designed to clarify the molecular and cellular mechanism(s) by which these mutant PS1 variants alters the homeostasis of hippocampal NPCs. Because the hippocampal NPCs provide a cellular reservoir for replacement of granule cells during normal aging, we suggest that in AD, mechanisms responsible for NPC division and neuronal differentiation are impaired and hence cannot fully compensate for the severe neuronal loss in disease. Indeed, recent studies have revealed a marked reduction in the numbers of NPC and their derivatives in brains of aged humans and patients with AD. As the hippocampus plays a central role in memory formation, understanding the physiological and molecular underpinnings of mutant PS1 on NPC proliferation and neurogenesis is critical. To this end, we have shown that proliferation and neurogenesis of hippocampal NPCs are controlled, at least in part, by microglial cells in the hippocampal niche by non-cell autonomous mechanisms. We now propose three Aims in which we will employ biochemical and genetic strategies to elucidate the cellular and molecular mechanisms underlying the effects of FAD-linked PS1 on proliferation and neuronal differentiation of adult hippocampal progenitors.

Project Summary: It is widely accepted that A? peptides, the principal component of senile plaques, play a causative role in the pathogenesis of Alzheimer's disease (AD). Thus, the majority of potential disease-modifying treatments of AD are directed against A?, and the most elaborated approach is immunotherapy. Although passive immunization with intact antibody against A? can attenuate amyloid deposition and improve cognitive function in vivo, administration of antibodies will require intravenous delivery, chronic dosing, and high quantities, which increase the probability of adverse effects and costs of the therapy. In this regard, somatic gene therapy provides a promising therapeutic approach for treatment of AD. As the largest surface organ, human skin serves an ideal site for tissue engineering, as well as the long-term and efficient delivery of therapeutic genes in vivo. Compared to conventional gene therapy approaches, including viral vectors, transplantation with autologous skin grafts derived from epidermal stem/progenitor cells is technically well-established, minimally invasive, and has been successfully used for decades in the treatment of burn wounds. It has also been well documented that therapeutic molecules, including large proteins secreted by skin epidermal cells, can cross the epidermal/dermal barrier and reach the circulation to achieve therapeutic effects in a systemic manner. We have recently resolved the long-standing technical obstacle in the field by establishing a novel mouse skin organotypic culture and transplantation model. With this key technical advancement, we have demonstrated that cutaneous gene therapy with engineered epidermal progenitor cells can serve as a safe and effective treatment for many human diseases, including diabetes and substance abuse. In this proposal, we will take advantage of this novel platform and explore the feasibility and clinical potential of cutaneous gene therapy for treatment of AD. Specifically, we will employ CRISPR (clustered regularly-interspaced short palindromic repeats) technology to engineer and develop skin epidermal progenitor cells with inducible expression of scFv (single-chain variable fragment) targeting A?. Upon transplantation of skin organoids derived from engineered cells, we will explore the potential therapeutic effects in AD model animals and examine the long-term stability and potential immune reactions of skin grafts. Together, our studies will establish a unique and powerful model of cutaneous gene therapy for treatment of AD, revealing the therapeutic potential for somatic gene therapy with epidermal progenitor cells.