Angele Parent - US grants

[unreadable] DESCRIPTION (provided by applicant): Synaptic dysfunction is a critical feature of cognitive decline and neurodegeneration associated with Alzheimer's disease (AD). Autosomal dominant mutations in PSEN genes cause familial early-onset AD. Presenilins (PS) form the catalytic center of gamma-secretase, an enzyme responsible for the generation of beta amyloid peptides, which accumulate in the brains of individuals with AD. In addition to intramembranous cleavage of APP, Notch ligands/Notch receptor, and several cell adhesion molecules (CAMs) including cadherins, protocadherins, DCC, ErbB4, ephrin/Eph receptors, nectin-1alpha and syndecan, are also processed by gamma-secretase. Notably, each of these proteins facilitates diverse neuronal functions during embryonic development such as axon guidance, neuronal outgrowth and synaptogenesis. Recently, we reported that loss of PS expression or function in cultured neurons enhances glutamatergic synaptic transmission, synapse formation, and activation of cAMP-dependent signaling cascades. Interestingly, using PS1 M146V knock-in mice we find that expression of FAD-linked mutant also elevates cAMP-dependent signaling in cultured neurons, and leads to increased spine density in CA1 area of hippocampus. Analysis of DCC (the netrin receptor) processing revealed that lack of gamma-secretase cleavage increases neurite outgrowth and cAMP-dependent signaling, illustrating a novel function for PS wherein cleavage by gamma-secretase terminates intracellular signaling cascades associated with certain CAM substrates. Taken together, we hypothesize that gamma-secretase processing of certain CAMs regulates signaling cascades that play pivotal roles in synapse formation, synaptic transmission and plasticity. We also hypothesize that FAD-linked PS1 variants promote AD pathogenesis by influencing Abeta generation as well as proteolysis of certain CAMs that are important for synaptic function. The specific aims of this investigation are to examine the influence of PS-dependent proteolysis of CAMs: 1) on cellular function, 2) on synapse formation, and 3) on cellular substrates of memory. Using PS1 null embryos, PS1 M146V knock-in mice, and cultured cell systems we propose to perform electrophysiological, biochemical and imaging studies to investigate how PS-dependent proteolysis of CAMs influences synaptic functions. In particular, we will focus our studies on the synaptic influence of APP, DCC and N-cadherin in normal and pathological conditions because of their known association with the pathogenesis of AD, synapse formation and signaling pathways related to synaptic functions. We are extremely confident that our complementary approaches outlined in our proposal will provide important mechanistic insights into the role of PS in health and disease. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is pathologically characterized by the accumulation of ?-amyloid peptides (A?) generated via sequential proteolysis of amyloid precursor protein (APP). Over the years several physiological functions have been ascribed to APP. It has been proposed that APP can affect synaptic function by its dual roles via its cell-adhesive properties or through its putative receptor-like function that mediates intracellular signaling. Cleavage of full-length APP by ? and ?-secretases releases the entire ectodomain, leaving behind membrane bound C-terminal fragments (CTF) capable of mediating intracellular signaling until they are further processed by ?-secretase. In order to activate in a constitutive manner putative signaling associated with APP-CTF, we have designed a membrane-tethered APP cytoplasmic domain (mAICD). We found that accumulation of APP-CTFs generated by processing of APP or expression of mAICD (but not AICD) results in adenylate cyclase-dependent activation of PKA, inhibition of GSK3?, and enhanced dendritic and axonal arborization in primary cortical neurons. We identified a novel interaction between APP intracellular domain and the heterotrimeric G-protein subunit G?S, and by mutagenesis of the interaction motif within APP as well as expression of a dominant negative G?S mutant, demonstrate that interaction with G?S and subsequent G?S coupling to adenylate cyclase are essential for membrane-bound APP intracellular domain-induced neurite outgrowth. Our study provides clear evidence that APP intracellular domain can have a non-transcriptional role in regulating neurite outgrowth through its membrane association. Moreover, it was previously reported that APP could also interact with G?O. Activation of cAMP/PKA pathway is known to impact several brain functions such as synaptic plasticity and memory formation, as well as A??production through non-amyloidogenic pathway. Based on these findings, we hypothesize that functional coupling of APP cytoplasmic domain with G-proteins could influence neurite outgrowth, synapse formation and A??production. In order to investigate this hypothesis, we propose to develop mouse models that overexpress mAICD or mAICD mutant lacking G-protein binding site(s). We will employ recombinant adeno-associated viral (AAV) delivery and also generate transgenic mouse models to assess the physiopathological relevance of mAICD expression in the brain and its putative value in gene therapy. Amyloid deposition, A??production, stimulation of neurite outgrowth and synapse formation will be monitored in APP-null mice, mice coexpressing familial AD-linked mutant of APP and presenilin, and their control littermates, following AAV injections or transgenic intercross breeding. Our investigation will address the importance of a previously unrecognized intracellular signaling pathway associated with APP-CTF. A better understanding of APP-CTF and its associated signaling partners might provide important insights into the cellular mechanisms by which APP-CTF affects synaptic function and A??production, which could potentially impact on AD pathogenesis.

Alzheimer's disease (AD) is pathologically characterized by the accumulation of ß-amyloid peptides (Aß) generated via sequential proteolysis of amyloid precursor protein (APP). Cleavage of APP by ?- and ß- secretases releases the entire ectodomain, leaving behind membrane bound C-terminal fragments (CTF) capable of mediating intracellular signaling until they are further processed by ?-secretase. In order to activate in a constitutive manner putative signaling associated with APP-CTF, we have designed a membrane-tethered APP cytoplasmic domain (mAICD). We found that accumulation of APP-CTFs generated by processing of APP or expression of mAICD (but not AICD) results in adenylate cyclase-dependent activation of PKA, inhibition of GSK3ß, and enhanced axondendritic arborization in primary cortical neurons. We identified a novel interaction between APP intracellular domain and the heterotrimeric G-protein subunit G?S. By mutagenesis of the interaction motif identified within APP as well as expression of a dominant negative G?S mutant, we demonstrated that interaction with G?S and subsequent G?S coupling to adenylate cyclase are essential for membrane-bound APP intracellular domain-induced neurite outgrowth. Moreover, our preliminary results indicate that mutation of a previously described G?O binding motif of APP-CTF also reduces dendritic outgrowth. Thus, by analogy to other G-protein coupled receptors, it is possible that G- protein-mediated signaling through APP-CTF involves stochastic or simultaneous binding of G?O and G?S on the same APP molecule allowing dynamic regulation of APP function in neuronal morphology and neuronal dysfunction. Interestingly, APP processing and Aß production is a highly regulated process under the control of a number of phosphorylation events that could be affected by G-protein coupled receptor signaling cascades. Based on these findings, we hypothesize that spatiotemporal signaling of APP cytoplasmic domain with G?S/G?O-proteins could selectively affect axodendritic development and impact on AD pathogenesis. In order to investigate this hypothesis, we propose to examine (1) if axodendritic localization of ß-CTF is regulated by G- protein interactions; (2) characterize how ß-CTFG-protein interactions selectively affect axodendritic signaling; and (3) elucidate how spatiotemporal localization of APP-CTF affects full-length APP processing and Aß production. Altogether, our study provides clear evidence that APP intracellular domain has a non- transcriptional role in regulating neurite outgrowth through its membrane association via cAMP-dependent signaling and GSK3ß inhibition - two processes that are known to have a role in memory consolidation, Aß production and Tau-associated pathology. Our investigation will address the importance of a previously unrecognized intracellular signaling pathway associated with APP-CTF. A better understanding of APP-CTF and its associated signaling partners might provide important insights into the cellular mechanisms by which APP-CTF affects synaptic function and Aß production, thus have an impact on AD pathogenesis.