Anastasios Georgakopoulos - US grants

Missence mutations in Presenilin-1 (PS1) are the most common cause of autosomal dominant familial Alzheimer's disease (FAD). PS1 controls the gamma-secretase cleavage of many type I transmembrane proteins. EphrinB proteins are type I transmembrane proteins that function as ligands for the ephrinB receptors (EphBs). The ephrinB-EphB system transmits cellular signals from both the receptor and the ligand thus constituting a bi-directional signaling system. The ephrinB-EphB interactions regulate important cellular processes in development and adulthood including cell migration, axon guidance, dendritic spine morphogenesis, angiogenesis and synaptic plasticity as well as cognitive processes regulating two forms of long-term synaptic plasticity that are important for information storage in the brain, the long-term potentiation (LTP) and the long-term depression (LTD). We found that PS1 controls the proteolytic processing of both ephrinB and ephB proteins by a gamma-secretase-like activity, producing carboxy terminal ephrinB and ephB fragments. Our data shows that ephrinB and ephB proteins are first processed by a metalloproteinase (MMP) activity to produce a membrane-bound carboxy terminal fragments termed CTF1s. These fragments are subsequently cleaved by the PS1/gamma-secretase system to produce carboxy terminal fragments termed CTF2s. We obtained data that cytoplasmic sequence of both ephB and ephrinB translocate to the nucleus where they may act as transcription factors. Nuclear localization of these sequences is regulated by the PS1/gamma-secretase system. We also observed that the PS1/gamma-secretase system regulates the ephB-induced phosphorylation of Src kinase, a process initiated by the eprhinB-ephB interaction. Src kinase acts as a second messenger regulating various cellular functions like phosphorylation of cytoskeletal proteins, assembly of focal adhesions, memory formation and neurodegeneration, functions severely impaired in AD. Thus, PS1 may control synaptic structure and function by affecting the physiological processing of ephrinB ligands and ephB receptors and the ephrinB-ephB-mediated signaling.

Missence mutations in Presenilin-1 (PS1) are the most common cause of autosomal dominant familial Alzheimer's disease (FAD). PS1 controls the gamma-secretase cleavage of many type I transmembrane proteins. EphrinB proteins are type I transmembrane proteins that function as ligands for the ephrinB receptors (EphBs). The ephrinB-EphB system transmits cellular signals from both the receptor and the ligand thus constituting a bi-directional signaling system. The ephrinB-EphB interactions regulate important cellular processes in development and adulthood including cell migration, axon guidance, dendritic spine morphogenesis, angiogenesis and synaptic plasticity as well as cognitive processes regulating two forms of long-term synaptic plasticity that are important for information storage in the brain, the long-term potentiation (LTP) and the long-term depression (LTD). We found that PS1 controls the proteolytic processing of both ephrinB and ephB proteins by a gamma-secretase-like activity, producing carboxy terminal ephrinB and ephB fragments. Our data shows that ephrinB and ephB proteins are first processed by a metalloproteinase (MMP) activity to produce a membrane-bound carboxy terminal fragments termed CTF1s. These fragments are subsequently cleaved by the PS1/gamma-secretase system to produce carboxy terminal fragments termed CTF2s. We obtained data that cytoplasmic sequence of both ephB and ephrinB translocate to the nucleus where they may act as transcription factors. Nuclear localization of these sequences is regulated by the PS1/gamma-secretase system. We also observed that the PS1/gamma-secretase system regulates the ephB-induced phosphorylation of Src kinase, a process initiated by the eprhinB-ephB interaction. Src kinase acts as a second messenger regulating various cellular functions like phosphorylation of cytoskeletal proteins, assembly of focal adhesions, memory formation and neurodegeneration, functions severely impaired in AD. Thus, PS1 may control synaptic structure and function by affecting the physiological processing of ephrinB ligands and ephB receptors and the ephrinB-ephB-mediated signaling.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by severe neurovascular disfunction. Increasing evidence implicates cerebral microvasculature abnormalities in the genesis of AD neuropathology. Presenilin 1 (PS1) is a protein of central importance to the neuropathology of AD. Mutations in this protein are linked to many cases of autosomal dominant forms of AD. PS1 controls the y-secretase cleavage of several type I transmembrane proteins producing cytoplasmic peptides with signaling and gene expression functions. We recently showed that PS1/y-secretase cleaves ephrinB proteins and promotes the EphB-induced phosphorylation of Src. Since both ephrinB and Src play a critical role in angiogenesis it is possible that PS1/y-secretase may regulate ephrinB-mediated angiogenesis. Here we show that y-secretase promotes the angiogenic response of endothelial cells to EphB in a Src-dependent manner and so does the product of y-secretase cleavage of ephrinB (ephrinB/CTF2 cytoplasmic peptide), whose production is stimulated by EphB receptor. This suggests that PSI/y-secretase may regulate the EphB-induced angiogenesis by cleaving ephrinB and activating Src via this cleavage. To elucidate the mechanism by which PS1/y-secretase promotes the EphB-induced Src activation we analyze the association of ephrinB/CTF2 peptide with the Src regulatory complex. This complex consists of the adaptor protein PAG/Cbp and the regulatory kinase Csk, which regulate the autophosphorylation and activation of Src. We found that ephrinB/CTF2 forms specific complexes with PAG/Cbp affecting its phosphorylation on tyrosine residues and its association with Csk kinase and that PAG/Cbp mediates the EphB-induced angiogenic response of endothelial cells in two in vitro angiogenesis assays. Our data suggest that PS1/y-secretase promotes EphBinduced angiogenesis by cleaving ephrinB, producing ephrinB/CTF2 peptide, which interacts with the Src regulatory machinery promoting Src activation and cell sprouting. Thus PS1/y-secretase may regulate development and integrity of the brain vasculature, and PS1 mutations found in Familial AD may impair it. We propose to examine the role of PSI/y-secretase in the EphB/ephrinB-mediated angiogenesis.

? DESCRIPTION (provided by applicant): Evidence in the last decade implicates cerebral microvasculature abnormalities in the genesis of AD neuropathology. Additional literature shows that the EphB4/ephrinB2 system is an important regulator of the development and function of the vascular system. Binding of the extracellular sequence of EphB4 receptor to its transmembrane ligand ephrinB2 protein on the surface of endothelial cells of blood vessels, stimulates angiogenesis and growth of new vessels from existing vasculature. Furthermore, in vitro assays show that treatment of ephrinB2expressing endothelial cells with the extracellular domain of EphB4 stimulates cell sprouting and tube formation, processes considered crucial initial steps in angiogenesis, while transgenic mouse experiments indicate that the intracellular (cytoplasmic) domain of ephrinB2 protein is necessary for ephrinB2dependent angiogenesis. We found that EphB4 stimulates the metalloproteinase (MP) and PS/?secretase processing of ephrinB2 producing cytosolic peptide ephrinB2/CTF2 and that the EphB4induced sprouting and tube formation of endothelial cells depends on ?secretase activity. These observations raise the possibility that the endothelial EphB4/ephrinB2 system regulates angiogenesis through PS/?secretase. In support of this hypothesis, we obtained data that peptide ephrinB2/CTF2 stimulates sprouting of endothelial cells in vitro. Recent literature shows that a crucial step in angiogenic factorinduced angiogenesis is formation of complexes between Raf1/ Rok? and Vascular Endothelial cadherin (VEcadherin) and we made the novel observation (preliminary data) that treatment of endothelial cell cultures with EphB4 increases these angiogenic complexes. Together, our observations suggest that PS1/?secretase may affect angiogenesis by regulating processing of transmembrane protein ephrinB2, a critical step in EphB4induced angiogenesis. Here we propose to explore the mechanisms via which the EphB4/ephrinB2 and PS1/?secretase systems interact to promote endothelial cell sprouting and angiogenesis and to examine whether any of these mechanisms are altered in Alzheimer disease (AD) brains. Furthermore, we and others reported that PS1 familial AD (FAD) mutations may affect the epsilon (?) cleavage of PS1/?secretase substrates thus decreasing production of CTF2 peptides including ephrinB2/CTF2 (see Significance). Thus, we will ask whether PS1 FAD mutants alter the EphB4/ephrinB2dependent angiogenesis.

Cerebral microvasculature abnormalities, such as degeneration of the capillary endothelium, are implicated in the genesis of Alzheimer's disease (AD) neuropathology. In addition, ischemic lesions that cause neuronal damage are often found in AD brains. The brain responds to ischemia by stimulating tissue neovascularization via sprouting angiogenesis; impairment of this function renders the brain vulnerable to the insult. It has been hypothesized that decreased angiogenesis in AD leads to insufficient blood flow and neuronal dysfunction in affected areas. The EphB4/ephrinB2 (efnB2) ligand-receptor system is known to regulate brain angiogenesis in response to ischemia. We present evidence that Presnilin1 (PS1), an important factor in familial AD (FAD), regulates angiogenic functions of EphB4/efnB2 such as stimulation of VE-cadherin angiogenic complexes in brain endothelial cells (ECs). We also found that this function is impaired by PS1 FAD mutants. In addition, we found that ischemia stimulates formation of the same angiogenic complexes in the brain and that this function is attenuated by PS1 FAD mutants together with ischemia-induced neovascularization and cerebral blood flow while neuronal death is increased. We also found that PS1 FAD mutants decrease the ?-secretase processing of efnB2 and production of the angiogenic peptide efnB2/CTF2 thus impairing the response of brain ECs to angiogenic factors. Together, our data support the hypothesis that PS1 FAD mutants inhibit ischemia-induced angiogenic functions of ECs by decreasing the ?-secretase processing of efnB2 and impairing the EphB4/efnB2 signaling, thus decreasing neovascularization in the adult brain and leading to increased vulnerability to this toxic insult and neuronal death. In this application we examine the roles of PS1 FAD mutants and ?-secretase on ischemia-induced angiogenic complexes, blood flow, angiogenesis and neuronal death. In addition, we propose to test a small peptide, which derives from the proteolytic processing of efnB2 by ?-secretase and which we found to rescue angiogenic functions of PS1 FAD ECs, for its pro-angiogenic and neuroprotective functions in brains expressing FAD mutants. In addition we aim to search for novel ischemia-induced angiogenic pathways that are impaired by PS1 FAD mutants and examine whether VE-cadherin angiogenic complexes and angiogenic protein expression are impaired in brains of human PS1 FAD and AD patients.