Amarantus Bioscience Holdings Inc (AMBS)

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NATIONAL HEART, LUNG, AND BLOOD INSTITUTE - MANF
Project Funding Information for 2013:
Total Funding: $304,756
2013 Award Notice Date: 13-SEP-2013
http://projectreporter.nih.gov/project_info_details.cfm?aid=8452819&icde=18489718

2013 Award Notice Date: 8-JUN-2013
Total Funding: $344,920
http://projectreporter.nih.gov/project_info_details.cfm?aid=8488313&icde=18489803


DESCRIPTION (provided by applicant): Synopsis: Our long-term objective is to determine the roles of ER stress in the heart. Using a genomics approach we identified a group of genes induced by the ATF6 branch of the ER stress response (ERSR) that encode proteins predicted to have para/autocrine effects on the heart. These proteins, which we call ER stress-inducible cardiomyokines (ERS-CMKs), are unique, since they are synthesized in, and secreted from the heart during stresses, e.g. ischemia, that impair synthesis and release of most other proteins. The focus of this proposal is the ERS-CMK, mesencephalic astrocyte-derived neurotrophic factor (MANF), which is novel since it functions intra- and extracellularly to affect cardioprotection. In this proposal cardiomyokines (CMKs) are defined as proteins secreted by the heart that may exert para/autocrine effects, in part, by direct binding to heart cells, as well as binding to resident and, perhaps non-cardiac-derived stem cells to affect their function. Most CMKs are synthesized and folded in the rough ER, routed to the Golgi, then to secretory vesicles before secretion. In the absence of ER stress, conditions are optimal for expression, folding and, thus, secretion of functional CMKs. However, some stresses impair protein folding in the ER, activating ER stress, which decreases CMK transcription, translation, folding and secretion, thus leading to a loss of CMK function. ER stress leads to activation of ATF6, a nodal sensor of ER stress that increases transcription of many known ERSR genes that encode ER-targeted proteins that directly augment ER-protein folding. We showed that the ATF6 branch of the ERSR, which is activated during ischemia, protects the heart from damage during ischemia and reperfusion, ex vivo and in vivo. Microarray analyses of mouse hearts revealed numerous ATF6-inducible genes that encode proteins predicted to be ER-targeted and secreted (ERS-CMKs). One ERS-CMK, MANF, is unusual, since it is either retained or secreted, depending on the stress. Overexpression of MANF, or addition of recombinant MANF (rMANF) to culture medium, protected cardiomyocytes, while MANF knock-down increased simulated I & I/R-mediated cell death. Hypothesis: The specific hypothesis addressed in this proposal is that ischemia, which is known to activate ER stress, induces the ERS-CMK, MANF, in an ATF6-dependent manner, and acts intra- and extracellularly to protect the heart from ischemic damage. The Specific Aims that address this hypothesis are to: 1. examine expression of MANF in the ischemic mouse heart subjected to ATF6 gain- and loss-of-function, 2. determine the effects MANF gain- and loss-of-function in the ischemic heart, in vivo, and 3. dissect the functions of intra- and extracellular MANF, delineate the structural features of MANF required for its activities, and identify signaling mechanisms responsible for MANF function in cardiomyocytes.

Abstract Text:
Proteins secreted by the heart are called cardiokines. After secretion, cardiokines, such as cytokines, growth promoters and stem cell homing factors affect ischemic damage, as well as stem cell survival and engraftment. But ischemia impairs protein folding and secretion, and negatively impacts stem cell-mediated regeneration. However, we discovered a secretion process that resists this inhibition, enabling the release of certain beneficial cardiokines, just when they are needed the most. The objectives of this study are to examine the functions of, and molecular mechanisms governing this secretion process in cardiac myocytes, in vitro and in vivo, and in cardiac stem cells. We discovered this process while studying the beneficial cardiokine, mesencephalic astrocyte-derived neurotrophic factor (MANF), which resides in the endoplasmic/sarcomplasmic reticulum (ER/SR). Our hypothesis is that 1) GRP78 regulates the secretion of beneficial ER stress cardiokines from cardiac myocytes and cardiac progenitor cells by mediating the conditional retention of proteins in the ER/SR, and 2) CPCs are specially configured with a novel cytosolic form of GRP78 that enhances survival, as well as cardiokine secretion during ER stress.We will address this hypothesis by using MANF as a model cardiokine, GRP78 gain- and loss-of-function, cultured cells and mouse hearts, /\AV9-mediated in vivo gene transfer, and zero-distance live cell cross linking in the following specific aims: 1- to determine the mechanism by which GRP78 regulates cardiokine secretion from cardiac myocytes, 2- to assess GRP78-regulated cardiokine secretion in the heart, in vivo, and determine the effects of disrupting this secretion on ischemic damage and regeneration, and 3- to examine the effects of GRP78 in the ER, as well as a novel, cytosolic form of GRP78 on cardiokine secretion, responses to ER stress and survival of cardiac stem cells. The results of these studies will facilitate the design of therapeutic strategies aimed at enhancing the secretion of beneficial cardiokines that minimize damage and maximize regeneration.


http://projectreporter.nih.gov/project_info_description.cfm?aid=8452819&icde=18489718


http://projectreporter.nih.gov/project_info_description.cfm?aid=8488313&icde=18489803&ddparam=&ddvalue=&ddsub=&cr=2&csb=default&cs=ASC

AMBS - MANF