Widespread cortical expression of MANF by AAV serotype 7: Localization and protection against ischemic brain injury
Introduction
The pathophysiological mechanisms of acute stroke involve multiple processes including oxygen and glucose deprivation, excessive glutamate release with corresponding excitoxicity, reactive oxygen species formation, protein and lipid modifications, Ca++ dysregulation, mitochondrial dysfunction, endoplasmic reticulum (ER) stress and apoptosis. These processes lead to neurodegeneration in the ischemic area which results in disruption of neural circuits that can affect many behavioral and cognitive functions. Evidence from animal models show that neuronal rewiring and synapse strengthening occurs during recovery from ischemic brain injury (for review see/(Murphy and Corbett, 2009). Thus, three therapeutic strategies for treating ischemic brain injury are: 1) to minimize the acute molecular mechanisms related to neuronal death e.g. endoplasmic reticulum (ER) –stress, mitochondrial dysfunction, excitoxicity and apoptosis; 2) to promote functional enhancement of remaining circuitry; and 3) to increase behavioral and cognitive recovery through “neural rewiring’ by promoting new functional neural connections. These latter compensations include neural precursor proliferation and migration, axonal pathfinding, neuritic outgrowth and synaptogenesis. Neurotrophic factors have demonstrated capacity for both of these therapeutic strategies. Viral vectors have been shown to provide sustained neurotrophic factor expression throughout the brain. However, the use of viral vectors encoding neurotrophic factors has not been extensively studied in the stroke literature (for review see/(Lim, et al., 2010).
Mesencephalic astrocyte-derived neurotrophic factor (MANF) was initially characterized as a trophic factor for cultured embryonic dopaminergic neurons (Petrova, et al., 2003). MANF and its homologue, cerebral dopamine neurotrophic factor (CDNF) have been shown to promote survival and recovery of midbrain dopamine neurons (Lindholm et al., 2007, Petrova et al., 2003, Voutilainen et al., 2009). MANF is endogenously expressed in neurons and in non-neuronal tissues (Lindholm et al., 2008, Mizobuchi et al., 2007). In the brain, the highest levels of MANF are detected in cerebral cortex, hippocampus and cerebellar Purkinje cells (Lindholm, et al., 2008). MANF expression has been shown to be upregulated by ischemia (Apostolou et al., 2008, Lindholm et al., 2008, Tadimalla et al., 2008, Yu et al., 2010) as well as by endoplasmic reticulum (ER) -stress (Apostolou et al., 2008, Lee et al., 2003, Mizobuchi et al., 2007). We have recently shown that intra- cortical delivery of recombinant MANF protein reduces cerebral infarction and ischemia-mediated apoptosis in stroke animals (Airavaara, et al., 2009). Additionally, MANF has been shown to protect cells against glucose deprivation and tunicamycin, a inhibitor of protein glycosylation that induces ER-stress (Apostolou, et al., 2008). Thus, increased MANF levels after injury may be a result of activation of endogenous protective processes against protein misfolding.
Adeno-associated viral (AAV) vectors are currently the predominant viral vector used in clinical trials for neurodegenerative diseases (Lim, et al., 2010). Although the transgene capacity of single (i.e., 4-5 kb) or double (2-3 kb) stranded (McCarty et al., 2003, Wang et al., 2003) AAV vectors is limited, the vector genome is amenable to relatively small cDNA sizes which makes them ideal for expressing functional neurotrophic factors such as MANF (< 1 kb). The advantage of using dsAAV vectors or self-complementary vectors is higher transduction efficiency and more rapid onset of transgene expression compared to single-stranded AAV vectors (McCarty et al., 2003, Wang et al., 2003).
In this study, we generated a serotype 7, dsAAV vector expressing human MANF cDNA for intracortical expression of MANF in a rat model of stroke. We devised a delivery scheme to provide wide spread transduction of cortical cells in the area affected by middle cerebral artery occlusion in rats. AAV-MANF reduced infarction volume and increased post-stroke recovery of locomotor activity and neurological score. Ischemic injury caused a redistribution of MANF in the AAV-MANF treated animals which was consistent with injury-induced secretion of MANF. Overall, our findings show that AAV-mediated delivery of MANF improves the outcome following ischemic brain injury in rats.
Section snippets
Animals
Adult male Sprague-Dawley rats (250-350 g, Charles River Laboratory) were maintained under a 12-h light–dark cycle. Food and water were freely available in the home cage. Experimental procedures followed the guidelines of the “Principles of Laboratory Care” (National Institutes of Health publication No. 86-23, 1996) and were approved by the NIDA Animal Care and Use Committee.
Primary cortical cell cultures
Neocortical tissue from E15 embryos of timed-pregnant Sprague-Dawley rats were used to prepare neuronal cultures as
Characterization of AAV-MANF in vitro
We successfully constructed a dsAAV vector packing plasmid containing the human MANF cDNA. Transfection of HEK293 cells with the AAV-MANF packaging plasmid for 24 hours resulted in increased MANF protein as analyzed by western blotting (Fig. 1A). Neuronal MANF immunoreactivity was found in rat primary cortical cultures transduced with AAV-MANF virus for 48 hours and immunostained for MANF (Fig. 1D). Minimal MANF-immunoreactivity was seen in no-virus treated cultures (1C) or cultures transduced by
Discussion
We report the first in vivo gene therapy study using AAV-mediated delivery of the human MANF in a rodent model of stroke. We show that an AAV vector (serotype 7) can be used to achieve widespread transduction of cells throughout the ischemic region in rats. Increased human MANF protein levels by AAV-MANF in ischemic rodent cortex reduces brain injury and promotes behavioral recovery in rats.
Increased expression of human MANF protein via AAV-MANF delivery to rat cortex did not alter locomotor
Acknowledgments
This research was supported by the intramural research program at NIDA, NIH, and DHHS. The authors declare that they do not have any conflicts of interest (financial or otherwise) related to the data presented in this manuscript.
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