Heat shock proteins, including Hsp70, are chaperones induced by heat shock and many stresses including ischemia and assist protein folding during protein synthesis and re-folding after protein denaturation. Hsp70 protein expression is neuroprotective in a variety of models using a number of different methods of over expression. This proposal will address protective, anti-apoptotic and anti-inflammatory roles of Hsp70 in: (1) primary cultures of neurons and glia;and (2) following focal cerebral ischemia. A novel aspect of the study will be to intravenously administer recombinant Hsp70, Hsp70C and mutant Hsp70C-DEVD proteins using a newly developed single-chain fragment of an anti-DNA antibody referred to as Fv. Fv-Hsp70 binds to the nucleoside salvage transporter ENT2 found on all cells and the Fv-Hsp70 enters these cells via the ATP independent ENT2 transporter. Our preliminary data show: (a) that Fv-Hsp70 protects neurons and glia in vitro;(b) FvHsp70 decreases infarct volumes and improves behavioral outcomes following middle cerebral artery occlusions (MCAO) in vivo;(c) and pro-inflammatory and pro-apoptotic genes induced in blood leukocytes following MCAO are decreased by treatment with FvHsp70 in vivo. Therefore, we propose the following aims.
Specific Aim #1 a: Demonstrate that Fv-Hsp70, Fv-Hsp70C and mutant Fv-Hsp70C-DEVD protect cultured neurons and astrocytes from oxygen glucose deprivation (OGD).
Specific Aim #1 b: Begin to explore the anti- apoptotic and anti-inflammatory mechanisms of protection by examining the interaction between Hsp70, NF:B and TNF in primary cells from brain;and show that Hsp70 blocks NF:B activation in primary brain cells.
Specific Aim #2 : Demonstrate that the Fv-Hsp70 constructs decrease infarct volumes and improve behavioral outcomes in rat models of focal cerebral ischemia. The effects of Fv-Hsp70, Fv-Hsp70C and Fv-Hsp70C-DEVD will be compared to each other and to vehicle, Hsp70 alone, Fv alone and Fv-Green Fluorescent Protein (GFP) controls.
Specific Aim #3 a. Perform genomic profiling of rat blood following the MCAO strokes produced in Aim #2 and demonstrate that MCAO induces a damaging set of pro-apoptotic and pro-inflammatory genes while suppressing pro-survival genes in leukocytes in blood.
Specific Aim #3 b: Demonstrate that treatment with Fv- Hsp70 constructs in rats following MCAO attenuates the deleterious gene response in blood leukocytes and will increase anti-apoptotic, increase anti-inflammatory and increase other pro-survival genes in rat blood leukocytes. Significance: These studies will provide a proof of principle that Hsp70, administered intravenously as a Fv- fusion protein, enters brain and improves outcome from stroke. More generally, the Fv protein delivery method used here could be useful for delivering any protein to treat stroke, other acute injuries to the brain and spinal cord, and possibly to treat degenerative neurological diseases. We also propose that monitoring gene expression changes in peripheral blood that correlate with effective stroke treatments in rodents can be used to assess potential treatment responsiveness in humans.
This proposal will examine the neuroprotective properties of the heat shock protein, Hsp70. A newly developed protein delivery method, termed Fv, will be used to deliver Fv-Hsp70 and Hsp70 mutants to primary brain cells and to brain. We will demonstrate that Fv-Hsp70 constructs protect neurons and glia from oxygen and glucose deprivation and that FvHsp70 constructs protect rat brain against stroke. The Fv-Hsp70 mediated protection will be due in part to blockade of inflammation and blockade of pro-apoptotic pathways in both blood and brain. It is proposed that FvHsp70 could be used to treat humans with stroke, and that changes of gene expression in blood of rats that correlate with improved outcome in rats can be used as biomarkers to predict improved outcomes in humans treated with FvHsp70.
|Liu, Da Zhi; Waldau, Ben; Ander, Bradley P et al. (2017) Inhibition of Src family kinases improves cognitive function after intraventricular hemorrhage or intraventricular thrombin. J Cereb Blood Flow Metab 37:2359-2367|
|Liu, Da Zhi; Jickling, Glen C; Ander, Bradley P et al. (2016) Elevating microRNA-122 in blood improves outcomes after temporary middle cerebral artery occlusion in rats. J Cereb Blood Flow Metab 36:1374-83|
|Jickling, Glen C; Sharp, Frank R (2015) Improving the translation of animal ischemic stroke studies to humans. Metab Brain Dis 30:461-7|
|Weis, S N; Toniazzo, A P; Ander, B P et al. (2014) Autophagy in the brain of neonates following hypoxia-ischemia shows sex- and region-specific effects. Neuroscience 256:201-9|
|Sharp, Frank R; Jickling, Glen C (2013) Whole genome expression of cellular response to stroke. Stroke 44:S23-5|
|Sharp, Frank R; Zhan, Xinhua; Liu, Da-Zhi (2013) Heat shock proteins in the brain: role of Hsp70, Hsp 27, and HO-1 (Hsp32) and their therapeutic potential. Transl Stroke Res 4:685-92|
|Hansen, James E; Chan, Grace; Liu, Yanfeng et al. (2012) Targeting cancer with a lupus autoantibody. Sci Transl Med 4:157ra142|
|del Zoppo, Gregory J; Sharp, Frank R; Heiss, Wolf-Dieter et al. (2011) Heterogeneity in the penumbra. J Cereb Blood Flow Metab 31:1836-51|
|Zhan, Xinhua; Ander, Bradley P; Liao, Isaac H et al. (2010) Recombinant Fv-Hsp70 protein mediates neuroprotection after focal cerebral ischemia in rats. Stroke 41:538-43|
|Lu, Aigang; Clark, Joseph F; Broderick, Joseph P et al. (2009) Mechanical reperfusion is associated with post-ischemic hemorrhage in rat brain. Exp Neurol 216:407-12|