Heat Shock Protein B8 (HSPB8) Reduces Oxygen-Glucose Deprivation/Reperfusion Injury via the Induction of Mitophagy.

BACKGROUND/AIMS: We have reported the neuroprotective properties of Heat shock protein B8(HSPB8) against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury by inhibiting the mitochondrial apoptotic pathway. However, the exact underlying mechanism of its protective effect on mitochondrial function remains unknown. Here we examined whether the beneficial effect of HSPB8 on OGD/R-induced cell death is associated with mitophagy in mouse neuroblastoma Neuro2a (N2a) cells. METHODS: Using the mouse transient middle cerebral artery occlusion (tMCAO) model and mouse neuroblastoma Neuro2a (N2a) cell cultures subjected to OGD/R, we employed western-blot, RT-PCR and immunostaining to analyze the change of expression pattern of HSPB8 and mitophagic flux after brain I/R both in vivo and in vitro. Moreover, via overexpressing HSPB8 or knocking down HSPB8 expression with siRNA in N2a cell, we evaluated the effect of HSPB8 on mitochondrial function during OGD/R. The impact of HSPB8 on mitophagic pathway was also assessed. Finally, mitotophagy inhibitors (CQ and Mdivi-1) were adopted to verify the involvement of mitophagy in HSPB8- induced neuroprotection. RESULTS: HSPB8 could be up-regulated by brain I/R both in vivo and in vitro. Mitophagy enhancement coincided with induction of HSPB8 during I/R. Overexpression of HSPB8 reinforced I/R-induced mitophagy in OGD/R-treated mouse N2a cells and HSPB8 silence suppressed mitophagy process. Inhibition of mitophagy compromised neuroprotection conferred by HSPB8 overexpression. CONCLUSIONS: HSPB8 promoted OGD/R-induced mitophagy, which restored the mitochondrial function and contributed to the decrease in cell apoptosis after OGD/R. Therefore, HSPB8 could be a favorable neuroprotective agent for cerebral I/R related disorders.

Blockade of Ser16-Hsp20 phosphorylation attenuates neuroprotection dependent upon Bcl-2 and Bax.

Ischemic stroke causes a significant amount of cell damage resulting from an insufficient supply of glucose and oxygen to central nervous system tissue and finding more effective therapeutic neuroprotective agents has become a priority in the treatment of ischemic stroke. Hsp20, one of the small heat shock proteins, has been implicated in multiple physiological and pathophysiological processes and is a potential neuroprotective agents. To investigate whether Hsp20 exerts protective effects on in vitro ischemia-reperfusion injury, mouse neuroblastoma cells were subjected to oxygen-glucose deprivation/reoxygenation (OGDR) insult. The N2a cells transfected with Hsp20 and constitutively phosphorylated Hsp20 (S16D) had significantly less cell loss and less proportion of apoptotic cells compared to N2a cells transfected with pEGFP-N1 after oxygen-glucose deprivation (OGD) 4 h plus 12 and 24 h reperfusion, which showed no difference in N2a cells transfected with nonphosphorylatable Hsp20 (S16A). Meanwhile, transfected with Hsp20 and constitutively phosphorylated Hsp20 (S16D) also significantly attenuated mitochondrial fragmentation and modulated Bcl-2 and Bax expression level after OGD 4 h plus 12 reperfusion, which were not affected in N2a cells transfected with Hsp20 (S16A). In conclusion, our data demonstrated that increased Hsp20 and Hsp20 (S16D) expression in mouse N2A neuroblastoma cells protected against ischemia-reperfusion injury, the neuroprotective mechanism may be related to regulate Bcl-2 and Bax expression. However, blockade of Ser16-Hsp20 phosphorylation attenuated the neuroprotective effects of Hsp20. Therefore, Hsp20 and factors that contribute to regulation of phosphorylation on Ser16 of Hsp20 are potential new therapeutic targets for the treatment of cerebral ischemia-reperfusion injury.

Developmental expression pattern of Hspb8 mRNA in the mouse brain: analysis through online databases.

Hspb8 is a member of the Hspb family of chaperone-like proteins. It is involved in several neural disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, hereditary distal motor neuropathy, and Charcot-Marie-Tooth's disease. In this work, we aimed to characterize its expression pattern in the mouse brain, by using the information available at online databases of high-throughput in situ hybridization. Therefore, we downloaded and analyzed the image series from these databases showing Hspb8 mRNA expression from embryonic to adult and aging stages. In early gestational embryos, Hspb8 was expressed in the hippocampal anlagen and in the ventricular layer of rhombomere 4. At perinatal stages, there appeared transitory expression in the dentate gyrus and the cerebellar cortex. From perinatal to aging stages, the neurons of the mesencephalic trigeminal nucleus and cranial motor nuclei displayed stable and strong Hspb8 expression. Additionally, along these stages there was moderate and relatively homogenous expression in the anterodorsal thalamic, lateral mammillary, arcuate hypothalamic and medial habenular nuclei, and in the locus coeruleus. In its turn, the basal ganglia, cerebellar inner granular layer and diverse sensory and reticular formation nuclei of the hindbrain contained scattered cells with strong expression. In conclusion, Hspb8 mRNA is constitutively expressed in specific brain structures across ontogeny, so that eventually they could be affected by the malfunction or deregulation of this molecule.

Small heat shock proteins in smooth muscle.

The small heat shock proteins (HSPs) HSP20, HSP27 and alphaB-crystallin are chaperone proteins that are abundantly expressed in smooth muscles are important modulators of muscle contraction, cell migration and cell survival. This review focuses on factors regulating expression of small HSPs in smooth muscle, signaling pathways that regulate macromolecular structure and the biochemical and cellular functions of small HSPs. Cellular processes regulated by small HSPs include chaperoning denatured proteins, maintaining cellular redox state and modifying filamentous actin polymerization. These processes influence smooth muscle proliferation, cell migration, cell survival, muscle contraction and synthesis of signaling proteins. Understanding functions of small heat shock proteins is relevant to mechanisms of disease in which dysfunctional smooth muscle causes symptoms, or is a target of drug therapy. One example is that secreted HSP27 may be a useful marker of inflammation during atherogenesis. Another is that phosphorylated HSP20 which relaxes smooth muscle may prove to be highly relevant to treatment of hypertension, vasospasm, asthma, premature labor and overactive bladder. Because small HSPs also modulate smooth muscle proliferation and cell migration they may prove to be targets for developing effective, novel treatments of clinical problems arising from remodeling of smooth muscle in vascular, respiratory and urogenital systems.