Role of HSP70 in motoneuron survival after excitotoxic stress in a rat spinal cord injury model in vitro

Abstract

The outcome for gait recovery from paralysis due to spinal lesion remains uncertain even when damage is limited. One critical factor is the survival of motoneurons, which are very vulnerable cells. To clarify the early pathophysiological mechanisms of spinal damage, an in vitro injury model of the rat spinal cord caused by moderate excitotoxicity was used. With this preparation we investigated whether motoneuron survival was dependent on the expression of the neuroprotective protein HSP70. In the present study excitotoxicity evoked by kainate induced delayed (24 h) loss (35%) of motoneurons, which became pyknotic with translocation of the cell death biomarker apoptosis-inducing factor (AIF) to the nucleus. This process was concomitant with suppression of locomotor network electrical activity. Surviving cells showed strong expression of HSP70 without nuclear AIF. The HSP70 inhibitor VER155008 per se induced neurotoxicity similar to that of kainate, while the HSP90 inhibitor geldanamycin did not damage spinal tissue. Electrophysiological recording following kainate or VER155008 indicated depression of motoneuron field potentials, with decreased excitability and impaired synaptic transmission. When these two drugs were applied together, more intense neurotoxicity emerged. Our data indicate that HSP70 was one important contributor to motoneuron survival and suggest that enhancing HSP70 activity is a potential future strategy for neuroprotecting these cells.

Nicotine protects rat hypoglossal motoneurons from excitotoxic death via downregulation of connexin 36

“…24, 25 Figures 5b and c show that, after TBOA or nicotine treatment, Hsp70 expression was unchanged compared with basal sham conditions. When nicotine and TBOA were co-applied, Hsp70 expression was significantly enhanced ( P =0.027 among groups; P <0.05 for sham versus nicotine + TBOA; n =5 brainstems).…”

“…In fact, protection of motoneurons from excitotoxicity depends to a large extent on the binding by Hsp70 of AIF, a mitochondrial factor released by metabolically damaged motoneurons. 24, 25 When, during excitotoxicity, Hsp70 expression is insufficient to bind AIF, the latter migrates to the cell nucleus and inactivates DNA. 31 We, therefore, studied immunohistochemical expression of AIF when TBOA was applied alone or together with nicotine.…”

“…As the intracellular expression of Hsp70 is an important biomarker of the ability of motoneurons to resist to excitotoxicity, 24 we surmise that surviving cells had an adequate Hsp70 expression although these data were obtained from brainstem tissue rather than single HMs. The perturbation triggered by TBOA plus the effects of nicotine probably synergized to raise Hsp70 expression and extend neuroprotection.…”

“…To this aim, we studied how nicotine could modulate Ca 2+ transients, Cx36 and Panx1, and compared it with the effects of the gap junction blocker carbenoxolone. 23 In analogy with previous reports, 24, 25 we used expression of heat shock protein 70 (Hsp70) and apoptosis-inducing factor (AIF) as indices of cell protection or death, respectively.…”

Pharmacological induction of Heat Shock Protein 70 by celastrol protects motoneurons from excitotoxicity in rat spinal cord in vitro

“…Thus, higher levels of HSPs have been detected in the cerebrospinal liquid of dogs with spinal cord injury (Awad et al., ). In our former report (Shabbir et al., ) using an in vitro spinal cord model simulating the excitotoxic damage developing as an early consequence of spinal injury, we observed that motoneurons able to survive this insult usually expressed high levels of HSP70. Furthermore, pharmacological inhibition of HSP70 intensified neuronal losses, especially motoneuronal death (Shabbir et al., ).…”