HSP70 attenuates neuronal necroptosis through the HSP90α-RIPK3 pathway following neuronal trauma

by Selleckchem | Nov 14 2023

Background: Necroptosis, a newly defined regulatable necrosis with membrane disruption, has been demonstrated to participate in trauma brain injury (TBI) related neuronal cell death. Heat shock protein 70 (HSP70) is a stress protein with neuroprotective activity, but the potential protective mechanisms are not fully understood.

Methods and results: Here, we investigated the effects of HSP70 regulators in a cellular TBI model induced by traumatic neuronal injury (TNI) and glutamate treatment. We found that necroptosis occurred in cortical neurons after TNI and glutamate treatment. Neuronal trauma markedly upregulated HSP70 protein expression within 24 h. The results of immunostaining and lactate dehydrogenase release assay showed that necroptosis following neuronal trauma was inhibited by HSP70 activator TRC051384 (TRC), but promoted by the HSP70 inhibitor 2-phenylethyenesulfonamide (PES). In congruent, the expression and phosphorylation of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) were differently regulated by HSP70. Furthermore, the expression of HSP90α induced by neuronal trauma was further promoted by PES but decreased by TRC. The data obtained from western blot showed that the phosphorylation of RIPK3 and MLKL induced by HSP70 inhibition were reduced by RIPK3 inhibitor GSK-872 and HSP90α inhibitor geldanamycin (GA). Similarly, inhibition of HSP90α with GA could partially prevented the increased necroptosis induced by PES.

Conclusions: Taken together, HSP70 activation exerted protective effects against neuronal trauma via inhibition of necroptosis. Mechanistically, the HSP90α-mediated activation of RIPK3 and MLKL is involved in these effects.

Comments:

The provided research investigates the role of heat shock protein 70 (HSP70) in regulating necroptosis, a form of cell death, in the context of traumatic brain injury (TBI) and glutamate-induced neuronal injury. Here's a breakdown of the methods and results:

**Methods**:
1. **Cellular TBI Model**: Traumatic neuronal injury (TNI) and glutamate treatment were used as cellular models to simulate neuronal trauma.

2. **Necroptosis Assessment**: Necroptosis, a regulated form of necrosis characterized by membrane disruption, was assessed as a mechanism of neuronal cell death following TNI and glutamate treatment.

3. **HSP70 Regulation**: The study investigated how regulators of HSP70, specifically an activator called TRC051384 (TRC) and an inhibitor known as 2-phenylethyenesulfonamide (PES), affected necroptosis in cortical neurons.

4. **Protein Expression**: Changes in the expression of HSP70, receptor interacting protein kinase 3 (RIPK3), and mixed lineage kinase domain-like protein (MLKL) were monitored following neuronal trauma.

5. **Immunostaining and Lactate Dehydrogenase Assay**: Immunostaining techniques and a lactate dehydrogenase release assay were employed to assess necroptosis and its modulation by HSP70 regulators.

6. **HSP90α Regulation**: The study also explored the impact of HSP70 on HSP90α expression and how HSP70 regulators influenced HSP90α levels.

7. **Phosphorylation Analysis**: Phosphorylation of RIPK3 and MLKL was assessed as these proteins are key players in necroptosis signaling.

8. **Inhibitor Intervention**: The study used specific inhibitors, such as GSK-872 (a RIPK3 inhibitor) and geldanamycin (GA, an HSP90α inhibitor), to further dissect the signaling pathways involved in necroptosis regulation.

**Results**:
1. **Necroptosis in Neuronal Trauma**: Necroptosis was observed in cortical neurons following TNI and glutamate treatment, indicating that this form of cell death is associated with neuronal trauma.

2. **Upregulation of HSP70**: HSP70 protein expression was significantly increased within 24 hours of neuronal trauma, suggesting a potential protective response to cellular stress.

3. **Effect of HSP70 Regulators**: The study found that the HSP70 activator TRC inhibited necroptosis, while the HSP70 inhibitor PES promoted necroptosis in response to neuronal trauma.

4. **Differential Regulation of RIPK3 and MLKL**: HSP70 had varying effects on the expression and phosphorylation of RIPK3 and MLKL, two key regulators of necroptosis.

5. **HSP90α Involvement**: HSP90α, another heat shock protein, was also affected by HSP70 modulation. PES increased HSP90α expression, while TRC decreased it. The study demonstrated that HSP90α mediated the activation of RIPK3 and MLKL.

6. **Inhibitor Effects**: Inhibition of RIPK3 with GSK-872 and HSP90α with GA reduced the phosphorylation of RIPK3 and MLKL induced by HSP70 inhibition. GA partially prevented the increased necroptosis induced by PES, suggesting that HSP90α plays a role in necroptosis regulation.

**Conclusions**:
In summary, this research suggests that HSP70 activation exerts protective effects against necroptosis in neuronal trauma. The study proposes that the HSP90α-mediated activation of RIPK3 and MLKL is involved in these protective effects. These findings provide insights into the potential mechanisms by which HSP70 may help protect neurons from cell death following traumatic brain injury.