Periodic Reporting for period 1 - NeuProHRI (NeuroProtective role of HRI following cerebral ischemia)
Reporting period: 2023-06-01 to 2025-05-31
Nowadays, cerebrovascular diseases are devastating conditions representing a large cause of mortality worldwide. It is estimated that 1.1 million people suffer a stroke every year in Europe. Due to the lack of a reliable treatment for stroke, it is important to keep looking for new treatments that stop the progression of the insult by providing neuroprotection, and even promote neuronal recovery for those neurons already affected by the condition. The goal of NeuProHRI is to study the potential of targeting the HRI kinase cellular cascade as a new therapeutic aim for stroke. To develop this, a combination of cutting-edge molecular techniques will be used to define the cellular pathways underlying the activation of the HRI kinase, and their effect on promoting neuroprotection in in vitro and in vivo models of stroke. First, molecular cascade will be deciphered following the activation of HRI in primary cultured neurons by pharmacological and genetic approaches. Then, in vitro models of stroke will be used to measure the impact of these treatments in cells with stroke-like phenotype. Finally, mouse models of stroke will allow to tackle the impact of pharmaceutical and genetic treatments on in vivo brain tissue.
Considering the aforementioned background, the overall objective of NeuProHRI is to deciphering the cellular pathway underlying HRI-induced eIF2α phosphorylation in healthy and stressed cells after ischemia; as well as its in vivo role as therapeutic target in mouse models of IS. Therefore, 3 specific objectives are proposed:
1. Identifying all players participating in the downstream signaling following HRI activation.
2. HRI as a potential therapeutic target for IS.
3. Determining the impact of HRI-induced eIF2α phosphorylation on mRNA translation in vivo after IS.
Considering the aforementioned background, the overall objective of NeuProHRI is to deciphering the cellular pathway underlying HRI-induced eIF2α phosphorylation in healthy and stressed cells after ischemia; as well as its in vivo role as therapeutic target in mouse models of IS. Therefore, 3 specific objectives are proposed:
1. Identifying all players participating in the downstream signaling following HRI activation.
2. HRI as a potential therapeutic target for IS.
3. Determining the impact of HRI-induced eIF2α phosphorylation on mRNA translation in vivo after IS.
In vitro experiments:
We use different cell cultures to determine the best dose of the HRI activator promoting the highest degree of cell viability in all cell types. These positive results were reversed when we treated cells with an HRI inhibitor. Further analysis of genetic expression confirmed these pharmacological results.
In vivo experiments:
We used two different animal models of cerebral ischemia. Mice suffering from cerebral ischemia were treated with the HRI activator at different time points after injury. Overall, the in vivo application of the HRI activator seems to promote neuroprotection and improve the behavioral outcome after cerebral ischemia.
We use different cell cultures to determine the best dose of the HRI activator promoting the highest degree of cell viability in all cell types. These positive results were reversed when we treated cells with an HRI inhibitor. Further analysis of genetic expression confirmed these pharmacological results.
In vivo experiments:
We used two different animal models of cerebral ischemia. Mice suffering from cerebral ischemia were treated with the HRI activator at different time points after injury. Overall, the in vivo application of the HRI activator seems to promote neuroprotection and improve the behavioral outcome after cerebral ischemia.
NeuProHRI project is the first study ever to address the novel application of eIF2α-mediated signaling as a neuroprotective pathway following ischemic stroke. Many compounds have been successfully developed as activators of UPR, such as PERK activators and inhibitors of eIF2α dephosphorylation. However, these approaches have caveats due to the irreversible inhibition of eIF2α dephosphorylation, and the multifactor and unspecific activation by PERK. NeuProHRI project proposes a new approach that would improve the previous ones, based on the facts that eIF2α is the sole substrate of HRI kinase, and BTdCPU is a specific and reversible activator of HRI kinase without causing toxicity in primary cultured neurons.
Overall, in vivo application of BTdCPU results in decreased lesion volumes in two different animal models of cerebral ischemia, targeting the HRI kinase as a promising target to promote neuroprotection following cerebral ischemia. Importantly, our experiments prove the necessity of the HRI kinase in the cellular response following cerebral ischemia, as inhibiting this kinase exacerbates the damage acutely. Of note, results from NeuProHRI can be also applied to other cellular stress-related diseases such as hemorrhagic stroke and Alzheimer´s disease (AD).
The results of the project were patented. Specifically, an EP24383003 patent application protecting BTdCPU, or pharmaceutical composition comprising thereof, for use in a method for the treatment of a neurological disorder or for use as a neuroprotective agent.
Overall, in vivo application of BTdCPU results in decreased lesion volumes in two different animal models of cerebral ischemia, targeting the HRI kinase as a promising target to promote neuroprotection following cerebral ischemia. Importantly, our experiments prove the necessity of the HRI kinase in the cellular response following cerebral ischemia, as inhibiting this kinase exacerbates the damage acutely. Of note, results from NeuProHRI can be also applied to other cellular stress-related diseases such as hemorrhagic stroke and Alzheimer´s disease (AD).
The results of the project were patented. Specifically, an EP24383003 patent application protecting BTdCPU, or pharmaceutical composition comprising thereof, for use in a method for the treatment of a neurological disorder or for use as a neuroprotective agent.