Maanasa Cherukuvada

Background: An ischemic stroke is a lack of blood flow due to occlusion of cerebral blood vessels, causing a core area of irreversible injury, and a surrounding area of potential damage, termed the penumbra.¹ Neurological function scales, non-contrast CT and CT-perfusion are performed to identify the nature and location of the stroke.² A thrombolytic agent, tPA, is the preferred treatment, but there is a narrow time window to utilize the treatment, driving the need for alternative neuroprotective treatments.^1,2 One important process after an ischemic stroke is excitotoxicity. There is an increase in Na⁺ and Ca²⁺ channel signaling coupled with NMDARs, excitatory receptors in the brain. This is associated with neuronal cell death. An important molecule, postsynaptic density protein-95 (PSD-95), co-localizes with NMDARs, and has been shown to play an important role in hippocampal neuron survival, synaptic maturation, and limiting reciprocal receptor potentiation between the D1 dopamine receptor and NMDARs.^3,4

Objective: During an ischemic stroke, PSD-95 is cleaved by the Ca²⁺ dependent protease, calpain, due to the increased calcium in excitotoxicity, presenting a clinical opportunity for intervention, specifically using a cell-penetrating peptide model (CPP).⁵ This review aims to understand the complexities of the calpain molecule and present a potential therapeutic CPP strategy.

Search Methods: An online search in the PubMed database was conducted from 2018 to 2025 with the following key words: “PSD-95”, “calpain”, “excitotoxicity”, and “cell-penetrating peptide.”

Results: Calpain upregulation plays a role in inflammatory pathways after an ischemic stroke. It was shown to act on the PARP-NF-kB, an inflammatory pathway, and application of MDL, a calpain inhibitor, showed a decrease in inflammatory cytokines. Calpain inhibition also resulted in preservation of the neurovasculature unit, with less astrocyte swelling, and perivascular edema.⁶ Calpain upregulation is also involved in lysosomal disruption, which leads to downstream apoptotic processes. With calpain knock-out, there is increased co-localization of lysosomal proteins LAMP1 and CTSB. There is also autophagic disruption, an important neuroprotective mechanism. This was shown through increased LC3II/LC3-I ratio with calpain inhibition, indicative of increased autophagic function.⁷ Calpain inhibition preserved tight junction protein levels, such as ZO-1 and occludin, indicating the role of calpain in disruption of tight junction proteins.⁸ Apart from these roles of calpain, it can also cleave PSD-95. With application of lithium as a neuroprotective treatment, PSD-95 levels were shown to increase, indicating a protective function.⁹ Calpain cleavage of PSD-95 can be targeted using a CPP, which has an enhanced ability to cross the blood brain barrier. TP95₄₁₄ is a CPP targeting calpain cleavage of the PDZ-SH3 linkage by acting as a potential mock substrate or preventing phosphorylation required for cleavage, and it was shown to decrease infarct size, and increase PSD-95 full length levels, without statistical significance. However, it showed statistically significant reduction of motor symptoms in the mouse model through a balance beam test.⁵

Conclusion: Calpain-I plays a significant role in ischemic stroke due to excitotoxic conditions. Calpain processing of PSD-95 can potentially be targeted with the cell penetrating peptide TP95_414. The cell penetrating peptide was able to successfully cross the blood brain barrier on the animal model, and it provided some reduction of infarct size in a severe mouse model of ischemia. Furthermore, even though the reduction in PSD-95 levels was present but not significant, there was already a significant reduction in motor deficits.⁵

Works Cited

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9. Tai SH, Huang SY, Chao LC, et al. Lithium upregulates growth-associated protein-43 (GAP-43) and postsynaptic density-95 (PSD-95) in cultured neurons exposed to oxygen-glucose deprivation and improves electrophysiological outcomes in rats subjected to transient focal cerebral ischemia following a long-term recovery period. Neurol Res. 2022;44(10):870-878. doi:10.1080/01616412.2022.205681