Investigating the Molecular Determinants of Neuroserpin as a Neuroprotective Network in the Central Nervous System

Neurodegenerative diseases, marked by the gradual loss of neurons, impair mental and physical abilities. Regardless of advances in medicine, these disorders have remained a significant concern, particularly for aging population. Alzheimer's disease (AD) is a prime example where a combination of neuroinflammation, mitochondrial dysfunction, excitotoxicity, and protein aggregation results in neurodegeneration. Consequently, neuroprotective strategies focus on blocking these events in both acute and chronic conditions. Neuroserpin (SERPINI1), a serine protease inhibitor that regulates tPA/plasmin in the nervous system, has emerged as a candidate for neuroprotection. Research from our group demonstrated the detrimental consequences of neuroserpin ablation in a mouse model of glaucoma, while its overexpression proved to be protective against injury. However, the specific mechanisms underlying these effects remain unclear. Here, we first explored the functions of neuroserpin by comparing (phospho)proteome changes between neuroserpin-deficient (Serpini1-/-) and wild type (Serpini1+/+) mice, in retina, optic nerve, frontal cortex, visual cortex and cerebellum. To achieve this, a multiple-plex quantitative approach using isobaric tandem mass tags technology was employed followed by comprehensive bioinformatic analysis. We detected around 5000 proteins in each tissue, resulting in a pool of more than 1,200 differentially expressed proteins. Functional pathway analysis unveiled region-specific changes, including visual perception, focal adhesion, apoptosis, glutamate receptor activation and supramolecular fibre organization in retina, optic nerve, frontal cortex, visual cortex and cerebellum, respectively. Phosphoproteomics analysis identified changes in approximately 800 phosphosites, mostly in the retina and the visual cortex. Bioinformatic analysis further discovered dysregulated phosphorylation of Rho/Grk1, Map2 and Nefm, which are involved in photoreceptor adaptation, cytoskeleton integrity and axonal calibre, respectively. To further understand the neuroserpin signaling in different brain regions, we analysed key proteins involved in BDNF-TrkB axis and downstream proteins between various brain regions from wild type (Serpini1+/+), neuroserpin-deficient (Serpini1-/-) and transgenic overexpression mice (Serpini1+/+Tg) using western blotting and immunofluorescence imaging. Our results showed that loss of neuroserpin resulted in downstream inactivation of Erk1/2 and Akt1/2 signaling mediated by BDNF/TrkB axis. This was followed by increased neuronal apoptotic markers, Bcl2 and Bax in a region-wise pattern. Moreover, modulation of neuroserpin impacted postsynaptic marker PSD-95 in frontal cortex of knockout mice while no changes were observed for the presynaptic marker, synaptophysin. Finally, we investigated how interacting partners of neuroserpin are impaired in AD as a neurodegenerative condition. Initially, proteomics analysis of retina, temporal cortex, hippocampus and cerebellum in AD revealed only a significant change in the expression levels of prion protein in temporal cortex. We further analysed the expression of SERPINI1 interactome via our in-house visualization tool, consisting of 7 AD brain cortex datasets. While neuroserpin itself did not exhibit a significant change in the meta-analysis, 11 out of 20 interactors showed consistent alteration across multiple datasets. These findings provide comprehensive insights into the functions of Serpini1 and how it potentially acts as a neuroprotective molecule in the nervous system. Future studies on the role of neuroserpin in other neurodegenerative diseases will expand our understanding of neuroserpin mediated neuroprotection.<p></p>