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Introduction/Objectives: Chronic mental illnesses, such as schizophrenia and major depressive disorder, affect millions of people every year, but their origins and biological basis remain unclear. We have recently proposed protein aggregation as a potential mechanism, due to the similarity of symptoms between neurodegenerative and mental illnesses. Several proteins have been implicated to aggregate in the brains of patients with mental illness, including Neuronal PAS domain protein 3 (NPAS3). NPAS3 is a transcription factor involved in the regulation of neurogenesis, metabolism, and circadian rhythms. It is implicated as aggregating in mental illness as a result of an aggregation-causing mutation, V304I, found in a family with diagnosed schizophrenia cases. New data from patient blood serum, however, suggests that NPAS3 aggregation may be more prevalent then this mutation.Participants, Materials/Methods: Plasmids encoding wild-type (WT) and mutated (V304I) NPAS3, or fragments of NPAS3, were expressed in SH-SY5Y neuroblastoma cells. Their expression was confirmed by Western blot, and their aggregation was investigated by fluorescent microscopy. The experiments were performed in normal conditions versus oxidative stress, induced by sodium arsenite treatment. Experiments were assessed quantitatively by a blinded researcher and statistically analyzed by Welch's t-test or ANOVA, as appropriate for the number of constructs used. Results: In neurodegenerative conditions such as amyotrophic lateral sclerosis, specific nuclear proteins mislocalize to the cytoplasm, where they go onto form aggregates. Both WT and V304I NPAS3 were seen principally in the nucleus, with some cytoplasmic expression, and there was a non-significant trend of V304I being seen less often in the nucleus. When cells were exposed to oxidative stress, a significant increase in mislocalization to the cytoplasm was seen. There was no significant effect of mutation status, nor the interaction between mutation and stress status. Examining fragments of NPAS3, the PAS1 domain influenced the localization, with plasmids lacking this domain having significantly more nuclear and less cytoplasmic localization. Conclusions: Based on the results of our model, we can conclude that the V304I mutation is not required for cytoplasmic localization or NPAS3 aggregation, however, it may be a contributing factor. In addition to genetic stress, the environmental stresses could affect NPAS3 cytoplasmic localization, and therefore aggregation. Furthermore, aggregation of NPAS3 may rely on the PAS1 domain in the absence of the V304I mutation. This is consistent with our data on blood serum of patients with schizophrenia, and we are now investigating NPAS3 aggregation further in postmortem brain samples. |
