nNOS alpha and nNOS beta localization to aggresome-like inclusions is dependent on HSP90 activity

Nitric oxide (NO) is a highly reactive gas that participates in many physiological processes including neuroplasticity and neuronal survival. In brain neurons, NO is produced by two variants of neuronal nitric oxide synthase (nNOS), nNOSalpha and nNOSbeta. The activity of nNOSalpha is tightly regulated at the transcriptional and post-transcriptional levels. Heat shock protein 90 (HSP90) regulates nNOSalpha activity by facilitating heme insertion into the nNOSalpha monomer, resulting in increased NO production. HSP90 also regulates nNOSalpha degradation through the proteasome pathway. Here, we show in vitro that inhibition of HSP90 with geldanamycin increases nNOS mobility and induces formation of aggresome-like inclusions containing both nNOSalpha and nNOSbeta in primary cortical neurons. We also report the formation of endogenous nNOS-containing aggresome-like inclusions in healthy, untreated, mature primary cortical neurons. We propose that nNOS aggregation may be an additional mechanism for regulating nNOS activity, as has been proposed for inducible nitric oxide synthase. These findings reveal a new role for HSP90 in regulating nNOS sub-cellular localization and underscore the complexity of nNOS regulatory mechanisms.