Synaptic localization of the SUMOylation‐regulating protease SENP5 in the adult mouse brain

Abstract

Covalent conjugation of small ubiquitin-like modifiers (SUMOs) or SUMOylation is a reversible post-translational modification that regulates the stability and function of target proteins. SUMOs are removed from substrate proteins by sentrin/SUMO-specific proteases (SENPs). Numerous studies have implicated SUMOylation in various physiological and pathological processes in neurons. To understand the functional roles of SUMOylation, it is necessary to determine the distribution of enzymes regulating SUMO conjugation and deconjugation; yet, the localization of SENPs has not been described in detail in intact brain tissue. Here, we report the distribution and subcellular localization of SENP3 and 5 in the adult murine brain. Immunohistochemical analyses revealed the ubiquitous distribution of both SENPs across different brain regions. Within individual cells, SENP3 was confined to the nucleus, consistent with the conventional view that SENPs regulate nuclear events. In contrast, SENP5 was detected in the neuropil but not in cell bodies. Moreover, strong SENP5 immunoreactivity was observed in regions with high numbers of synapses such as the cerebellar glomeruli, suggesting that SENP5 localizes to pre- and/or postsynaptic structures. We performed double immunolabeling in cultured neurons and found that SENP5 co-localized with pre- and post-synaptic markers, as well as a mitochondrial marker. Immunoelectron microscopy confirmed this finding and revealed that SENP5 was localized to presynaptic terminals, postsynaptic spines, and mitochondria in axon terminals. These findings advance the current understanding of the functional roles of SUMOylation in neurons, especially in synaptic regulation, and have implications for future therapeutic strategies in neurodegenerative disorders mediated by mitochondrial dysfunction.

Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

“…Our previous immunoelectron microscopy study revealed that in the adult brain, Senp5 protein localizes to presynaptic terminals, postsynaptic spines, and mitochondria 27 , suggesting a functional relevance of Senp5S to synaptogenesis and synaptic transmission in the mature neuron. Since we confirmed the expression of the SENP5S isoform in humans (UniProt number; Q96HI0-2), which lacks His-646 and Asp-663 in the C-terminal catalytic triad 41 , it is likely that SENP5S plays an evolutionarily conserved role in the development and maintenance of the mammalian nervous system.…”

“…Because of the technical difficulty of designing effective shRNAs specific to Senp5S, we used shRNA constructs (shRNA#01 and #02) that knocked down both Senp5L and 5S (Supplementary Fig. 5a) 27 . In control embryos electroporated with scrambled shRNA, new-born turboRFP + neurons radially migrated away from the VZ toward the pial surface.…”

“…The 5′-GCACTACCAGAGCTAACTCAGATAGTACT-3′ (scrambled non-targeting control), 5′-GCAGGTGAAGCATTTCCAGTGTAATAAGG-3′ (Senp5 shRNA#1), and 5′-CTGGAGCAGACAAATCTGTGAGTAGTGTA-3′ (Senp5 shRNA#2). The shRNA #1 and shRNA #2 target both isoforms of Senp5 mRNA 27 .…”

“…Plasmid constructs containing short hairpin RNA (shRNA) cassettes in the pRFP-C-RS vector were purchased from OriGene Technologies. Plasmid-transfected cells expressed shRNA, which was controlled by the U6 polymerase III promoter and RFP fluorescent protein27 . The silencing effect of the Senp5 shRNAs was established in our previous report.…”

SUMOylation, aging and autophagy in neurodegeneration

“…Hsc70interacting protein (CHIP) facilitates the ubiquitination of SENP3 followed by its degradation under normal conditions. SENPs show variable inducibility, cellular and subcellular distribution, thereby fine-tuning SUMO status, with SENP5 showing largely synaptic expression (Mendes et al, 2016;Akiyama et al, 2018). However, it has been reported that under cell stress SENP3 levels are increased, due to its association with the molecular chaperone, heat shock protein 90 (HSP90), which protects it from CHIP-mediated ubiquitination (Guo and Henley, 2014;Yan et al, 2010).…”