Biology subjects

Insinna, C.

Publications and source records attributed to Insinna, C..

4 recordsLinked to original sources

bioRxiv2026

WDR44 drives de novo α-synuclein aggregation at the lysosomal membrane and promotes neuronal dysfunction in Parkinson's Disease

The aggregation of -synuclein (-SYN) into Lewy bodies (LBs) is a central event in the pathogenesis of Parkinsons disease (PD) and related synucleinopathies1,2. Despite significant advances in understanding -SYN self-assembly, the precise sequence of early aggregation steps has not been directly visualized in living neurons. Here, we use an optogenetic-induced protein aggregation system with a high temporal resolution to monitor the onset of -SYN assembly in neurons. We found that the initiation and accumulation of -SYN aggregates occur predominantly at the lysosomal membrane, an event driven by the -SYN N-terminus and modulated by the membrane-associated adaptor protein WD repeat-containing protein 44 (WDR44). Remarkably, we demonstrate that WDR44 knockdown markedly reduced de novo -SYN aggregation in both neuronal cultures and in vivo, whereas WDR44 overexpression enhances -SYN aggregation in PD patient-derived iPSC neurons. Consistent with its potential pathogenic involvement, WDR44 aberrantly accumulates in vivo and in the brains of PD patients, where it colocalizes with LB inclusions. Finally, we show that lysosome-associated -SYN aggregates compromised lysosomal structure and function, leading to neuronal impairment, a phenotype worsened by WDR44 overexpression, linking early aggregation events to downstream toxicity. Together, these findings reveal the earliest dynamic stages of -SYN oligomerization in living neurons and identify the WDR44--SYN interaction as a promising therapeutic target for reducing -SYN pathology and enabling early intervention in PD.

neuroscience↗

bioRxiv2021

RASopathy Mutations Demonstrate the Critical Function of the Cysteine-rich Domain in Maintaining BRAF in an Autoinhibited State

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well-characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity in zebrafish models. Further, we show that CRD-mediated autoinhibition is essential for preventing the constitutive plasma membrane localization of BRAF and increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

cell biology↗

bioRxiv2019

The Deubiquitinating Enzyme USP37 Stabilizes CHK1 to Promote the Cellular Response to Replication Stress

The deubiquitinating enzyme USP37 is known to contribute to timely S-phase onset. However, it is not clear if USP37 is essential beyond S-phase entry despite expression and activity of USP37 peaking within S-phase. Here, we determine that USP37-depleted cells exhibited altered S-phase kinetics and harbored increased levels of the replication stress and DNA damage markers {gamma}H2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase CHK1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubquitinates and stabilizes CHK1. Our data contribute to an evolving understanding of UPS37 as a multifaceted regulator of genome regulation.

cell biology↗

bioRxiv2018

Kif17 phosphorylation regulates its ciliary localization and photoreceptor outer segment turnover

BackgroundKIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown.\n\nResultsUsing transgenic expression in both mammalian cells and zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization to cilia. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding.\n\nConclusionsTaken together, our data support a model in which phosphorylation of KIF17 promotes its ciliary localization. In cone photoreceptor outer segments, this promotes disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.

cell biology↗