Lara Ordonez, A. J. | bioRxiv Science

bioRxiv2026

The phosphorylation status of LRRK2 at the S910/S935 cluster determines its sensitivity to activation by RAB29

Leucine-Rich Repeat Kinase 2 (LRRK2) is a signaling molecule involved in Parkinsons disease pathomechanisms. In disease, the LRRK2 protein displays both a toxic gain of kinase function and a loss of phosphorylation at heterophosphosites found in an extended loop of the LRR domain. RAB GTPases, such as RAB29, have been identified as upstream activators of LRRK2. Indeed, co-expression of LRRK2 with RAB29 induces a hyperactivation of LRRK2 kinase activity, however the role of the LRRK2 heterologous phosphorylation status in its activation remains unknown. Here, our aim was to determine the role of LRRK2 heterologous phosphorylation on its activation by RAB29. Using single and compound phosphodead or phosphomimetic mutants of LRRK2 we show differential sensitivity of LRRK2 phosphomutants to activation by RAB29, with phosphodead mutants being more susceptible to be activated than phosphomimetic mutants. Interestingly, we find that the single phosphodead S910A LRRK2 mutant displays an activation of LRRK2 kinase activity similar to that observed for the compound phosphodead 6xS>A LRRK2 mutant (S860A/S910A/S935A/S955A/S973A/S976A). Time-course analysis revealed that phosphodead mutants displayed higher but also faster activation by RAB29. In addition, both physical interaction between LRRK2 and RAB29 as well as RAB29-induced recruitment of LRRK2 to the trans-Golgi network (TGN) was enhanced by phosphodead compared to phosphomimetic mutants. To confirm effects on native LRRK2, we tested a panel of ten nanobodies targeting LRRK2 that stabilized LRRK2 phosphorylation at varying levels. Nanobodies stabilizing LRRK2 at low S935 phosphorylation levels showed enhanced RAB29-induced activation compared to nanobodies not affecting pS935 LRRK2. Finally, we tested whether LRRK2 heterologous phosphorylation could affect centrosome cohesion deficits, a phenotype that has been linked to LRRK2 hyperactivation, and found that both the phosphodead LRRK2 as well as a nanobody stabilizing dephosphorylated LRRK2 enhanced the centrosome cohesion deficit. Our findings indicate that hyperactivability of LRRK2 is directly related to its heterologous phosphorylation status, with dephosphorylation leading to strong hyperactivation of LRRK2 by upstream activating RABs, and phosphorylated LRRK2 showing the opposite. This implies that strategies favoring LRRK2 phosphorylation will have therapeutic benefit.

neuroscience↗

bioRxiv2024

Maternal Immune Activation imprints translational dysregulation and differential MAP2 phosphorylation in descendant neural stem cells

Alterations induced by maternal immune activation (MIA) during gestation impact the subsequent neurodevelopment of progeny, a process that in humans, has been linked to the development of several neuropsychiatric conditions. To undertake a comprehensive examination of the molecular mechanisms governing MIA, we have devised an in vitro model based on neural stem cells (NSCs) sourced from fetuses carried by animals subjected to Poly I:C treatment. These neural progenitors demonstrate proliferative capacity and can be effectively differentiated into both neurons and glial cells. Transcriptomic, proteomic, and phosphoproteomic analyses conducted on these cellular models, in conjunction with counterparts from control treatments, revealed discernible shifts in the expression levels of a specific subset of proteins implicated in neuronal function. Noteworthy, we found an absence of congruence between these alterations at the transcriptomic level, suggesting that differences in protein translation contribute to the observed dysregulation. Furthermore, the phosphoproteomic data highlighted a discernible discrepancy in the basal phosphorylation of proteins between differentiated cells from both experimental groups, particularly within proteins associated with cytoskeletal architecture and synaptic functionality, notably those belonging to the MAP family. Observed alterations in MAP phosphorylation were found to potentially have functional consequences as they correlate with changes in neuronal plasticity and the establishment of neuronal synapses. Our data agrees with previous published observations and further underscore the importance of MAP2 phosphorylation state on its function and the impact that this protein has in neuronal structure and function.

neuroscience↗