Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation.

Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein.

Engineering memory with an extrinsically disordered kinase.

Synaptic plasticity plays a crucial role in memory formation by regulating the communication between neurons. Although actin polymerization has been linked to synaptic plasticity and dendritic spine stability, the causal link between actin polymerization and memory encoding has not been identified yet. It is not clear whether actin polymerization and structural changes in dendritic spines are a driver or a consequence of learning and memory. Using an extrinsically disordered form of the protein kinase LIMK1, which rapidly and precisely acts on ADF/cofilin, a direct modifier of actin, we induced long-term enlargement of dendritic spines and enhancement of synaptic transmission in the hippocampus on command. The activation of extrinsically disordered LIMK1 in vivo improved memory encoding and slowed cognitive decline in aged mice exhibiting reduced cofilin phosphorylation. The engineered memory by an extrinsically disordered LIMK1 supports a direct causal link between actin-mediated synaptic transmission and memory.

A Bioengineering Strategy to Control ADAM10 Activity in Living Cells.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer's disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-ß precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.

Neural Stem Cell-Derived Extracellular Vesicles Counteract Insulin Resistance-Induced Senescence of Neurogenic Niche.

Neural stem and progenitor cell (NSPC) depletion may play a crucial role in the cognitive impairment observed in many age-related non-communicable diseases. Insulin resistance affects brain functions through a plethora of mechanisms that remain poorly understood. In an experimental model of insulin resistant NSPCs, we identified a novel molecular circuit relying on insulin receptor substrate-1 (IRS-1)/ Forkhead box O (FoxO) signaling cascade and inhibiting the recruitment of transcription factors FoxO1 and FoxO3a on the promoters of genes regulating proliferation and self-renewal. Insulin resistance also epigenetically increased the expression of cyclin-dependent kinase inhibitor 1 (p21) and accelerated NSPC senescence. Of note, we found that stimulation of NSPCs with NSPC-derived exosomes (exo-NSPC) rescued IRS-1/FoxO activation and counteracted both the reduced proliferation and senescence of stem cells. Accordingly, intranasal administration of exo-NSPC counteracted the high-fat diet-dependent impairment of adult hippocampal neurogenesis in mice by restoring the balance between proliferating and senescent NSPCs in the hippocampus. Our findings suggest a novel mechanism underlying the metabolic control of NSPC fate potentially involved in the detrimental effects of metabolic disorders on brain plasticity. In addition, our data highlight the role of extracellular vesicle-mediated signals in the regulation of cell fate within the adult neurogenic niche.