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

Neural stem cell properties and adult hippocampal neurogenesis in a knock-in mice model, expressing an autism-associated mutation.

Alteration of adult neurogenesis has been associated with neuropsychiatric disorders, including autism spectrum disorders (ASDs). Particularly, in the hippocampus of few ASD mice models, the properties of adult neural stem/progenitor cells (aNSPC) pool and the formation of new neurons have been found altered, suggesting a link between deregulated neurogenesis and some of the behavioural deficits found in these mice. In order to investigate neurogenesis in association to ASDs, we have been using the R451C Neuroligin3 (NLG3) knock-in mice, a model of a monogenic form of ASDs carrying the R451C substitution found in autistic patients. NLG3 is a postsynaptic protein involved in maturation, specification and plasticity of neural networks and the R451C knock-in mice display excitatory/inhibitory balance alterations in different brain regions, behavioural deficits, and structural brain abnormalities. We focused our study on the subgranular zone of the hippocampal dentate gyrus (DG), a neurogenic niche of the adult brain. Specifically, we compared proliferation and differentiation of new neurons between knock-in and wild-type mice, both in vivo and in vitro. In vitro data demonstrate that NSPC cultures derived from the DG of two-month-old knock-in mice contained a higher number of cells compared to the wild-type. However, BrdU cell number in the DGs was unchanged between KI and WT mice. In vivo data also show a decrease in the number of newly formed differentiated mature neurons (BrdU-NeuN double positive) in the hippocampus of the knock-in compared to wild-type mice. The mechanisms underlying the neurogenesis reduction in the knock-in mice are currently under investigation

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-β 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