Synapses connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. Synaptic function and plasticity, the neuronal process of adapting to diverse and variable inputs, depend on the dynamic nature of synaptic molecular components, which is mediated in part by cell adhesion signaling pathways. Here, we found that the enzyme biliverdin reductase (BVR) physically links together key focal adhesion signaling molecules at the synapse. BVR -null ( BVR −/− ) mice exhibited substantial deficits in learning and memory on neurocognitive tests, and hippocampal slices in which BVR was postsynaptically depleted showed deficits in electrophysiological responses to stimuli. RNA sequencing, biochemistry, and pathway analyses suggested that these deficits were mediated through the loss of focal adhesion signaling at both the transcriptional and biochemical level in the hippocampus. Independently of its catalytic function, BVR acted as a bridge between the primary focal adhesion signaling kinases FAK and Pyk2 and the effector kinase Src. Without BVR, FAK and Pyk2 did not bind to and stimulate Src, which then did not phosphorylate the N -methyl- d -aspartate (NMDA) receptor, a critical posttranslational modification for synaptic plasticity. Src itself is a molecular hub on which many signaling pathways converge to stimulate NMDAR-mediated neurotransmission, thus positioning BVR at a prominent intersection of synaptic signaling.

Aims: Several studies reported that astrocytes support neuronal communication by the release of gliotransmitters, including ATP and glutamate. Astrocytes also play a fundamental role in buffering extracellular glutamate in the synaptic cleft, thus limiting the risk of excitotoxicity in neurons. We previously demonstrated that extracellular tau oligomers (ex-oTau), by specifically targeting astrocytes, affect glutamate-dependent synaptic transmission via a reduction in gliotransmitter release. The aim of this work was to determine if ex-oTau also impair the ability of astrocytes to uptake extracellular glutamate, thus further contributing to ex-oTau-dependent neuronal dysfunction.Methods: Primary cultures of astrocytes and organotypic brain slices were exposed to ex-oTau (200 nM) for 1 h. Extracellular glutamate buffering by astrocytes was studied by: Na + imaging; electrophysiological recordings; high-performance liquid chromatography; Western blot and immunofluorescence. Experimental paradigms avoiding ex-oTau internalisation (i.e. heparin pre-treatment and amyloid precursor protein knockout astrocytes) were used to dissect intracellular vs extracellular effects of oTau.Results: Ex-oTau uploading in astrocytes significantly affected glutamate-transporter-1 expression and function, thus impinging on glutamate buffering activity. Ex-oTau also reduced Na-K-ATPase activity because of pump mislocalisation on the plasma membrane, with no significant changes in expression. This effect was independent of oTau internalisation and it caused Na + overload and membrane depolarisation in ex-oTautargeted astrocytes.Conclusions: Ex-oTau exerted a complex action on astrocytes, at both intracellular and extracellular levels. The net effect was dysregulated glutamate signalling in terms of both release and uptake that relied on reduced expression of glutamate-transporter-1, altered function and localisation of NKA1A1, and NKA1A2. Consequently, Na + gradients and all Na + -dependent transports were affected. Domenica Donatella Li Puma and Cristian Ripoli equally contributed to this work.

Engineered proteases are promising tools to address physiological and pathophysiological questions as well as to develop new therapeutic approaches. Here we introduce a new genetically encoded engineered single‐chain tobacco etch virus protease, allowing to control proprotein cleavage in different compartments of living mammalian cells. We demonstrated a set of controllable proteolytic effects, including cytosolic protein cleavage, inducible gene expression, and maturation of brain‐derived neurotrophic factor (BDNF) in the secretory pathway thus showing the versatility of this technique. Of note, the secretory pathway exhibits different characteristics from the cytosol and it is difficult to target because inaccessible to some small molecules. We were able to induce ligand‐mediated BDNF maturation and monitor its effects on dendritic spines in hippocampal pyramidal cells and in the mouse brain. This strategy paves the way to dissect proteolytic cleavage product signaling in various processes as well as for future therapeutic applications.

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [ 14 C]-tetraethylamonium or [ 3 H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MβCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MβCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [ 14 C]-tetraethylamonium or [ 3 H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

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.