Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells

Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including Bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain non-neuronal proteins LL5β and KANK1, which in migrating cells organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. While previous analyses in vitro and in neurons suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

Recent advances in CRISPR/Cas9-mediated knock-in methods enable labeling of individual endogenous proteins with fluorophores, to determine their spatiotemporal expression in intact biological preparations. However, multiplex knock-in methods remain limited, particularly in postmitotic cells, due to a high degree of crosstalk between genome editing events. We present Conditional Activation of Knock-in Expression (CAKE), which delivers efficient, flexible and accurate multiplex genome editing in neurons. CAKE is based on sequential gRNA expression operated by a Cre- or Flp-recombinase to control the time window for genomic integration of each donor sequence, which diminishes crosstalk between genome editing events. Importantly, CAKE is compatible with multiple CRISPR/Cas9 strategies, and we show the utilization of CAKE for co-localization of various endogenous proteins, including synaptic scaffolds, ion channels and neurotransmitter receptor subunits. Knock-in efficacy was highly sensitive to DNA vector amount, while knock-in crosstalk was dependent on the rate of donor DNA integration and timing of Cre activation. We applied CAKE to study the co-distribution of endogenous synaptic proteins using dual-color single-molecule localization microscopy, and we introduced dimerization modules to acutely control synaptic receptor dynamics in living neurons. Taken together, CAKE is a versatile method for multiplex protein labeling, enabling accurate detection, precise localization and acute manipulation of endogenous proteins in single cells.

Pathophysiological interplay between O-GlcNAc transferase and the Machado–Joseph disease protein ataxin-3

Aberrant O-GlcNAcylation, a protein posttranslational modification defined by the O-linked attachment of the monosaccharide N-acetylglucosamine (O-GlcNAc), has been implicated in neurodegenerative diseases. However, although many neuronal proteins are substrates for O-GlcNAcylation, this process has not been extensively investigated in polyglutamine disorders. We aimed to evaluate the enzyme O-GlcNAc transferase (OGT), which attaches O-GlcNAc to target proteins, in Machado–Joseph disease (MJD). MJD is a neurodegenerative condition characterized by ataxia and caused by the expansion of a polyglutamine stretch within the deubiquitinase ataxin-3, which then present increased propensity to aggregate. By analyzing MJD cell and animal models, we provide evidence that OGT is dysregulated in MJD, therefore compromising the O-GlcNAc cycle. Moreover, we demonstrate that wild-type ataxin-3 modulates OGT protein levels in a proteasome-dependent manner, and we present OGT as a substrate for ataxin-3. Targeting OGT levels and activity reduced ataxin-3 aggregates, improved protein clearance and cell viability, and alleviated motor impairment reminiscent of ataxia of MJD patients in zebrafish model of the disease. Taken together, our results point to a direct interaction between OGT and ataxin-3 in health and disease and propose the O-GlcNAc cycle as a promising target for the development of therapeutics in the yet incurable MJD.

PDZ-Containing Proteins Targeted by the ACE2 Receptor

Angiotensin-converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike (S) protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residue segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein-interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high-throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants’ values ranging from 3 to 81 μM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role in ACE2 internalization and recycling that could be of benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in the neurological symptoms of COVID-19.

The role of dendritic cells and their interactions in the pathogenesis of antibody-associated autoimmune encephalitis

Autoimmune encephalitis (AE) is an inflammatory brain disease which is frequently associated with antibodies (Abs) against cell-surface, synaptic or intracellular neuronal proteins. There is increasing evidence that dendritic cells (DCs) are implicated as key modulators in keeping the balance between immune response and tolerance in the CNS. Migratory features of DCs to and from the brain are linked to initiating and maintaining of neuroinflammation. Genetic polymorphisms together with other triggers such as systemic or cerebral viral infection, or systemic malignancies could contribute to the dysbalance of “regulatory” and “encephalitogenic” DCs with subsequent dysregulated T and B cell reactions in AE. Novel in vivo models with implantation of mature DCs containing neuronal antigens could help to study the pathogenesis and perhaps to understand the origin of AE. Investigations of DCs in human blood, lymphoid tissues, CSF, and brain parenchyma of patients with AE are necessary to deepen our knowledge about the complex interactions between DCs, T and B cells during neuroinflammation in AE. This can support developing new therapy strategies.

Paraneoplastic and Other Autoimmune Neurologic Disorders

A paraneoplastic disorder arises from remote effects of malignancy, rather than direct tumor invasion. Paraneoplastic neurologic disorders occur because of the vigorous immune responses directed against antigens expressed in tumors. The vigor of the immune response usually ensures that neoplasm is confined to the primary organ and regional lymph nodes. However, in the clinical setting of paraneoplastic disease, the immune response may attack any part of the nervous system (brain, spinal cord, nerve, neuromuscular junction, or muscle), and antibodies are either directed at intracellular neuronal proteins or against neuronal cell surface or synaptic proteins. Treatment is often aimed at the cancer and suppression of the abnormal immune response.

A sticky situation: regulation and function of protein palmitoylation with a spotlight on the axon and axon initial segment

In neurons, the axon and axon initial segment (AIS) are critical structures for action potential initiation and propagation. Their formation and function rely on tight compartmentalisation, a process where specific proteins are trafficked to and retained at distinct subcellular locations. One mechanism which regulates protein trafficking and association with lipid membranes is the modification of protein cysteine residues with the 16-carbon palmitic acid, known as S-acylation or palmitoylation. Palmitoylation, akin to phosphorylation, is reversible, with palmitate cycling being mediated by substrate-specific enzymes. Palmitoylation is well-known to be highly prevalent among neuronal proteins and is well studied in the context of the synapse. Comparatively, how palmitoylation regulates trafficking and clustering of axonal and AIS proteins remains less understood. This review provides an overview of the current understanding of the biochemical regulation of palmitoylation, its involvement in various neurological diseases, and the most up-to-date perspective on axonal palmitoylation. Through a palmitoylation analysis of the AIS proteome, we also report that an overwhelming proportion of AIS proteins are likely palmitoylated. Overall, our review and analysis confirm a central role for palmitoylation in the formation and function of the axon and AIS and provide a resource for further exploration of palmitoylation-dependent protein targeting to and function at the AIS.

HuD Regulates mRNA-circRNA-miRNA Networks in the Mouse Striatum Linked to Neuronal Development and Drug Addiction

The RNA-binding protein HuD (a.k.a., ELAVL4) is involved in neuronal development and synaptic plasticity mechanisms, including addiction-related processes such as cocaine conditioned-place preference (CPP) and food reward. The most studied function of this protein is mRNA stabilization; however, we have recently shown that HuD also regulates the levels of circular RNAs (circRNAs) in neurons. To examine the role of HuD in the control of coding and non-coding RNA networks associated with substance use, we identified sets of differentially expressed mRNAs, circRNAs and miRNAs in the striatum of HuD knockout (KO) mice. Our findings indicate that significantly downregulated mRNAs are enriched in biological pathways related to cell morphology and behavior. Furthermore, deletion of HuD altered the levels of 15 miRNAs associated with drug seeking. Using these sets of data, we predicted that a large number of upregulated miRNAs form competing endogenous RNA (ceRNA) networks with circRNAs and mRNAs associated with the neuronal development and synaptic plasticity proteins LSAMP and MARK3. Additionally, several downregulated miRNAs form ceRNA networks with mRNAs and circRNAs from MEF2D, PIK3R3, PTRPM and other neuronal proteins. Together, our results indicate that HuD regulates ceRNA networks controlling the levels of mRNAs associated with neuronal differentiation and synaptic physiology.

Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells.

Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including Bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain non-neuronal proteins LL5β and KANK1, which in migrating cells organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. While previous analyses in vitro and in neurons suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

PDZ-containing proteins targeted by the ACE2 receptor

Angiotensin converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residues segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants values ranging from 3 to 81 μM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role on ACE2 internalization and recycling that could benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in neurological symptoms of COVID-19.