The Multitasking Potential of Alarmins and Atypical Chemokines

When the human genome was sequenced, it came as a surprise that it contains “only” 21,306 protein-coding genes. However, complexity and diversity are multiplied by alternative splicing, non-protein-coding transcripts, or post-translational modifications (PTMs) on proteome level. Here, we discuss how the multi-tasking potential of proteins can substantially enhance the complexity of the proteome further, while at the same time offering mechanisms for the fine-regulation of cell responses. Discoveries over the past two decades have led to the identification of “surprising” and previously unrecognized functionalities of long known cytokines, inflammatory mediators, and intracellular proteins that have established novel molecular networks in physiology, inflammation, and cardiovascular disease. In this mini-review, we focus on alarmins and atypical chemokines such as high-mobility group box protein-1 (HMGB-1) and macrophage migration-inhibitory factor (MIF)-type proteins that are prototypical examples of these classes, featuring a remarkable multitasking potential that allows for an elaborate fine-tuning of molecular networks in the extra- and intracellular space that may eventually give rise to novel “task”-based precision medicine intervention strategies

High fidelity hypothermic preservation of primary tissues in organ transplant preservative for single cell transcriptome analysis

Background: High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards. Results: Here we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4 days (6 h, 1, 2, 3, and 4 days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3 days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution. Conclusions: Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis

Autism-Associated Neuroligin-3 Mutations Commonly Impair Striatal Circuits to Boost Repetitive Behaviors

In humans, neuroligin-3 mutations are associated with autism, while in mice the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum, but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse, and thereby provide a plausible circuit substrate for autism pathophysiology

Autism-Associated Neuroligin-3 Mutations Commonly Impair Striatal Circuits to Boost Repetitive Behaviors

In humans, neuroligin-3 mutations are associated with autism, while in mice the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum, but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse, and thereby provide a plausible circuit substrate for autism pathophysiology

ADAM10 and ADAM17 promote SARS‐CoV‐2 cell entry and spike protein‐mediated lung cell fusion

The severe‐acute‐respiratory‐syndrome‐coronavirus‐2 (SARS‐CoV‐2) is the causative agent of COVID‐19, but host cell factors contributing to COVID‐19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS‐CoV‐2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID‐19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS‐CoV‐2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease‐targeted inhibitors severely impair lung cell infection by the SARS‐CoV‐2 variants of concern alpha, beta, delta, and omicron and also reduce SARS‐CoV‐2 infection of primary human lung cells in a TMPRSS2 protease‐independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development

A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntington's disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD

Decreased Striatal RGS2 Expression Is Neuroprotective in Huntington's Disease (HD) and Exemplifies a Compensatory Aspect of HD-Induced Gene Regulation

The molecular phenotype of Huntington's disease (HD) is known to comprise highly reproducible changes in gene expression involving striatal signaling genes. Here we test whether individual changes in striatal gene expression are capable of mitigating HD-related neurotoxicity.We used protein-encoding and shRNA-expressing lentiviral vectors to evaluate the effects of RGS2, RASD2, STEP and NNAT downregulation in HD. Of these four genes, only RGS2 and RASD2 modified mutant htt fragment toxicity in cultured rat primary striatal neurons. In both cases, disease modulation was in the opposite of the predicted direction: whereas decreased expression of RGS2 and RASD2 was associated with the HD condition, restoring expression enhanced degeneration of striatal cells. Conversely, silencing of RGS2 or RASD2 enhanced disease-related changes in gene expression and resulted in significant neuroprotection. These results indicate that RGS2 and RASD2 downregulation comprises a compensatory response that allows neurons to better tolerate huntingtin toxicity. Assessment of the possible mechanism of RGS2-mediated neuroprotection showed that RGS2 downregulation enhanced ERK activation. These results establish a novel link between the inhibition of RGS2 and neuroprotective modulation of ERK activity.Our findings both identify RGS2 downregulation as a novel compensatory response in HD neurons and suggest that RGS2 inhibition might be considered as an innovative target for neuroprotective drug development

Short-Term Striatal Gene Expression Responses to Brain-Derived Neurotrophic Factor Are Dependent on MEK and ERK Activation

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is believed to be an important regulator of striatal neuron survival, differentiation, and plasticity. Moreover, reduction of BDNF delivery to the striatum has been implicated in the pathophysiology of Huntington's disease. Nevertheless, many essential aspects of BDNF responses in striatal neurons remain to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we assessed the relative contributions of multipartite intracellular signaling pathways to the short-term induction of striatal gene expression by BDNF. To identify genes regulated by BDNF in these GABAergic cells, we first used DNA microarrays to quantify their transcriptomic responses following 3 h of BDNF exposure. The signal transduction pathways underlying gene induction were subsequently dissected using pharmacological agents and quantitative real-time PCR. Gene expression responses to BDNF were abolished by inhibitors of TrkB (K252a) and calcium (chelator BAPTA-AM and transient receptor potential cation channel [TRPC] antagonist SKF-96365). Interestingly, inhibitors of mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) and extracellular signal-regulated kinase ERK also blocked the BDNF-mediated induction of all tested BDNF-responsive genes. In contrast, inhibitors of nitric oxide synthase (NOS), phosphotidylinositol-3-kinase (PI3K), and CAMK exhibited less prevalent, gene-specific effects on BDNF-induced RNA expression. At the nuclear level, the activation of both Elk-1 and CREB showed MEK dependence. Importantly, MEK-dependent activation of transcription was shown to be required for BDNF-induced striatal neurite outgrowth, providing evidence for its contribution to striatal neuron plasticity. CONCLUSIONS: These results show that the MEK/ERK pathway is a major mediator of neuronal plasticity and other important BDNF-dependent striatal functions that are fulfilled through the positive regulation of gene expression