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Background Dysregulation of microRNA (miRNA) expression in the brain is a common feature of neurodegenerative diseases. Beyond their conventional role in regulating gene expression at the post-transcriptional level, certain miRNAs can act extracellularly as signaling molecules. Our study elucidates the identity of such miRNA species serving as ligands for membrane receptors expressed in central nervous system (CNS) neurons and the impact of such miRNAs on neurons in the context of neurodegenerative disease. Methods We combined a machine learning approach with the analysis of disease-associated miRNA databases to predict Alzheimer’s disease (AD)-associated miRNAs as potential signaling molecules for single-stranded RNA-sensing Toll-like receptors (TLRs) 7 and 8. TLR-expressing HEK-Blue reporter cells, primary murine microglia, and human THP-1 macrophages were used to validate the AD miRNAs as ligands for human and mouse TLR7 and/or TLR8. Interaction between mouse cortical neurons and extracellularly applied AD miRNAs was analyzed by live cell imaging and confocal microscopy. Transcriptome changes in cortical neurons exposed to AD miRNAs were assessed by RNAseq and RT-qPCR. The extracellular AD miRNAs’ effects on CNS neuron structure were investigated in cell cultures of murine primary cortical neurons and iPSC-derived human cortical neurons by immunocytochemistry. We employed a mouse model of intrathecal injection to assess effects of AD miRNAs acting as signaling molecules on neurons in vivo. Results We identified the AD-associated miRNAs miR-124-5p, miR-92a-1-5p, miR-9-5p, and miR-501-3p as novel endogenous ligands for TLR7 and/or TLR8. These miRNAs being extracellularly stable and active were taken up by murine cortical neurons via endocytosis and induced changes in neuronal inflammation-, proliferation-, and apoptosis-related gene expression. Exposure of both murine and human cortical neurons to the AD-associated miRNAs led to alterations of dendrite and axon structure, synapse protein expression, and cell viability in a sequence-dependent fashion. Extracellular introduction of the AD miRNAs into the cerebrospinal fluid of mice resulted in both changes in neuronal structure and synapses, and neuronal loss in the cerebral cortex. Most of the observed extracellular miRNA-induced effects on cortical neurons involved TLR7/8 signaling. Conclusion Neurodegenerative disease-associated miRNAs in extracellular form act as signaling molecules for CNS neurons including human cortical neurons, thereby modulating their structure and viability.

N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) and long-term depression (LTD) of signal transmission form neural circuits and thus are thought to underlie learning and memory. These mechanisms are mediated by AMPA receptor (AMPAR) trafficking in postsynaptic neurons. However, the regulatory mechanism of bidirectional plasticity at excitatory synapses remains unclear. We present a network model of AMPAR trafficking for adult hippocampal pyramidal neurons, which reproduces both LTP and LTD. We show that the induction of both LTP and LTD is regulated by the competition between exocytosis and endocytosis of AMPARs, which are mediated by the calcium-sensors synaptotagmin 1/7 (Syt1/7) and protein interacting with C-kinase 1 (PICK1), respectively. Our result indicates that recycling endosomes containing AMPAR are always ready for Syt1/7-dependent exocytosis of AMPAR at peri-synaptic/synaptic membranes. This is because molecular motor myosin V b constitutively transports the recycling endosome toward the membrane in a Ca 2+ -independent manner.

Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease worldwide, characterized by progressive neuron degeneration or loss due to excessive accumulation of β-amyloid (Aβ) peptides, formation of neurofibrillary tangles (NFTs), and hyperphosphorylated tau. The treatment of AD has been only partially successful as the majority of the pharmacotherapies on the market may alleviate some of the symptoms. In the occurrence of AD, increasing attention has been paid to neurodegeneration, while the resident glial cells, like microglia are also observed. Microglia, a kind of crucial glial cells associated with the innate immune response, functions as double-edge sword role in CNS. They exert a beneficial or detrimental influence on the adjacent neurons through secretion of both pro-inflammatory cytokines as well as neurotrophic factors. In addition, their endocytosis of debris and toxic protein like Aβ and tau ensures homeostasis of the neuronal microenvironment. In this review, we will systematically summarize recent research regarding the roles of microglia in AD pathology and latest microglia-associated therapeutic targets mainly including pro-inflammatory genes, anti-inflammatory genes and phagocytosis at length, some of which are contradictory and controversial and warrant to further be investigated.

Folate supplementation reduces the occurrence of neural tube defects (NTDs), birth defects consisting in the failure of the neural tube to form and close. The mechanisms underlying NTDs and their prevention by folate remain unclear. Here we show that folate receptor 1 (FOLR1) is necessary for the formation of neural tube-like structures in human-cell derived neural organoids. FOLR1 knockdown in neural organoids and in Xenopus laevis embryos leads to NTDs that are rescued by pteroate, a folate precursor that is unable to participate in metabolism. We demonstrate that FOLR1 interacts with and opposes the function of CD2-associated protein, molecule essential for apical endocytosis and turnover of C-cadherin in neural plate cells. In addition, folates increase Ca 2+ transient frequency, suggesting that folate and FOLR1 signal intracellularly to regulate neural plate folding. This study identifies a mechanism of action of folate distinct from its vitamin function during neural tube formation. Neural tube defects are common birth defects that have been shown to be reduced through periconceptional folate supplementation, though the mechanism for this effect is unclear. Here they show that FOLR1 is necessary for neural tube formation in human neural organoids and Xenopus laevis embryos, and that this role is independent of its folate transport function.

Quantitative abnormalities of the von Willebrand factor-factor VIII (VWF-FVIII) complex associate with inherited bleeding or thrombotic disorders. Receptor-mediated interactions between plasma VWF-FVIII and phagocytic or immune cells can influence their hemostatic and immunogenic activities. Genetic association studies have demonstrated that variants in the STAB2 gene, which encodes the scavenger receptor stabilin-2, associate with plasma levels of VWF-FVIII. However, the mechanistic basis and pathophysiological consequences of this association are unknown. We have demonstrated that stabilin-2-expressing cells bind and internalize human VWF and FVIII in a VWF-dependent manner, and stabilin-2-deficient mice displayed prolonged human VWF-FVIII half-life compared with controls. The stabilin-2 variant p.E2377K significantly decreased stabilin-2 expression and impaired VWF endocytosis in a heterologous expression system, and common STAB2 variants associated with plasma VWF levels in type 1 von Willebrand disease patients. STAB2-deficient mice displayed a decreased immunogenic response to human VWF-FVIII complex, while coinfusion of human VWF-FVIII with the stabilin-2 ligand hyaluronic acid attenuated the immune response to exogenous FVIII. Collectively, these data suggest that stabilin-2 functions as both a clearance and an immunoregulatory receptor for VWF-FVIII, making stabilin-2 a novel molecular target for modification of the half-life of VWF-FVIII and the immune response to VWF-FVIII concentrates.

In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young's modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.

SARS-CoV-2 infection primarily targets the lung but may also damage other organs, including the brain, heart, kidney, and intestine. Central nervous system (CNS) pathologies include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Coronavirus disease 2019 (COVID-19) is frequently associated with neurological deficits, but how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces these effects remains unclear. Here, we show that astrocytes are readily infected by SARS-CoV-2, but surprisingly, neuropilin-1, not angiotensin-converting enzyme 2 (ACE2), serves as the principal receptor mediating cell entry. Infection is further positively modulated by the two-pore segment channel 2 (TPC2) protein that regulates membrane trafficking and endocytosis. Astrocyte infection produces a pathological response closely resembling reactive astrogliosis characterized by elevated type I interferon (IFN) production, increased inflammation, and the decreased expression of transporters of water, ions, choline, and neurotransmitters. These combined events initiated within astrocytes produce a hostile microenvironment that promotes the dysfunction and death of uninfected bystander neurons. IMPORTANCE SARS-CoV-2 infection primarily targets the lung but may also damage other organs, including the brain, heart, kidney, and intestine. Central nervous system (CNS) pathologies include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection, but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Here, we used induced pluripotent stem cell (iPSC)-derived brain organoids and primary human astrocytes from the cerebral cortex to study direct SARS-CoV-2 infection. Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. The deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

A strong genetic predisposition (60–80% of attributable risk) is present in Alzheimer’s disease (AD). In view of this major genetic component, identification of the genetic risk factors has been a major objective in the AD field with the ultimate aim to better understand the pathological processes. In this review, we present how the genetic risk factors are involved in APP metabolism, β-amyloid peptide production, degradation, aggregation and toxicity, innate immunity, and Tau toxicity. In addition, on the basis of the new genetic landscape, resulting from the recent high-throughput genomic approaches and emerging neurobiological information, we propose an over-arching model in which the focal adhesion pathway and the related cell signalling are key elements in AD pathogenesis. The core of the focal adhesion pathway links the physiological functions of amyloid precursor protein and Tau with the pathophysiological processes they are involved in. This model includes several entry points, fitting with the different origins for the disease, and supports the notion that dysregulation of synaptic plasticity is a central node in AD. Notably, our interpretation of the latest data from genome wide association studies complements other hypotheses already developed in the AD field, i.e., amyloid cascade, cellular phase or propagation hypotheses. Genetically driven synaptic failure hypothesis will need to be further tested experimentally within the general AD framework.

At postsynaptic sites of neurons, a prominent clathrin-coated structure, the endocytic zone (EZ), controls the trafficking of glutamate receptors and is essential for synaptic plasticity. Despite its importance, little is known about how this clathrin structure is organized to mediate endocytosis. We used live-cell and super-resolution microscopy to reveal the dynamic organization of this poorly understood clathrin structure in rat hippocampal neurons. We found that a subset of endocytic proteins only transiently appeared at postsynaptic sites. In contrast, other proteins were persistently enriched and partitioned at the edge of the EZ. We found that uncoupling the EZ from the synapse led to the loss of most of these components, while disrupting interactions with the actin cytoskeleton or membrane did not alter EZ positioning. Finally, we found that plasticity-inducing stimuli promoted the reorganization of the EZ. We conclude that the EZ is a stable, highly organized molecular platform where components are differentially recruited and positioned to orchestrate the endocytosis of synaptic receptors.