Depletion of Mettl3 in cholinergic neurons causes adult-onset neuromuscular degeneration.

Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA's fate, and defects in RNA processing are critical determinants of MN degeneration. N6-methyladenosine (m6A) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the m6A requirement in MNs, we depleted the m6A methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey m6A effects, and their silencing phenocopies METTL3 depletion. Among the m6A targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired m6A signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation.

Caloric restriction leads to druggable LSD1-dependent cancer stem cells expansion.

Caloric Restriction (CR) has established anti-cancer effects, but its clinical relevance and molecular mechanism remain largely undefined. Here, we investigate CR's impact on several mouse models of Acute Myeloid Leukemias, including Acute Promyelocytic Leukemia, a subtype strongly affected by obesity. After an initial marked anti-tumor effect, lethal disease invariably re-emerges. Initially, CR leads to cell-cycle restriction, apoptosis, and inhibition of TOR and insulin/IGF1 signaling. The relapse, instead, is associated with the non-genetic selection of Leukemia Initiating Cells and the downregulation of double-stranded RNA (dsRNA) sensing and Interferon (IFN) signaling genes. The CR-induced adaptive phenotype is highly sensitive to pharmacological or genetic ablation of LSD1, a lysine demethylase regulating both stem cells and dsRNA/ IFN signaling. CR + LSD1 inhibition leads to the re-activation of dsRNA/IFN signaling, massive RNASEL-dependent apoptosis, and complete leukemia eradication in ~90% of mice. Importantly, CR-LSD1 interaction can be modeled in vivo and in vitro by combining LSD1 ablation with pharmacological inhibitors of insulin/IGF1 or dual PI3K/MEK blockade. Mechanistically, insulin/IGF1 inhibition sensitizes blasts to LSD1-induced death by inhibiting the anti-apoptotic factor CFLAR. CR and LSD1 inhibition also synergize in patient-derived AML and triple-negative breast cancer xenografts. Our data provide a rationale for epi-metabolic pharmacologic combinations across multiple tumors.

Inhibition of the lysine demethylase LSD1 modulates the balance between inflammatory and antiviral responses against coronaviruses.

Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

SUMOylation promotes survival and integration of neural stem cell grafts in ischemic stroke.

BACKGROUND: Neural stem cell (NSC)-based therapies hold great promise for treating diseases of the central nervous system (CNS). However, several fundamental problems still need to be overcome to fully exploit the clinical potential of NSC therapeutics. Chief among them is the limited survival of NSC grafts within hostile microenvironments. METHODS: Herein, we sought to engineer NSCs in an effort to increase graft survival within ischemic brain lesions via upregulation of global SUMOylation, a post-translational modification critically involved in mediating tolerance to ischemia/reperfusion. FINDINGS: NSCs overexpressing the SUMO E2-conjugase Ubc9 displayed resistance to oxygen-glucose-deprivation/restoration of oxygen/glucose (OGD/ROG) and enhanced neuronal differentiation in vitro, as well as increased survival and neuronal differentiation when transplanted in mice with transient middle cerebral artery occlusion in vivo. INTERPRETATION: Our work highlights a critical role for SUMOylation in NSC biology and identifies a biological pathway that can be targeted to increase the effectiveness of exogenous stem cell medicines in ischemic stroke. FUND: Intramural Research Program of the NINDS/NIH, the Italian Multiple Sclerosis Foundation (FISM), the Bascule Charitable Trust, NIH-IRTA-OxCam and Wellcome Trust Research Training Fellowships.

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.

BACKGROUND: The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. RESULTS: We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. CONCLUSIONS: This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation.

Neural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain.

Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles.

Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in) EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Extracellular membrane vesicles and immune regulation in the brain.

The brain is characterized by a complex and integrated network of interacting cells in which cell-to-cell communication is critical for proper development and function. Initially considered as an immune privileged site, the brain is now regarded as an immune specialized system. Accumulating evidence reveals the presence of immune components in the brain, as well as extensive bidirectional communication that takes place between the nervous and the immune system both under homeostatic and pathological conditions. In recent years the secretion of extracellular membrane vesicles (EMVs) has been described as a new and evolutionary well-conserved mechanism of cell-to-cell communication, with EMVs influencing the microenvironment through the traffic of bioactive molecules that include proteins and nucleic acids, such as DNA, protein coding, and non-coding RNAs. Increasing evidence suggests that EMVs are a promising candidate to study cross-boundary cell-to-cell communication pathways. Herein we review the role of EMVs secreted by neural cells in modulating the immune response(s) within the brain under physiological and pathological circumstances.