SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) to facilitate ...viral-host membrane fusion. ACE2 and TMPRSS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq levels. However, transcriptomic data and actual protein validation convey conflicting information regarding the distribution of the biologically relevant protein receptor in whole tissues. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRSS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals was stained using antibodies against ACE2 and TMPRSS2, combined with SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize their expression and related infection patterns. The data demonstrate that infection is restricted to sites containing both ACE2 and TMPRSS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the bronchioles and alveoli. Conversely, infection completely overlaps where ACE2 and TMPRSS2 co-localize in the tertiary bronchi, bronchioles, and alveoli.
Abstract Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to ...investigate the early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments.
MicroRNAs (miRNAs) are evolutionarily conserved non-coding RNAs of ∼22 nucleotides that regulate gene expression at the level of translation and play vital roles in hippocampal neuron development, ...function and plasticity. Here, we performed a systematic and in-depth analysis of miRNA expression profiles in cultured hippocampal neurons during development and after induction of neuronal activity. MiRNA profiling of primary hippocampal cultures was carried out using locked nucleic-acid-based miRNA arrays. The expression of 264 different miRNAs was tested in young neurons, at various developmental stages (stage 2-4) and in mature fully differentiated neurons (stage 5) following the induction of neuronal activity using chemical stimulation protocols. We identified 210 miRNAs in mature hippocampal neurons; the expression of most neuronal miRNAs is low at early stages of development and steadily increases during neuronal differentiation. We found a specific subset of 14 miRNAs with reduced expression at stage 3 and showed that sustained expression of these miRNAs stimulates axonal outgrowth. Expression profiling following induction of neuronal activity demonstrates that 51 miRNAs, including miR-134, miR-146, miR-181, miR-185, miR-191 and miR-200a show altered patterns of expression after NMDA receptor-dependent plasticity, and 31 miRNAs, including miR-107, miR-134, miR-470 and miR-546 were upregulated by homeostatic plasticity protocols. Our results indicate that specific miRNA expression profiles correlate with changes in neuronal development and neuronal activity. Identification and characterization of miRNA targets may further elucidate translational control mechanisms involved in hippocampal development, differentiation and activity-depended processes.
Hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, but the pathogenic mechanism of this mutation remains ...unresolved. Haploinsufficiency has been proposed as one potential mechanism. However, insights if and how reduced C9orf72 proteins levels might contribute to disease pathogenesis are still limited because C9orf72 expression, localization and functions in the central nervous system (CNS) are uncertain, in part due to the poor specificity of currently available C9orf72 antibodies.Here, we generated and characterized novel knock-out validated monoclonal rat and mouse antibodies against C9orf72. We found that C9orf72 is a low abundant, cytoplasmic, highly soluble protein with the long 481 amino acid isoform being the predominant, if not exclusively, expressed protein isoform in mouse tissues and human brain. As consequence of the C9orf72 repeat expansion, C9orf72 protein levels in the cerebellum were reduced to 80% in our series of C9orf72 mutation carriers (n = 17) compared to controls (n = 26). However, no associations between cerebellar protein levels and clinical phenotypes were seen. Finally, by utilizing complementary immunohistochemical and biochemical approaches including analysis of human iPSC derived motor neurons, we identified C9orf72, in addition to its association to lysosomes, to be localized to the presynapses and able to interact with all members of the RAB3 protein family, suggestive of a role for C9orf72 in regulating synaptic vesicle functions by potentially acting as guanine nucleotide exchange factor for RAB3 proteins.In conclusion, our findings provide further evidence for haploinsufficiency as potential mechanism in C9orf72 pathogenesis by demonstrating reduced protein levels in C9orf72 mutation carriers and important novel insights into the physiological role of C9orf72 in the CNS. Moreover, the described novel monoclonal C9orf72 antibodies will be useful tools to further dissect the cellular and molecular functions of C9orf72.
Semaphorins constitute one of the largest families of repulsive and attractive growth cone guidance proteins. They affect the growth cone’s actin cytoskeleton through interactions with receptor ...complexes composed of ligand-binding, signal-transducing, and modulatory subunits. Our understanding of the intracellular signal transduction machinery linking semaphorins to actin dynamics is limited; however, recent advances provide a more comprehensive view of the molecular basis of neuronal semaphorin signaling.
MICALs form an evolutionary conserved family of multidomain signal transduction proteins characterized by a flavoprotein monooxygenase domain. MICALs are being implicated in the regulation of an ...increasing number of molecular and cellular processes including cytoskeletal dynamics and intracellular trafficking. Intriguingly, some of these effects are dependent on the MICAL monooxygenase enzyme and redox signaling, while other functions rely on other parts of the MICAL protein. Recent breakthroughs in our understanding of MICAL signaling identify the ability of MICALs to bind and directly modify the actin cytoskeleton, link MICALs to the docking and fusion of exocytotic vesicles, and uncover MICALs as anti-apoptotic proteins. These discoveries could lead to therapeutic advances in neural regeneration, cancer, and other diseases.
Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we ...showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson's disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons. To investigate whether RGMa might model aspects of the neuropathology of Parkinson's disease in mouse, we targeted RGMa to adult midbrain dopaminergic neurons using adeno-associated viral vectors. Overexpression of RGMa resulted in a progressive movement disorder, including motor coordination and imbalance, which is typical for a loss of DA release in the striatum. In line with this, RGMa induced selective degeneration of dopaminergic neurons in the substantia nigra (SN) and affected the integrity of the nigrostriatal system. The degeneration of dopaminergic neurons was accompanied by a strong microglia and astrocyte activation. The behavioral, molecular, and anatomical changes induced by RGMa in mice are remarkably similar to the clinical and neuropathological hallmarks of Parkinson's disease. Our data indicate that dysregulation of RGMa plays an important role in the pathology of Parkinson's disease, and antibody-mediated functional interference with RGMa may be a disease modifying treatment option.
Parkinson's disease (PD) is a neurodegenerative disease characterized by severe motor dysfunction due to progressive degeneration of mesencephalic dopaminergic (DA) neurons in the substantia nigra. To date, there is no regenerative treatment available. We previously showed that repulsive guidance molecule member a (RGMa) is upregulated in the substantia nigra of PD patients. Adeno-associated virus-mediated targeting of RGMa to mouse DA neurons showed that overexpression of this repulsive axon guidance and cell patterning cue models the behavioral and neuropathological characteristics of PD in a remarkable way. These findings have implications for therapy development as interfering with the function of this specific axon guidance cue may be beneficial to the survival of DA neurons.
Additionally, circRNAs are involved in circRNA-protein (circRNP) complex formation and localization, sponging of other RNAs (e.g., miRNAs) and RNA-binding proteins (RBPs), and may be translated into ...proteins/peptides (3). circRNAs may control oscillations in transcriptional networks (e.g., as molecular components of the circadian clock) and influence processes like pluripotency maintenance and neuronal differentiation (You et al., 2015; Yu et al., 2017). FUS regulates several RNA metabolic processes, including RNA splicing, and is linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (Robberecht and Philips, 2013; Scotti and Swanson, 2016; van Es et al., 2017). ...insight into the roles of FUS on circRNA expression could provide new clues on how circRNAs are formed and how circRNAs contribute to FUS-associated neurological disorders. In their study, Errichelli et al. identified circRNAs in in vitro-derived mouse motor neurons (purified mouse embryonic stem cell (mESC)-derived motor neurons, obtained from wild-type (FUS+/+) or knockout (FUS−/−) mice) and demonstrate that FUS, through its control of back-splicing reactions, regulates the production of a considerable number of circRNAs. New insights into context-specific circRNA biogenesis via RBPs will probably not only contribute to our understanding of circRNAs in neuronal disorders and lead to the identification of novel molecular biomarkers and therapeutic targets, but may also provide clues about their physiological functions, e.g., in RNA-based mechanisms underlying neuronal development.
Abstract A large genome-wide screen in patients with sporadic amyotrophic lateral sclerosis (ALS) showed that the common variant rs12608932 in gene UNC13A was associated with disease susceptibility. ...UNC13A regulates the release of neurotransmitters, including glutamate. Genetic risk factors that, in addition, modify survival, provide promising therapeutic targets in ALS, a disease whose etiology remains largely elusive. We examined whether UNC13A was associated with survival of ALS patients in a cohort of 450 sporadic ALS patients and 524 unaffected controls from a population-based study of ALS in The Netherlands. Additionally, survival data were collected from individuals of Dutch, Belgian, or Swedish descent (1767 cases, 1817 controls) who had participated in a previously published genome-wide association study of ALS. We related survival to rs12608932 genotype. In both cohorts, the minor allele of rs12608932 in UNC13A was not only associated with susceptibility but also with shorter survival of ALS patients. Our results further corroborate the role of UNC13A in ALS pathogenesis.
Mutations in FOXP1 have been linked to neurodevelopmental disorders including intellectual disability and autism; however, the underlying molecular mechanisms remain ill-defined. Here, we demonstrate ...with RNA and chromatin immunoprecipitation sequencing that FOXP1 directly regulates genes controlling neurogenesis. We show that FOXP1 is expressed in embryonic neural stem cells (NSCs), and modulation of FOXP1 expression affects both neuron and astrocyte differentiation. Using a murine model of cortical development, FOXP1-knockdown in utero was found to reduce NSC differentiation and migration during corticogenesis. Furthermore, transplantation of FOXP1-knockdown NSCs in neonatal mice after hypoxia-ischemia challenge demonstrated that FOXP1 is also required for neuronal differentiation and functionality in vivo. FOXP1 was found to repress the expression of Notch pathway genes including the Notch-ligand Jagged1, resulting in inhibition of Notch signaling. Finally, blockade of Jagged1 in FOXP1-knockdown NSCs rescued neuronal differentiation in vitro. Together, these data support a role for FOXP1 in regulating embryonic NSC differentiation by modulating Notch signaling.
•FOXP1 promotes astrocyte and neuronal differentiation of NSCs in vitro•FOXP1 promotes neuronal differentiation of NSCs in vivo•FOXP1 transcriptionally regulates pro-neural genes and represses Notch pathway genes•FOXP1 promotes neuronal differentiation by limiting Jagged1 expression
In this issue of Stem Cell Reports, Braccioli et al. describe how FOXP1 promotes embryonic neural stem cell differentiation both in vitro and in vivo by transcriptionally regulating pro-neural genes and by repressing the Notch-ligand Jagged1.
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