MicroRNA profiling reveals marker of motor neuron disease in ALS models

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type to elucidate underlying disease mechanisms and to identify novel targets that inform on MN health during ALS disease time course. microRNAs (miRNAs) are short, noncoding RNAs that can shape the expression profile of a cell and thus often exhibit cell-type-enriched expression. To determine MN-enriched miRNA expression, we used Cre recombinase-dependent miRNA tagging and affinity purification in mice. By defining thein vivomiRNA expression of MNs, all neurons, astrocytes, and microglia, we then focused on MN-enriched miRNAs via a comparative analysis and found that they may functionally distinguish MNs postnatally from other spinal neurons. Characterizing the levels of the MN-enriched miRNAs in CSF harvested from ALS models of MN disease demonstrated that one miRNA (miR-218) tracked with MN loss and was responsive to an ALS therapy in rodent models. Therefore, we have used cellular expression profiling tools to define the distinct miRNA expression of MNs, which is likely to enrich future studies of MN disease. This approach enabled the development of a novel, drug-responsive marker of MN disease in ALS rodents.SIGNIFICANCE STATEMENTAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons (MNs) in the brain and spinal cord are selectively lost. To develop tools to aid in our understanding of the distinct expression profiles of MNs and, ultimately, to monitor MN disease progression, we identified small regulatory microRNAs (miRNAs) that were highly enriched or exclusive in MNs. The signal for one of these MN-enriched miRNAs is detectable in spinal tap biofluid from an ALS rat model, where its levels change as disease progresses, suggesting that it may be a clinically useful marker of disease status. Furthermore, rats treated with ALS therapy have restored expression of this MN RNA marker, making it an MN-specific and drug-responsive marker for ALS rodents.</jats:p

Defining Motor Neuron-Enriched microRNAs Reveals Novel Biomarkers and Genetic Causes of Motor Neuron Disease and Uncovers New Mechanisms of Glial Dysfunction in ALS

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons (MNs) in the brain and spinal cord. However, the pathophysiology underlying MN selective vulnerability is unclear, especially considering ALS-causative mutations are in ubiquitously expressed proteins. Moreover, despite the preferential degeneration of MNs in ALS, the surrounding cell types are not bystanders, but greatly contribute to disease progression. Thus, to better understand MN vulnerability in ALS, we characterized the distinct microRNA (miRNAs) expression profile of MNs as compared to other central nervous system cell types. We focused on miRNAs because they have broad regulatory capacity and often exhibit cell type-enriched expression, and thus, may be important determinants of MN identity. Additionally, we predicted that MN-enriched miRNA profiles would inform on novel markers of MN disease and mechanisms of ALS pathogenesis. To this end, we identified MN-enriched miRNA expression and found they may functionally define postnatal MN identity. Consistent with a critical role in MNs, we found that single nucleotide polymorphisms (SNPs) in MN-enriched miR-218 were significantly more prevalent in singleton ALS patients than controls, suggesting SNPs in MN-enriched miRNAs may confer a genetic pre-disposition to MN disease. Importantly, we also demonstrated that the levels of MN-enriched miR-218 are elevated in ALS rodent model cerebrospinal fluid (CSF) and are highly correlated with MN loss. Treatment of ALS model rats with a MN-sparing therapy prevented the increase in miR-218 CSF levels, demonstrating that miR-218 is a MN-specific and pharmacodynamic biomarker of MN disease. We further validated that miR-218 was also applicable to human MN disease, as the miR-218 CSF levels of Spinal Muscular Atrophy patients also responded to a MN-sparing therapy. Finally, we unexpectedly observed that extracellular, MN-derived miR-218 also contributes to ALS disease progression, as it can be taken up by astrocytes and functionally contribute to their dysfunction in ALS model mice. These data define a novel mechanism in neurodegeneration whereby diseased neurons can communicate, through extracellular miRNAs, with surrounding glia and deleteriously modify their phenotype. Overall, this work demonstrates that applying a cell-type specific focus to diseases that exhibit selective vulnerability can successfully uncover novel biomarkers, genetic risk factors, and mechanisms of disease

Aberrant cortical development is driven by impaired cell cycle and translational control in a DDX3X syndrome model.

Mutations in the RNA helicase, DDX3X, are a leading cause of Intellectual Disability and present as DDX3X syndrome, a neurodevelopmental disorder associated with cortical malformations and autism. Yet, the cellular and molecular mechanisms by which DDX3X controls cortical development are largely unknown. Here, using a mouse model of Ddx3x loss-of-function we demonstrate that DDX3X directs translational and cell cycle control of neural progenitors, which underlies precise corticogenesis. First, we show brain development is sensitive to Ddx3x dosage; complete Ddx3x loss from neural progenitors causes microcephaly in females, whereas hemizygous males and heterozygous females show reduced neurogenesis without marked microcephaly. In addition, Ddx3x loss is sexually dimorphic, as its paralog, Ddx3y, compensates for Ddx3x in the developing male neocortex. Using live imaging of progenitors, we show that DDX3X promotes neuronal generation by regulating both cell cycle duration and neurogenic divisions. Finally, we use ribosome profiling in vivo to discover the repertoire of translated transcripts in neural progenitors, including those which are DDX3X-dependent and essential for neurogenesis. Our study reveals invaluable new insights into the etiology of DDX3X syndrome, implicating dysregulated progenitor cell cycle dynamics and translation as pathogenic mechanisms

Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development

De novo germline mutations in the RNA helicase DDX3X account for 1%–3% of unexplained intellectual disability (ID) cases in females and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here, we use human and mouse genetics and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n = 107), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuron generation. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity, induce ectopic RNA-protein granules in neural progenitors and neurons, and impair translation. Together, these results uncover key mechanisms underlying DDX3X syndrome and highlight aberrant RNA metabolism in the pathogenesis of neurodevelopmental disease.Using human and mouse genetics, Lennox et al. identify 107 mutations in DDX3X, demonstrating DDX3X is essential for cortical development. A striking correlation between the severity of clinical mutations and abnormal RNA metabolism highlights unappreciated mechanisms of DDX3X syndrome

Pathogenic DDX3X Mutations Impair RNA Metabolism and Neurogenesis during Fetal Cortical Development

International audienceDe novo germline mutations in the RNA helicase DDX3X account for 1%-3% of unexplained intellectual disability (ID) cases in females and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here, we use human and mouse genetics and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n = 107), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuron generation. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity, induce ectopic RNA-protein granules in neural progenitors and neurons, and impair translation. Together, these results uncover key mechanisms underlying DDX3X syndrome and highlight aberrant RNA metabolism in the pathogenesis of neurodevelopmental disease

Human genetics and neuropathology suggest a link between miR-218 and amyotrophic lateral sclerosis pathophysiology

Motor neuron-specific microRNA-218 (miR-218) has recently received attention because of its roles in mouse development. However, miR-218 relevance to human motor neuron disease was not yet explored. Here, we demonstrate by neuropathology that miR-218 is abundant in healthy human motor neurons. However, in amyotrophic lateral sclerosis (ALS) motor neurons, miR-218 is down-regulated and its mRNA targets are reciprocally up-regulated (derepressed). We further identify the potassium channel Kv10.1 as a new miR-218 direct target that controls neuronal activity. In addition, we screened thousands of ALS genomes and identified six rare variants in the human miR-218-2 sequence. miR-218 gene variants fail to regulate neuron activity, suggesting the importance of this small endogenous RNA for neuronal robustness. The underlying mechanisms involve inhibition of miR-218 biogenesis and reduced processing by DICER. Therefore, miR-218 activity in motor neurons may be susceptible to failure in human ALS, suggesting that miR-218 may be a potential therapeutic target in motor neuron disease