Abstract
Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of ...human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of
ELP1
exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in
CFTR
,
LIPA
,
MLH1
and
MAPT
. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.
Familial dysautonomia (FD) is a rare neurodevelopmental and neurodegenerative disease caused by a splicing mutation in the Elongator Acetyltransferase Complex Subunit 1 (ELP1) gene. The reduction in ...ELP1 mRNA and protein leads to the death of retinal ganglion cells (RGCs) and visual impairment in all FD patients. Currently patient symptoms are managed, but there is no treatment for the disease. We sought to test the hypothesis that restoring levels of Elp1 would thwart the death of RGCs in FD. To this end, we tested the effectiveness of two therapeutic strategies for rescuing RGCs. Here we provide proof-of-concept data that gene replacement therapy and small molecule splicing modifiers effectively reduce the death of RGCs in mouse models for FD and provide pre-clinical foundational data for translation to FD patients.
Familial dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice mutation in the Elongator acetyltransferase complex subunit 1 (ELP1) gene. This mutation results in a ...tissue-specific reduction of ELP1 protein, with the lowest levels in the central and peripheral nervous systems (CNS and PNS, respectively). FD patients exhibit complex neurological phenotypes due to the loss of sensory and autonomic neurons. Disease symptoms include decreased pain and temperature perception, impaired or absent myotatic reflexes, proprioceptive ataxia, and progressive retinal degeneration. While the involvement of the PNS in FD pathogenesis has been clearly recognized, the underlying mechanisms responsible for the preferential neuronal loss remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying FD by conducting a comprehensive transcriptome analysis of neuronal tissues from the phenotypic mouse model TgFD9; Elp1
. This mouse recapitulates the same tissue-specific ELP1 mis-splicing observed in patients while modeling many of the disease manifestations. Comparison of FD and control transcriptomes from dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC) showed significantly more differentially expressed genes (DEGs) in the PNS than the CNS. We then identified genes that were tightly co-expressed and functionally dependent on the level of full-length ELP1 transcript. These genes, defined as ELP1 dose-responsive genes, were combined with the DEGs to generate tissue-specific dysregulated FD signature genes and networks. Within the PNS networks, we observed direct connections between Elp1 and genes involved in tRNA synthesis and genes related to amine metabolism and synaptic signaling. Importantly, transcriptomic dysregulation in PNS tissues exhibited enrichment for neuronal subtype markers associated with peptidergic nociceptors and myelinated sensory neurons, which are known to be affected in FD. In summary, this study has identified critical tissue-specific gene networks underlying the etiology of FD and provides new insights into the molecular basis of the disease.
Chromosome 16p11.2 reciprocal genomic disorder, resulting from recurrent copy-number variants (CNVs), involves intellectual disability, autism spectrum disorder (ASD), and schizophrenia, but the ...responsible mechanisms are not known. To systemically dissect molecular effects, we performed transcriptome profiling of 350 libraries from six tissues (cortex, cerebellum, striatum, liver, brown fat, and white fat) in mouse models harboring CNVs of the syntenic 7qF3 region, as well as cellular, transcriptional, and single-cell analyses in 54 isogenic neural stem cell, induced neuron, and cerebral organoid models of CRISPR-engineered 16p11.2 CNVs. Transcriptome-wide differentially expressed genes were largely tissue-, cell-type-, and dosage-specific, although more effects were shared between deletion and duplication and across tissue than expected by chance. The broadest effects were observed in the cerebellum (2,163 differentially expressed genes), and the greatest enrichments were associated with synaptic pathways in mouse cerebellum and human induced neurons. Pathway and co-expression analyses identified energy and RNA metabolism as shared processes and enrichment for ASD-associated, loss-of-function constraint, and fragile X messenger ribonucleoprotein target gene sets. Intriguingly, reciprocal 16p11.2 dosage changes resulted in consistent decrements in neurite and electrophysiological features, and single-cell profiling of organoids showed reciprocal alterations to the proportions of excitatory and inhibitory GABAergic neurons. Changes both in neuronal ratios and in gene expression in our organoid analyses point most directly to calretinin GABAergic inhibitory neurons and the excitatory/inhibitory balance as targets of disruption that might contribute to changes in neurodevelopmental and cognitive function in 16p11.2 carriers. Collectively, our data indicate the genomic disorder involves disruption of multiple contributing biological processes and that this disruption has relative impacts that are context specific.
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The 16p11.2 genomic disorder involves deletion and duplication of a contiguous set of genes, resulting in neurodevelopmental and other abnormalities. On the basis of comparisons of mouse models and human neuronal cells, the effects of these lesions on genome-wide gene expression and cellular function are highly dependent on cellular context.
Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a splice mutation in Elongator complex protein 1 (ELP1, also known as IKBKAP); this mutation leads to variable skipping ...of exon 20 and to a drastic reduction of ELP1 in the nervous system. Clinically, many of the debilitating aspects of the disease are related to a progressive loss of proprioception; this loss leads to severe gait ataxia, spinal deformities, and respiratory insufficiency due to neuromuscular incoordination. There is currently no effective treatment for FD, and the disease is ultimately fatal. The development of a drug that targets the underlying molecular defect provides hope that the drastic peripheral neurodegeneration characteristic of FD can be halted. We demonstrate herein that the FD mouse TgFD9;IkbkapΔ20/flox recapitulates the proprioceptive impairment observed in individuals with FD, and we provide the in vivo evidence that postnatal correction, promoted by the small molecule kinetin, of the mutant ELP1 splicing can rescue neurological phenotypes in FD. Daily administration of kinetin starting at birth improves sensory-motor coordination and prevents the onset of spinal abnormalities by stopping the loss of proprioceptive neurons. These phenotypic improvements correlate with increased amounts of full-length ELP1 mRNA and protein in multiple tissues, including in the peripheral nervous system (PNS). Our results show that postnatal correction of the underlying ELP1 splicing defect can rescue devastating disease phenotypes and is therefore a viable therapeutic approach for persons with FD.
Background and objective Disorders of consciousness include coma (cannot be aroused, eye remain closed), vegetative state-VS (can appear to be awake, but unable to purposefully interact) and ...minimally conscious state-MCS (minimal but definite awareness). The objective of this study is to assess the impact of the SARS-CoV-2 infection on the Disorder of Consciousness (DOC) Rehabilitation Unit. Methods This is a retrospective, longitudinal, descriptive, observational, pilot study. We consecutively enrolled 18 patients (age range: 40-72 years, 9 females and 9 males), from three to five months after a brain injury. They were grouped into VS (n = 8) and MCS (n = 10). A confirmed case of COVID-19 was defined as a positive result on high-throughput sequencing or real-time reverse-transcription polymerase chain reaction analysis of throat swab specimens. We collected data of lung Computed Tomography (CT) and laboratory exams. DOC patients who were positive for SARS-CoV-2 were classified into severe and no severe infected group, according to the American Thoracic Society guidelines. Results A total of 18 hospitalized patients with (16) and without confirmed (2) SARS-CoV-2 infection were included in the analysis. After one month, a follow-up clinical evaluation reported that one patient died, one patient was transferred from Covid Unit to Emergency Unit and 3 patients were resulted negative to double swab and they returned to Rehabilitative Unit. Significant differences were reported about hypertension, cardiac disease and respiratory problems between the patients with severe infection and patients without severe infection (P< 0.001). The laboratory findings, such as blood cell counts (P < 0.001), C-reactive protein, D-dimer, potassium and vitamin D levels, seemed to be considered as useful prognostic predictors. Conclusions To our knowledge, this is the first longitudinal study on a sample of chronic DOC patients affected by SARS-CoV-2. This study may offer important new clinical information on COVID-19 for management of DOC patients. Our findings showed that for the subjects with severe infection due to COVID-19, rapid clinical deterioration or worsening could be associated with clinical and laboratory findings, which could contribute to high mortality rate. During the COVID-19 epidemic period, the clinicians should consider all the reported risk factors to avoid delayed diagnosis or misdiagnosis and to prevent the infection transmission in DOC Rehabilitation Unit.
Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed ...for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.
Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease that affects the development and survival of sensory and autonomic neurons. FD is caused by an mRNA splicing mutation in ...intron 20 of the IKBKAP gene that results in a tissue-specific skipping of exon 20 and a corresponding reduction of the inhibitor of kappaB kinase complex-associated protein (IKAP), also known as Elongator complex protein 1. To date, several promising therapeutic candidates for FD have been identified that target the underlying mRNA splicing defect, and increase functional IKAP protein. Despite these remarkable advances in drug discovery for FD, we lacked a phenotypic mouse model in which we could manipulate IKBKAP mRNA splicing to evaluate potential efficacy. We have, therefore, engineered a new mouse model that, for the first time, will permit to evaluate the phenotypic effects of splicing modulators and provide a crucial platform for preclinical testing of new therapies. This new mouse model, TgFD9; Ikbkap(Δ20/flox) was created by introducing the complete human IKBKAP transgene with the major FD splice mutation (TgFD9) into a mouse that expresses extremely low levels of endogenous Ikbkap (Ikbkap(Δ20/flox)). The TgFD9; Ikbkap(Δ20/flox) mouse recapitulates many phenotypic features of the human disease, including reduced growth rate, reduced number of fungiform papillae, spinal abnormalities, and sensory and sympathetic impairments, and recreates the same tissue-specific mis-splicing defect seen in FD patients. This is the first mouse model that can be used to evaluate in vivo the therapeutic effect of increasing IKAP levels by correcting the underlying FD splicing defect.
: Normal human sexual functioning is a complex integration of an intact neuroanatomic substrate, vascular supply, a balanced hormonal profile, and a predominance of excitatory over inhibitory ...psychological mechanisms. However, sexual functioning in Parkinson's disease (PD) is often overlooked in clinical practice, especially in female patients.
: In this cross-sectional study, we have investigated the frequency of sexual dysfunction and the possible correlation with psycho-endocrinological factors in a sample of women with idiopathic PD. Patients were assessed using a semi-structured sexual interview, in addition to psychometric tools, including the Hamilton Rating Scale for Anxiety and for Depression and the Coping Orientation to the Problems Experiences-New Italian Version. Specific blood tests, including testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen E2, prolactin (PRL), and vitamin D3 were also evaluated.
: Our results reported a statistical difference in sexual intercourse frequency before and after the onset of PD (
< 0.001). The percentage of women who complained about reduced sexual desire increased after diagnosis (52.7%) compared to the period before the onset of the illness (36.8%). The endocrinological profile in females with PD revealed statistically significant differences regarding testosterone (
< 0.0006), estradiol (
< 0.00), vitamin D3 (
< 0.006), and calcium (0.002). Depression (44% characterized by perceived feelings of anger and frustration during sexual intercourse) and anxiety symptoms (29.5% reported feelings of fear and anxiety for not satisfying the partner) with abnormal coping strategies (48.14% experienced feelings of anger and intolerance) were also found to be statistically significant. This study showed a high frequency of sexual dysfunction in female patients with PD, which correlated with sexual hormone abnormalities, mood/anxiety, and coping strategies alterations. This supports the idea that there is a need to better investigate the sexual function of female patients with PD to provide them with an adequate therapeutic approach and potentially improve quality of life.
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