Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific ...nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs
, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref.
) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca
handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.
Abstract The aim of the present study was to further explore the in vivo function of the Leucine-rich repeat kinase 2 ( LRRK2 )-gene, which is mutated in certain familial forms of Parkinson's disease ...(PD). We generated a mouse model harboring the disease-associated point mutation R1441C in the GTPase domain of the endogenous murine LRRK2 gene (LRRK2 R1441C line) and performed a comprehensive analysis of these animals throughout lifespan in comparison with an existing knockdown line of LRRK2 (LRRK2 knockdown line). Animals of both lines do not exhibit severe motor dysfunction or pathological signs of neurodegeneration neither at young nor old age. However, at old age the homozygous LRRK2 R1441C animals exhibit clear phenotypes related to the prodromal phase of PD such as impairments in fine motor tasks, gait, and olfaction. These phenotypes are only marginally observable in the LRRK2 knockdown animals, possibly due to activation of compensatory mechanisms as suggested by in vitro studies of synaptic transmission. Thus, at the organismal level the LRRK2 R1441C mutation does not emerge as a loss of function of the protein, but induces mutation specific deficits. Furthermore, judged by the phenotypes presented, the LRRK2-R1441C knock-in line is a valid preclinical model for the prodromal phase of PD.
Abstract The oncogene DJ -1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 ...has an important function in cellular antioxidant responses, but its role in central metabolism of neurons is still elusive. We applied stable isotope assisted metabolic profiling to investigate the effect of a functional loss of DJ-1 and show that DJ-1 deficient neuronal cells exhibit decreased glutamine influx and reduced serine biosynthesis. By providing precursors for GSH synthesis, these two metabolic pathways are important contributors to cellular antioxidant response. Down-regulation of these pathways, as a result of loss of DJ-1 leads to an impaired antioxidant response. Furthermore, DJ-1 deficient mouse microglia showed a weak but constitutive pro-inflammatory activation. The combined effects of altered central metabolism and constitutive activation of glia cells raise the susceptibility of dopaminergic neurons towards degeneration in patients harboring mutated DJ-1 . Our work reveals metabolic alterations leading to increased cellular instability and identifies potential new intervention points that can further be studied in the light of novel translational medicine approaches.
Loss of function of DJ‐1 (PARK7) is associated with autosomal recessive early‐onset Parkinson's disease (PD), one of the major age‐related neurological diseases. In this study, we extended former ...studies on DJ‐1 knockout mice by identifying subtle morphological and behavioural phenotypes. The DJ‐1 gene trap‐induced null mutants exhibit less dopamine‐producing neurons in the ventral tegmental area (VTA). They also exhibit slight changes in behaviour, i.e. diminished rearing behaviour and impairments in object recognition. Furthermore, we detected subtle phenotypes, which suggest that these animals compensate for the loss of DJ‐1. First, we found a significant upregulation of mitochondrial respiratory enzyme activities, a mechanism known to protect against oxidative stress. Second, a close to significant increase in c‐Jun N‐terminal kinase 1 phosphorylation in old DJ‐1‐deficient mice hints at a differential activation of neuronal cell survival pathways. Third, as no change in the density of tyrosine hydroxylase (TH)‐positive terminals in the striatum was observed, the remaining dopamine‐producing neurons likely compensate by increasing axonal sprouting. In summary, the present data suggest that DJ‐1 is implicated in major non‐motor symptoms of PD appearing in the early phases of the disease—such as subtle impairments in motivated behaviour and cognition—and that under basal conditions the loss of DJ‐1 is compensated
Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. ...This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common genetic cause of Parkinson's disease (PD). However, LRRK2 function and molecular mechanisms causing the ...parkinsonian phenotype remain widely unknown. Most of LRRK2 knockdown and overexpression models strengthen the relevance of LRRK2 in regulating neurite outgrowth. We have recently identified ARHGEF7 as the first guanine nucleotide exchange factor (GEF) of LRRK2. This GEF is influencing neurite outgrowth through regulation of actin polymerization. Here, we examined the expression profile of neuroblastoma cells with reduced LRRK2 and ARHGEF7 levels to identify additional partners of LRRK2 in this process. Tropomyosins (TPMs), and in particular TPM4, were the most interesting candidates next to other actin cytoskeleton regulating transcripts in this dataset. Subsequently, enhanced neurite branching was shown using primary hippocampal neurons of LRRK2 knockdown animals. Furthermore, we observed an enhanced number of growth cones per neuron and a mislocalization and dysregulation of ARHGEF7 and TPM4 in these neuronal compartments. Our results reveal a fascinating connection between the neurite outgrowth phenotype of LRRK2 models and the regulation of actin polymerization directing further investigations of LRRK2-related pathogenesis.
•Co-knockdown of LRRK2 and its guanine nucleotide exchange factor ARHGEF7•Expression profiling reveals the F-actin stabilizing tropomyosin 4 as a key player.•Process phenotype of LRRK2 knockdown cells is correlated with ARHGEF7 deregulation.•Growth cone and neurite phenotype of LRRK2 knockdown neurons could be identified.•Tropomyosin 4 and ARHGEF7 are mislocalized in LRRK2 knockdown neurite growth cones.
encodes a developmental transcription factor and has been linked to restless legs syndrome (RLS) in genome-wide association studies. RLS is a movement disorder leading to severe sleep reduction and ...has a substantial impact on the quality of life of patients. In genome-wide association studies,
has consistently been the gene with the highest effect size and functional studies suggest a disease-relevant downregulation. Therefore, haploinsufficiency of
could be the system with the most potential for modeling RLS in animals. We used heterozygous
-knockout mice to study the effects of
haploinsufficiency on mouse behavioral and neurological phenotypes, and to relate the findings to human RLS. We exposed the
-deficient mice to assays of motor, sensorimotor and cognitive ability, and assessed the effect of a dopaminergic receptor 2/3 agonist commonly used in the treatment of RLS. The mutant mice showed a pattern of circadian hyperactivity, which is compatible with human RLS. Moreover, we discovered a replicable prepulse inhibition (PPI) deficit in the
-deficient animals. In addition, these mice were hyposensitive to the PPI-reducing effect of the dopaminergic receptor agonist, highlighting a role of Meis1 in the dopaminergic system. Other reported phenotypes include enhanced social recognition at an older age that was not related to alterations in adult olfactory bulb neurogenesis previously shown to be implicated in this behavior. In conclusion, the
-deficient mice fulfill some of the hallmarks of an RLS animal model, and revealed the role of Meis1 in sensorimotor gating and in the dopaminergic systems modulating it.
The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in ...early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca
signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.
Parkinson's Disease (PD) is the most common neurodegenerative movement disorder. Autosomal-recessive mutations in the mitochondrial protein kinase PINK1 (PTEN-induced kinase 1) account for 1–2% of ...the hereditary early-onset cases. To study the mechanisms underlying disease development, we generated Pink1-deficient mice. In analogy to other genetic loss-of-function mouse models, Pink1−/− mice did not show morphological alterations in the dopaminergic system. As a consequence, no gross motor dysfunctions were observed indicating that these mice do not develop the cardinal symptoms of PD. Nonetheless, symptoms which develop mainly before bradykinesia, rigidity and resting tremor were clearly evident in Pink1-deficient mice. These symptoms were gait alterations and olfactory dysfunctions. Remarkably in the glomerular layer of the olfactory bulb the density of serotonergic fibers was significantly reduced. Concerning mitochondrial morphology, neurons in Pink1−/− mice had less fragmented mitochondria. In contrast, upon acute knock-down of Pink1 increased mitochondrial fragmentation was observed in neuronal cultures. This fragmentation was, however, evened out within days. Taken together, we demonstrate that Pink1-deficient mice exhibit behavioral symptoms of early phases of PD and present systematic experimental evidence for compensation of Pink1-deficiency at the cellular level. Thus, Pink1-deficient mice represent a model for the early phases of PD in which compensation may still impede the onset of neurodegeneration. Consequently, these mice are a valuable tool for studying Pink1-related PD development, as well as for searching for reliable PD biomarkers.
►Pink1 deficient animals show olfactory impairments. ►Aged Pink1 deficient animals exhibit gait alterations. ►Aged Pink1 deficient animals exhibit alterations in the serotonergic system. ►Mitochondrial fragmentation induced by acute loss of Pink1 is compensated over time. ►Pink1 deficient mice are models for early phases of Parkinson's Disease.
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