GGC expansions in NOTCH2NLC contribute to Parkinson disease and dopaminergic neuron degeneration.

BACKGROUND AND PURPOSE: The role of GGC repeat expansions within NOTCH2NLC in Parkinson's disease (PD) and the substantia nigra (SN) dopaminergic neuron remains unclear. Here, we profile the NOTCH2NLC GGC repeat expansions in a large cohort of patients with PD. We also investigate the role of GGC repeat expansions within NOTCH2NLC in the dopaminergic neurodegeneration of SN. METHODS: A total of 2,522 patients diagnosed with PD and 1,085 health controls were analyzed for the repeat expansions of NOTCH2NLC by repeat-primed PCR and GC-rich PCR assay. Furthermore, the effects of GGC repeat expansions in NOTCH2NLC on dopaminergic neurons were investigated by using recombinant adeno-associated virus (AAV)-mediated overexpression of NOTCH2NLC with 98 GGC repeats in the SN of mice by stereotactic injection. RESULTS: Four PD pedigrees (4/333, 1.2%) and three sporadic PD patients (3/2189, 0.14%) were identified with pathogenic GGC repeat expansions (larger than 60 GGC repeats) in the NOTCH2NLC gene, while eight PD patients and one healthy control were identified with intermediate GGC repeat expansions ranging from 41 to 60 repeats. No significant difference was observed in the distribution of intermediate NOTCH2NLC GGC repeat expansions between PD cases and controls (Fisher's exact test p-value = 0.29). Skin biopsy showed P62-positive intranuclear NOTCH2NLC-polyGlycine (polyG) inclusions in the skin nerve fibers of patient. Expanded GGC repeats in NOTCH2NLC produced widespread intranuclear and perinuclear polyG inclusions, which led to a severe loss of dopaminergic neurons in the SN. Consistently, polyG inclusions were presented in the SN of EIIa-NOTCH2NLC-(GGC)98 transgenic mice and also led to dopaminergic neuron loss in the SN. CONCLUSIONS: Overall, our findings provide strong evidence that GGC repeat expansions within NOTCH2NLC contribute to the pathogenesis of PD and cause degeneration of nigral dopaminergic neurons.

A homozygous variant in INTS11 links mitosis and neurogenesis defects to a severe neurodevelopmental disorder.

The INTS11 endonuclease is crucial in modulating gene expression and has only recently been linked to human neurodevelopmental disorders (NDDs). However, how INTS11 participates in human development and disease remains unclear. Here, we identify a homozygous INTS11 variant in two siblings with a severe NDD. The variant impairs INTS11 catalytic activity, supported by its substrate's accumulation, and causes G2/M arrest in patient cells with length-dependent dysregulation of genes involved in mitosis and neural development, including the NDD gene CDKL5. The mutant knockin (KI) in induced pluripotent stem cells (iPSCs) disturbs their mitotic spindle organization and thus leads to slow proliferation and increased apoptosis, possibly through the decreased neurally functional CDKL5-induced extracellular signal-regulated kinase (ERK) pathway inhibition. The generation of neural progenitor cells (NPCs) from the mutant iPSCs is also delayed, with long transcript loss concerning neurogenesis. Our work reveals a mechanism underlying INTS11 dysfunction-caused human NDD and provides an iPSC model for this disease.

The genetic and clinical spectrum in a cohort of 39 families with complex inherited peripheral neuropathies.

With complicated conditions and a large number of potentially causative genes, the diagnosis of a patient with complex inherited peripheral neuropathies (IPNs) is challenging. To provide an overview of the genetic and clinical features of 39 families with complex IPNs from central south China and to optimize the molecular diagnosis approach to this group of heterogeneous diseases, a total of 39 index patients from unrelated families were enrolled, and detailed clinical data were collected. TTR Sanger sequencing, hereditary spastic paraplegia (HSP) gene panel, and dynamic mutation detection in spinocerebellar ataxia (SCAs) were performed according to the respective additional clinical features. Whole-exome sequencing (WES) was used in patients with negative or unclear results. Dynamic mutation detection in NOTCH2NLC and RCF1 was applied as a supplement to WES. As a result, an overall molecular diagnosis rate of 89.7% was achieved. All 21 patients with predominant autonomic dysfunction and multiple organ system involvement carried pathogenic variants in TTR, among which nine had c.349G > T (p.A97S) hotspot variants. Five out of 7 patients (71.4%) with muscle involvement harbored biallelic pathogenic variants in GNE. Five out of 6 patients (83.3%) with spasticity reached definite genetic causes in SACS, KIF5A, BSCL2, and KIAA0196, respectively. NOTCH2NLC GGC repeat expansions were identified in all three cases accompanied by chronic coughing and in one patient accompanied by cognitive impairment. The pathogenic variants, p.F284S and p.G111R in GNE, and p.K4326E in SACS, were first reported. In conclusion, transthyretin amyloidosis with polyneuropathy (ATTR-PN), GNE myopathy, and neuronal intranuclear inclusion disease (NIID) were the most common genotypes in this cohort of complex IPNs. NOTCH2NLC dynamic mutation testing should be added to the molecular diagnostic workflow. We expanded the genetic and related clinical spectrum of GNE myopathy and ARSACS by reporting novel variants.

Clinical features of NOTCH2NLC-related neuronal intranuclear inclusion disease.

BACKGROUND: Abnormal expanded GGC repeats within the NOTCH2HLC gene has been confirmed as the genetic mechanism for most Asian patients with neuronal intranuclear inclusion disease (NIID). This cross-sectional observational study aimed to characterise the clinical features of NOTCH2NLC-related NIID in China. METHODS: Patients with NOTCH2NLC-related NIID underwent an evaluation of clinical symptoms, a neuropsychological assessment, electrophysiological examination, MRI and skin biopsy. RESULTS: In the 247 patients with NOTCH2NLC-related NIID, 149 cases were sporadic, while 98 had a positive family history. The most common manifestations were paroxysmal symptoms (66.8%), autonomic dysfunction (64.0%), movement disorders (50.2%), cognitive impairment (49.4%) and muscle weakness (30.8%). Based on the initial presentation and main symptomology, NIID was divided into four subgroups: dementia dominant (n=94), movement disorder dominant (n=63), paroxysmal symptom dominant (n=61) and muscle weakness dominant (n=29). Clinical (42.7%) and subclinical (49.1%) peripheral neuropathies were common in all types. Typical diffusion-weighted imaging subcortical lace signs were more frequent in patients with dementia (93.9%) and paroxysmal symptoms types (94.9%) than in those with muscle weakness (50.0%) and movement disorders types (86.4%). GGC repeat sizes were negatively correlated with age of onset (r=-0.196, p<0.05), and in the muscle weakness-dominant type (median 155.00), the number of repeats was much higher than in the other three groups (p<0.05). In NIID pedigrees, significant genetic anticipation was observed (p<0.05) without repeat instability (p=0.454) during transmission. CONCLUSIONS: NIID is not rare; however, it is usually misdiagnosed as other diseases. Our results help to extend the known clinical spectrum of NOTCH2NLC-related NIID.

N6-methyladenosine dynamics in neurodevelopment and aging, and its potential role in Alzheimer's disease.

BACKGROUND: N6-methyladenosine (m6A) modification is known to impact many aspects of RNA metabolism, including mRNA stability and translation, and is highly prevalent in the brain. RESULTS: We show that m6A modification displays temporal and spatial dynamics during neurodevelopment and aging. Genes that are temporally differentially methylated are more prone to have mRNA expression changes and affect many pathways associated with nervous system development. Furthermore, m6A shows a distinct tissue-specific methylation profile, which is most pronounced in the hypothalamus. Tissue-specific methylation is associated with an increase in mRNA expression and is associated with tissue-specific developmental processes. During the aging process, we observe significantly more m6A sites as age increases, in both mouse and human. We show a high level of overlap between mouse and human; however, humans at both young and old ages consistently show more m6A sites compared to mice. Differential m6A sites are found to be enriched in alternative untranslated regions of genes that affect aging-related pathways. These m6A sites are associated with a strong negative effect on mRNA expression. We also show that many Alzheimer-related transcripts exhibit decreased m6A methylation in a mouse model of Alzheimer's disease, which is correlated with reduced protein levels. CONCLUSIONS: Our results suggest that m6A exerts a critical function in both early and late brain development in a spatio-temporal fashion. Furthermore, m6A controls protein levels of key genes involved in Alzheimer's disease-associated pathways, suggesting that m6A plays an important role in aging and neurodegenerative disease.

Identification of GGC repeat expansion in the NOTCH2NLC gene in amyotrophic lateral sclerosis.

OBJECTIVE: To determine whether the GGC repeats in the NOTCH2NLC gene contribute to amyotrophic lateral sclerosis (ALS). METHODS: In this study, 545 patients with ALS and 1,305 healthy controls from mainland China were recruited. Several pathogenic mutations in known ALS-causative genes (including C9ORF72 and ATXN2) and polynucleotide repeat expansions in NOP56 and AR genes were excluded. Repeat-primed PCR and GC-rich PCR were performed to determine the GGC repeat size in NOTCH2NLC. Systematic and targeted clinical evaluations and investigations, including skin biopsy and dynamic electrophysiologic studies, were conducted in the genetically affected patients. RESULTS: GGC repeat expansion was observed in 4 patients (numbers of repeats 44, 54, 96, and 143), accounting for ≈0.73% (4 of 545) of all patients with ALS. A comparison with 1,305 healthy controls revealed that GGC repeat expansion in NOTCH2NLC was associated with ALS (Fisher exact test, 4 of 545 vs 0 of 1,305, p = 0.007). Compared to patients with the neuronal intranuclear inclusion disease (NIID) muscle weakness-dominant subtype, patients with ALS phenotype carrying the abnormal repeat expansion tended to have a severe phenotype and rapid deterioration. CONCLUSION: Our results suggest that ALS is a specific phenotype of NIID or that GGC expansion in NOTCH2NLC is a factor that modifies ALS. These findings may help clarify the pathogenic mechanism of ALS and may expand the known clinical spectrum of NIID.

UBA5 Mutations Cause a New Form of Autosomal Recessive Cerebellar Ataxia.

Autosomal recessive cerebellar ataxia (ARCA) comprises a large and heterogeneous group of neurodegenerative disorders. For many affected patients, the genetic cause remains undetermined. Through whole-exome sequencing, we identified compound heterozygous mutations in ubiquitin-like modifier activating enzyme 5 gene (UBA5) in two Chinese siblings presenting with ARCA. Moreover, copy number variations in UBA5 or ubiquitin-fold modifier 1 gene (UFM1) were documented with the phenotypes of global developmental delays and gait disturbances in the ClinVar database. UBA5 encodes UBA5, the ubiquitin-activating enzyme of UFM1. However, a crucial role for UBA5 in human neurological disease remains to be reported. Our molecular study of UBA5-R246X revealed a dramatically decreased half-life and loss of UFM1 activation due to the absence of the catalytic cysteine Cys250. UBA5-K310E maintained its interaction with UFM1, although with less stability, which may affect the ability of this UBA5 mutant to activate UFM1. Drosophila modeling revealed that UBA5 knockdown induced locomotive defects and a shortened lifespan accompanied by aberrant neuromuscular junctions (NMJs). Strikingly, we found that UFM1 and E2 cofactor knockdown induced markedly similar phenotypes. Wild-type UBA5, but not mutant UBA5, significantly restored neural lesions caused by the absence of UBA5. The finding of a UBA5 mutation in cerebellar ataxia suggests that impairment of the UFM1 pathway may contribute to the neurological phenotypes of ARCA.

A novel mutation of GATA4 (K300T) associated with familial atrial septal defect.

The GATA-binding protein 4 gene (GATA4) encodes a zinc-finger transcription factor that plays a key role in embryogenesis and cardiac development. Variants in the GATA4 gene have been implicated in several congenital heart diseases (CHD), such as the tetralogy of Fallot (ToF), atrial septal defect (ASD), ventricular septal defect (VSD), atrioventricular septal defect (AVSD), and dilated cardiomyopathy (DCM). We studied a four-generation Chinese ASD family and identified a novel GATA4 mutation (c.A899C, p.K300T) in all surviving affected members and two carriers with incomplete penetrance. Bioinformatics programs (PolyPhen-2, SIFT, and MutationTaster) predicted the mutation to be deleterious. The lysine at the mutation position was highly conserved from Drosophila to humans and was recognized as a methylation location in the GATA4 protein. The involvement of the lysine methylation in cardiogenesis by attenuating the transcriptional activity of GATA4 in mice has been previously examined. Our study broadens the mutation spectrum of the GATA4 gene and reveals for the first time a mutation at the methylation position of GATA4 associated with ASD.

Decreased expression of MicroRNA-200 family in human breast cancer is associated with lymph node metastasis.

OBJECTIVES: MicroRNA-200 family (miR-200f) has been consistently reported to be deregulated and modulate the metastatic process in multiple cancers. In the present study, we detected the expression of miR-200f in breast cancer (BC) tissue and explored its relationships with clinicopathological characteristics, especially with lymph node metastasis. METHODS: Expression levels of miR-200a, miR-200b, miR-200c, miR-141, and miR-429 in 99 pairs of BC tissues and adjacent normal tissues were measured by real-time quantitative PCR. The correlation between miR-200f level and multiple clinicopathological factors was then examined by Mann-Whitney test, ANOVA, and operating characteristic (ROC) analysis. RESULTS: All members of the miR-200f were down-regulated in BC tissue compared with that in normal adjacent tissue; miR-200a, miR-200b, and miR-200c were highly decreased (p < 0.05), while the differences of miR-141 and miR-429 between patients and the control group were not statistically significant. Furthermore, all five members were found to be distinctly decreased with the incidence of lymph node metastasis (p < 0.05); When the patients were divided into three groups according to the number of lymph node metastasis (0; 1-3; ≥4), a gradual decrease of miR-200f expression was observed with the increasing number of lymph node metastasis; ROC revealed that miR-200b can differentiate patients with lymph node metastasis from those without lymph node metastasis. CONCLUSION: These observations imply that the down-regulation of miR-200f in human BC is associated with an invasive phenotype, and miR-200b may be useful to estimate the likelihood of the presence of pathologically positive lymph nodes.

Inhibition of transglutaminase exacerbates polyglutamine-induced neurotoxicity by increasing the aggregation of mutant ataxin-3 in an SCA3 Drosophila model.

Transglutaminases (TGs) comprise a family of Ca(2+)-dependent enzymes that catalyze protein cross-linking, which include nine family members in humans but only a single homolog in Drosophila with three conserved domains. Drosophila Tg plays important roles in cuticle morphogenesis, hemolymph clotting, and innate immunity. Mammalian tissue TG (TG2) is involved in polyglutamine diseases (polyQ diseases), and TG6 has been identified as a causative gene of a novel spinocerebellar ataxia, SCA35. Using a well-established SCA3 fly model, we found that RNA interference-mediated suppression of Tg aggravated polyQ-induced neurodegenerative phenotypes. The administration of cystamine, a known effective Tg inhibitor, enhanced ommatidial degeneration in SCA3 flies. We also demonstrated that the aggregates of pathogenic ataxin-3 increased greatly, when the Tg activity was repressed. These findings indicate that Tg is crucial for polyQ-induced neurotoxicity because Tg ablation resulted in more severe neurodegeneration due to the elevated accumulation of insoluble ataxin-3 complexes in the SCA3 Drosophila model.

miR-25 alleviates polyQ-mediated cytotoxicity by silencing ATXN3.

MicroRNAs (miRNAs) have been reported to play significant roles in the pathogenesis of various polyQ diseases. This study aims to investigate the regulation of ATXN3 gene expression by miRNA. We found that miR-25 reduced both wild-type and polyQ-expanded mutant ataxin-3 protein levels by interacting with the 3'UTR of ATXN3 mRNA. miR-25 also increased cell viability, decreased early apoptosis, and downregulated the accumulation of mutant ataxin-3 protein aggregates in SCA3/MJD cells. These novel results shed light on the potential role of miR-25 in the pathogenesis of SCA3/MJD, and provide a possible therapeutic intervention for this disorder.

Lithium chloride alleviates neurodegeneration partly by inhibiting activity of GSK3ß in a SCA3 Drosophila model.

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucelotide repeat that encodes an abnormal polyglutamine (PolyQ) tract in the disease protein, ataxin-3. The formation of neuronal intranuclear inclusions in the specific brain regions is one of the pathological hallmarks of SCA3. Acceleration of the degradation of the mutant protein aggregates is proven to produce beneficial effects in SCA3 and other PolyQ diseases. Lithium is known to be neuroprotective in various models of neurodegenerative disease and can reduce the mutant protein aggregates by inducing autophagy. In this study, we explored the therapeutic potential of lithium in a SCA3 Drosophila model. We showed that chronic treatment with lithium chloride at specific doses notably prevented eye depigmentation, alleviated locomotor disability, and extended the median life spans of SCA3 transgenic Drosophila. By means of genetic approaches, we showed that co-expressing the mutant S9E, which mimicked the phosphorylated S9 state of Shaggy as done by lithium, also partly decreased toxicity of gmr-SCA3tr-Q78. Taken together, our findings suggest that lithium is a promising therapeutic agent for the treatment of SCA3 and other PolyQ diseases.

Argonaute 1 regulates the fate of germline stem cells in Drosophila.

The Argonaute-family proteins play crucial roles in small-RNA-mediated gene regulation. In Drosophila, previous studies have demonstrated that Piwi, one member of the PIWI subfamily of Argonaute proteins, plays an essential role in regulating the fate of germline stem cells (GSCs). However, whether other Argonaute proteins also play similar roles remains elusive. Here, we show that overexpression of Argonaute 1 (AGO1) protein, another subfamily (AGO) of the Argonaute proteins, leads to GSC overproliferation, whereas loss of Ago1 results in the loss of GSCs. Combined with germline clonal analyses of Ago1, these findings strongly support the argument that Ago1 plays an essential and intrinsic role in the maintenance of GSCs. In contrast to previous observations of Piwi function in the maintenance of GSCs, we show that AGO1 is not required for bag of marbles (bam) silencing and probably acts downstream or parallel of bam in the regulation of GSC fate. Given that AGO1 serves as a key component of the miRNA pathway, we propose that an AGO1-dependent miRNA pathway probably plays an instructive role in repressing GSC/cystoblast differentiation.