Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy

Objective: We used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease. Methods: Motor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons. Results: We show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA. Conclusions: The strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.Peer reviewe

NEFL mRNA Expression Level Is a Prognostic Factor for Early-Stage Breast Cancer Patients

Neurofilament, light polypeptide (NEFL) was demonstrated to be ectopically expressed in breast cancer tissues and decreased in lymph node metastases compared to the paired primary breast cancers in our previous study. Moreover, in several studies, NEFL was regarded as a tumor suppressor gene, and its loss of heterozygosity (LOH) was related to carcinogenesis and metastasis in several types of cancer. To explore the role of NEFL in the progression of breast cancer and to evaluate its clinical significance, we detected the NEFL mRNA level in normal breast tissues, primary breast cancer samples and lymph node metastases, and then analyzed the association between the NEFL expression level and several clinicopathological parameters and disease-free survival (DFS). NEFL mRNA was found to be expressed in 92.3% of breast malignancies and down-regulated in lymph node metastases compared to the paired primary tumors. NEFL mRNA level was lower in primary breast cancers with positive lymph nodes than in cancers with negative lymph nodes. Moreover, a low expression level of NEFL mRNA indicated a poor five-year DFS for early-stage breast cancer patients. Thus, NEFL mRNA is ectopically expressed in breast malignancies and could be a potential prognostic factor for early-stage breast cancer patients

NeFL: Nested Federated Learning for Heterogeneous Clients

Federated learning (FL) is a promising approach in distributed learning keeping privacy. However, during the training pipeline of FL, slow or incapable clients (i.e., stragglers) slow down the total training time and degrade performance. System heterogeneity, including heterogeneous computing and network bandwidth, has been addressed to mitigate the impact of stragglers. Previous studies split models to tackle the issue, but with less degree-of-freedom in terms of model architecture. We propose nested federated learning (NeFL), a generalized framework that efficiently divides a model into submodels using both depthwise and widthwise scaling. NeFL is implemented by interpreting models as solving ordinary differential equations (ODEs) with adaptive step sizes. To address the inconsistency that arises when training multiple submodels with different architecture, we decouple a few parameters. NeFL enables resource-constrained clients to effectively join the FL pipeline and the model to be trained with a larger amount of data. Through a series of experiments, we demonstrate that NeFL leads to significant gains, especially for the worst-case submodel (e.g., 8.33 improvement on CIFAR-10). Furthermore, we demonstrate NeFL aligns with recent studies in FL.Comment: 21 page

Effects of impurity scattering on electron-phonon resonances in semiconductor superlattice high-field transport

A non-equilibrium Green's function method is applied to model high-field quantum transport and electron-phonon resonances in semiconductor superlattices. The field-dependent density of states for elastic (impurity) scattering is found non-perturbatively in an approach which can be applied to both high and low electric fields. I-V curves, and specifically electron-phonon resonances, are calculated by treating the inelastic (LO phonon) scattering perturbatively. Calculations show how strong impurity scattering suppresses the electron-phonon resonance peaks in I-V curves, and their detailed sensitivity to the size, strength and concentration of impurities.Comment: 7 figures, 1 tabl

The role of neurofilament-light polipeptide (NEFL) in the onset and progression of hepatocellular carcinoma

Background: Hepatocellular Carcinoma (HCC) is the third cause of cancer-related deaths worldwide and a major health problem. Although many factors, including chromosomal anomalies, genetic polymorphisms, genetic and epigenetic alterations, contribute to HCC onset and progression, the exact mechanisms remain largely unclear. Recently, it has been shown that NEFL gene (Neurofilament-light polypeptide), whose mutations are responsible of motor-neuron disease such as Charcot-Marie-Tooth disease type 2E, is also involved in some types of malignancies. However, no information about NEFL and HCC are available. Aim: Aim of this study was to investigate the role of NEFL in the several stages of HCC development in the Resistant-Hepatocyte (R-H) rat model of hepatocarcinogenesis. Results: microarray analysis and qRT-PCR analysis showed that the expression of NEFL was strongly up-regulated at all stage of the multistep process of hepatocarcinogenesis, including the very early ones, in particular NEFL mRNA was strongly up-regulated in KRT-19 positive preneoplastic lesions and in fully malignant HCCs. In agreement with mRNA levels, immunofluorescence studies identified the neurofilament only in the preneoplastic lesions positive for KRT-19 and in early HCCs. Since the presence of this neurofilament has never been described in the liver, we wished to investigate whether NEFL expression in the carcinogenic process could be the result of a re-acquisition of fetal/neonatal life status or of increased proliferative capacity of the hepatocytes. However, almost undetectable levels of mRNA were observed in the liver of 19 days fetuses or 2 days pups. Moreover, no NEFL up-regulation was observed in actively dividing hepatocytes in liver regeneration occurring following two/third partial hepatectomy (PH), suggesting that NEFL expression in the liver is a specific feature of cancer onset and development. The ectopic expression of NEFL at all stages of rat carcinogenesis implies that NEFL may function as an oncogene in the onset and development of HCC. To assess the clinical significance of NEFL expression in human HCC patients, we determined NEFL mRNA levels in 14 HCC patients. The results showed that, similarly to what observed in the rat model, while NEFL expression was undetectable in normal human liver, it was readily detectable in HCCs and in matched cirrhotic liver. This demonstrates that aberrant expression of NEFL takes place at the onset and progression in human hepatocarcinogenesis, translating our findings to human pathology. Interestingly, although not statistically significant, a clear trend towards an up-regulation of NEFL in HCCs when compared to matched cirrhotic tissue was observed. Notably, when patients were divided into two groups, NEFL low- or high-expressors, based on the median expression level, the results showed that time of recurrence was significantly shortened in high-NEFL expression compared to low-NEFL expression group, thus demonstrating that NEFL expression levels are a predictive factor for HCC prognosis. Searching for possible mechanisms underlying NEFL enhanced expression, and to determine if the up-regulation of NEFL in the carcinogenic process could be related to promoter methylation status, we analyze the CpG island described in UCSC Genome browser in the 5’ flanking genomic region of NEFL gene. Analysis of the methylation status of 10 consecutive CpG dinucleotides in 5 HCC and 3 normal rat livers exhibited no differences in the methylation status in HCC and controls. These results suggest that DNA hypomethylation of the NEFL promoter region is not involved in the regulation of the expression of this gene. Moreover, no change in NEFL-targeting microRNAs could be found in pre- or neoplastic lesions compared to normal liver. Conclusions: These results unveil the yet unknown role of NEFL in the onset and development of HCC and suggest that NEFL could act as an oncogene in the liver. Further studies are required to identify the mechanism(s) involved in regulating the expression of the NEFL gene and their exact role in HCC development. A better knowledge of the role of NEFL might hopefully provide a novel therapeutic target in HCC progression

In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS61K in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation

The role of neurofilament-light polipeptide (NEFL) in the onset and progression of hepatocellular carcinoma

Background: Hepatocellular Carcinoma (HCC) is the third cause of cancer-related deaths worldwide and a major health problem. Although many factors, including chromosomal anomalies, genetic polymorphisms, genetic and epigenetic alterations, contribute to HCC onset and progression, the exact mechanisms remain largely unclear. Recently, it has been shown that NEFL gene (Neurofilament-light polypeptide), whose mutations are responsible of motor-neuron disease such as Charcot-Marie-Tooth disease type 2E, is also involved in some types of malignancies. However, no information about NEFL and HCC are available. Aim: Aim of this study was to investigate the role of NEFL in the several stages of HCC development in the Resistant-Hepatocyte (R-H) rat model of hepatocarcinogenesis. Results: microarray analysis and qRT-PCR analysis showed that the expression of NEFL was strongly up-regulated at all stage of the multistep process of hepatocarcinogenesis, including the very early ones, in particular NEFL mRNA was strongly up-regulated in KRT-19 positive preneoplastic lesions and in fully malignant HCCs. In agreement with mRNA levels, immunofluorescence studies identified the neurofilament only in the preneoplastic lesions positive for KRT-19 and in early HCCs. Since the presence of this neurofilament has never been described in the liver, we wished to investigate whether NEFL expression in the carcinogenic process could be the result of a re-acquisition of fetal/neonatal life status or of increased proliferative capacity of the hepatocytes. However, almost undetectable levels of mRNA were observed in the liver of 19 days fetuses or 2 days pups. Moreover, no NEFL up-regulation was observed in actively dividing hepatocytes in liver regeneration occurring following two/third partial hepatectomy (PH), suggesting that NEFL expression in the liver is a specific feature of cancer onset and development. The ectopic expression of NEFL at all stages of rat carcinogenesis implies that NEFL may function as an oncogene in the onset and development of HCC. To assess the clinical significance of NEFL expression in human HCC patients, we determined NEFL mRNA levels in 14 HCC patients. The results showed that, similarly to what observed in the rat model, while NEFL expression was undetectable in normal human liver, it was readily detectable in HCCs and in matched cirrhotic liver. This demonstrates that aberrant expression of NEFL takes place at the onset and progression in human hepatocarcinogenesis, translating our findings to human pathology. Interestingly, although not statistically significant, a clear trend towards an up-regulation of NEFL in HCCs when compared to matched cirrhotic tissue was observed. Notably, when patients were divided into two groups, NEFL low- or high-expressors, based on the median expression level, the results showed that time of recurrence was significantly shortened in high-NEFL expression compared to low-NEFL expression group, thus demonstrating that NEFL expression levels are a predictive factor for HCC prognosis. Searching for possible mechanisms underlying NEFL enhanced expression, and to determine if the up-regulation of NEFL in the carcinogenic process could be related to promoter methylation status, we analyze the CpG island described in UCSC Genome browser in the 5’ flanking genomic region of NEFL gene. Analysis of the methylation status of 10 consecutive CpG dinucleotides in 5 HCC and 3 normal rat livers exhibited no differences in the methylation status in HCC and controls. These results suggest that DNA hypomethylation of the NEFL promoter region is not involved in the regulation of the expression of this gene. Moreover, no change in NEFL-targeting microRNAs could be found in pre- or neoplastic lesions compared to normal liver. Conclusions: These results unveil the yet unknown role of NEFL in the onset and development of HCC and suggest that NEFL could act as an oncogene in the liver. Further studies are required to identify the mechanism(s) involved in regulating the expression of the NEFL gene and their exact role in HCC development. A better knowledge of the role of NEFL might hopefully provide a novel therapeutic target in HCC progression

Genetic and clinical characteristics of NEFL-related Charcot-Marie-Tooth disease

OBJECTIVES: To analyse and describe the clinical and genetic spectrum of Charcot-Marie-Tooth disease (CMT) caused by mutations in the neurofilament light polypeptide gene (NEFL). METHODS: Combined analysis of newly identified patients with NEFL-related CMT and all previously reported cases from the literature. RESULTS: Five new unrelated patients with CMT carrying the NEFL mutations P8R and N98S and the novel variant L311P were identified. Combined data from these cases and 62 kindreds from the literature revealed four common mutations (P8R, P22S, N98S and E396K) and three mutational hotspots accounting for 37 (55%) and 50 (75%) kindreds, respectively. Eight patients had de novo mutations. Loss of large-myelinated fibres was a uniform feature in a total of 21 sural nerve biopsies and 'onion bulb' formations and/or thin myelin sheaths were observed in 14 (67%) of them. The neurophysiological phenotype was broad but most patients with E90K and N98S had upper limb motor conduction velocities <38 m/s. Age of onset was ≤3 years in 25 cases. Pyramidal tract signs were described in 13 patients and 7 patients were initially diagnosed with or tested for inherited ataxia. Patients with E90K and N98S frequently presented before age 3 years and developed hearing loss or other neurological features including ataxia and/or cerebellar atrophy on brain MRI. CONCLUSIONS: NEFL-related CMT is clinically and genetically heterogeneous. Based on this study, however, we propose mutational hotspots and relevant clinical-genetic associations that may be helpful in the evaluation of NEFL sequence variants and the differential diagnosis with other forms of CMT

Microarray gene expression profiling of neural tissues in bovine spastic paresis

Abstract: Background: Bovine Spastic Paresis (BSP) is a neuromuscular disorder which affects both male and female cattle. BSP is characterized by spastic contraction and overextension of the gastrocnemious muscle of one or both limbs and is associated with a scarce increase in body weight. This disease seems to be caused by an autosomal and recessive gene, with incomplete penetration, although no genes clearly involved with its onset have been so far identified. We employed cDNA microarrays to identify metabolic pathways affected by BSP in Romagnola cattle breed. Investigation of those pathways at the genome level can help to understand this disease. Results: Microarray analysis of control and affected individuals resulted in 268 differentially expressed genes. These genes were subjected to KEGG pathway functional clustering analysis, revealing that they are predominantly involved in Cell Communication, Signalling Molecules and Interaction and Signal Transduction, Diseases and Nervous System classes. Significantly enriched KEGG pathway's classes for the differentially expressed genes were calculated; interestingly, all those significantly under-expressed in the affected samples are included in Neurodegenerative Diseases. To identify genome locations possibly harbouring gene(s) involved in the disease, the chromosome distribution of the differentially expressed genes was also investigated. Conclusions: The cDNA microarray we used in this study contains a brain library and, even if carrying an incomplete transcriptome representation, it has proven to be a valuable tool allowing us to add useful and new information to a poorly studied disease. By using this tool, we examined nearly 15000 transcripts and analysed gene pathways affected by the disease. Particularly, our data suggest also a defective glycinergic synaptic transmission in the development of the disease and an alteration of calcium signalling proteins. We provide data to acquire knowledge of a genetic disease for which literature still presents poor results and that could be further and specifically analysed in the next future. Moreover this study, performed in livestock, may also harbour molecular information useful for understanding human diseases

Translatable plasma and CSF biomarkers for use in mouse models of Huntington’s disease

Huntington's disease is an inherited neurodegenerative disorder for which a wide range of disease-modifying therapies are in development and the availability of biomarkers to monitor treatment response is essential for the success of clinical trials. Baseline levels of neurofilament light chain in CSF and plasma have been shown to be effective in predicting clinical disease status, subsequent clinical progression and brain atrophy. The identification of further sensitive prognostic fluid biomarkers is an active research area, and total-Tau and YKL-40 levels have been shown to be increased in CSF from Huntington's disease mutation carriers. The use of readouts with clinical utility in the preclinical assessment of potential therapeutics should aid in the translation of new treatments. Here, we set out to determine how the concentrations of these three proteins change in plasma and CSF with disease progression in representative, well-established mouse models of Huntington's disease. Plasma and CSF were collected throughout disease progression from R6/2 transgenic mice with CAG repeats of 200 or 90 codons (R6/2:Q200 and R6/2:Q90), zQ175 knock-in mice and YAC128 transgenic mice, along with their respective wild-type littermates. Neurofilament light chain and total-Tau concentrations were quantified in CSF and plasma using ultrasensitive single-molecule array (Quanterix) assays, and a novel Quanterix assay was developed for breast regression protein 39 (mouse homologue of YKL-40) and used to quantify breast regression protein 39 levels in plasma. CSF levels of neurofilament light chain and plasma levels of neurofilament light chain and breast regression protein 39 increased in wild-type biofluids with age, whereas total-Tau remained constant. Neurofilament light chain and breast regression protein 39 were elevated in the plasma and CSF from Huntington's disease mouse models, as compared with wild-type littermates, at presymptomatic stages, whereas total-Tau was only increased at the latest disease stages analysed. Levels of biomarkers that had been measured in the same CSF or plasma samples taken at the latest stages of disease were correlated. The demonstration that breast regression protein 39 constitutes a robust plasma biomarker in Huntington's disease mouse models supports the further investigation of YKL-40 as a CSF biomarker for Huntington's disease mutation carriers. Neurofilament light chain and Tau are considered markers of neuronal damage, and breast regression protein 39 is a marker of inflammation; the similarities and differences in the levels of these proteins between mouse models may provide future insights into their underlying pathology. These data will facilitate the use of fluid biomarkers in the preclinical assessment of therapeutic agents for Huntington's disease, providing readouts with direct relevance to clinical trials