Accumulation Of Neurofilaments Research Articles

HEREDITARY NEUROPATHIES & ALS: P.112 Description of muscular involvement in a NEFL-caused neurological disease

Neurofilament light chain (NEFL) protein is one of the neurofilament-core-subunits, forming heterodimers. Toxic neurofilament-accumulation is a hallmark of many neurodegenerative disorders and the potential of NEFL to serve as a serum biomarker in different neurological disorders was investigated. NEFL mutations cause demyelinating, axonal and intermediate forms of Charcot-Marie-Tooth disease (CMT), whereby most of the mutations are dominant missense variants functioning through a gain-of-function mechanism by perturbing neurofilament assembly and organelle transportation in axons. Muscular involvement is described as a clinical feature in CMT2E patients mostly recognized as a secondary effect but overlapping myopathic and neurogenic findings were described in some cases suggesting that NEFL-mutations might be also causative for heterogeneous neuromuscular diseases. However, studies focusing on NEFL-based myopathological changes are still rare. Molecular genetic testing, histopathological, ultra-morphological and muscle proteome studies were done in an 8-year-old boy presenting with muscle weakness, myalgia and cramps. Phenotyping revealed reduced endurance (walking distance <500m), muscle stiffness accompanied by slowly progressive gait disturbances, brisk reflex levels, sensory ataxia, pes cavus, small CMAP, slightly reduced NCV and prolonged distal latencies. CK was 205 U/l. A heterozygous de novo NEFL variant (c310.T>G, p.Phe104Val) was identified. Electron microscopy showed focal Z-band alterations and minicores in some myofibers indicative for myofibrillar disintegration. Proteomic findings are indicative for a vulnerability of proteins beyond cytoskeleton. Our findings provide new morphological and biochemical insights into vulnerability of (proximal) muscle upon the presence of a dominant NEFL missense mutation and support the concept that NEFL might be causative for heterogeneous neuromuscular diseases.

Heterochrony of postnatal accumulation of nonphosphorylated heavy-chain neurofilament by neurons of the cat dorsal lateral geniculate nucleus.

Accumulation of the heavy-chain neurofilaments reflects the maturation status of neuronal structures. The spatial distribution and postnatal developmental dynamic of neurons expressing nonphosphorylated heavy-chain neurofilaments (labeled by SMI-32 antibody) were analyzed in the dorsal lateral geniculate nucleus (LGNd) of the cat. Both interlaminar and intralaminar differences in the dynamic of SMI-32 staining were observed. The following results were obtained: (a) Ascending dorsoventral gradient in the density of SMI-32 immunopositive (SMI-32(+)) neurons (the greatest neuronal density in layer Cm, the minor in the top sublayer of layer A). This gradient was most prominent at the earliest stages of postnatal development (1st-2nd weeks) and slowly flattened up to adulthood; (b) Layer A1 exhibits increases in SMI-32-positive cells earlier than layer A; (c) The general transient increment in the number and density of SMI-32(+) neurons around 2-5 postnatal weeks. Since SMI-32 antibody is considered to be a putative marker for Y cells forming a motion processing stream, we suggest that peculiarities of SMI-32 staining at geniculate level could reflect the heterogeneity of Y cell subpopulations and the heterochrony of their postnatal maturation.

Functional Characterization of Neurofilament Light Splicing and Misbalance in Zebrafish.

Neurofilaments (NFs), a major cytoskeletal component of motor neurons, play a key role in the differentiation, establishment and maintenance of their morphology and mechanical strength. The de novo assembly of these neuronal intermediate filaments requires the presence of the neurofilament light subunit (NEFL), whose expression is reduced in motor neurons in amyotrophic lateral sclerosis (ALS). This study used zebrafish as a model to characterize the NEFL homologue neflb, which encodes two different isoforms via a splicing of the primary transcript (neflbE4 and neflbE3). In vivo imaging showed that neflb is crucial for proper neuronal development, and that disrupting the balance between its two isoforms specifically affects the NF assembly and motor axon growth, with resultant motor deficits. This equilibrium is also disrupted upon the partial depletion of TDP-43 (TAR DNA-binding protein 43), an RNA-binding protein encoded by the gene TARDBP that is mislocalized into cytoplasmic inclusions in ALS. The study supports the interaction of the NEFL expression and splicing with TDP-43 in a common pathway, both biologically and pathogenetically.

Neurofilament Proteins as Prognostic Biomarkers in Neurological Disorders.

Neurofilaments: light, medium, and heavy (abbreviated as NF-L, NF-M, and NF-H, respectively), which belong to Type IV intermediate filament family (IF), are neuron-specific cytoskeletal components. Neurofilaments are axonal structural components and integral components of synapses, which are important for neuronal electric signal transmissions along the axons and post-translational modification. Abnormal assembly of neurofilaments is found in several human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy (SMA), and hereditary sensory-motor neuropathy (HSMN). In addition, those pathological neurofilament accumulations are known in α-synuclein in Parkinson's disease (PD), Aβ and tau in Alzheimer's disease (AD), polyglutamine in CAG trinucleotide repeat disorders, superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP43), neuronal FUS proteins, optineurin (OPTN), ubiquilin 2 (UBQLN2), and dipeptide repeat protein (DRP) in amyotrophic lateral sclerosis (ALS). When axon damage occurs in central nervous disorders, neurofilament proteins are released and delivered into cerebrospinal fluid (CSF), which are then circulated into blood. New quantitative analyses and assay techniques are well-developed for the detection of neurofilament proteins, particularly NF-L and the phosphorylated NF-H (pNF-H) in CSF and serum. This review discusses the potential of using peripheral blood NF quantities and evaluating the severity of damage in the nervous system. Intermediate filaments could be promising biomarkers for evaluating disease progression in different nervous system disorders.

Charcot-Marie-Tooth disease Type 2E/1F mutant neurofilament proteins assemble into neurofilaments.

Charcot-Marie-Tooth disease Type 2E/1F (CMT2E/1F) is a peripheral neuropathy caused by mutations in neurofilament protein L (NFL), which is one of five neurofilament subunit proteins that co-assemble to form neurofilaments in vivo. Prior studies on cultured cells have shown that CMT2E/1F mutations disrupt neurofilament assembly and lead to protein aggregation, suggesting a possible disease mechanism. However, electron microscopy of axons in peripheral nerve biopsies from patients has revealed accumulations of neurofilament polymers of normal appearance and no evidence of protein aggregates. To reconcile these observations, we reexamined the assembly of seven CMT2E/1F NFL mutants in cultured cells. None of the mutants assembled into homopolymers in SW13vim- cells, but P8R, P22S, L268/269P, and P440/441L mutant NFL assembled into heteropolymers in the presence of neurofilament protein M (NFM) alone, and N98S, Q332/333P, and E396/397K mutant NFL assembled in the presence of NFM and peripherin. P8R, P22S, N98S, L268/269P, E396/397K, and P440/441L mutant NFL co-assembled into neurofilaments with endogenous NFL, NFM, and α-internexin in cultured neurons, although the N98S and E396/397K mutants showed reduced filament incorporation, and the Q332/333P mutant showed limited incorporation. We conclude that all the mutants are capable of assembling into neurofilaments, but for some of the mutants this was dependent on the identity of the other neurofilament proteins available for co-assembly, and most likely also their relative expression level. Thus, caution should be exercised when drawing conclusions about the assembly capacity of CMT2E/1F mutants based on transient transfections in cultured cells.

Correct expression and localization of collagen XIII are crucial for the normal formation and function of the neuromuscular system.

Transmembrane collagen XIII has been linked to maturation of the musculoskeletal system. Its absence in mice (Col13a1-/- ) results in impaired neuromuscular junction (NMJ) differentiation and function, while transgenic overexpression (Col13a1oe ) leads to abnormally high bone mass. Similarly, loss-of-function mutations in COL13A1 in humans produce muscle weakness, decreased motor synapse function and mild dysmorphic skeletal features. Here, analysis of the exogenous overexpression of collagen XIII in various muscles revealed highly increased transcript and protein levels, especially in the diaphragm. Unexpectedly, the main location of exogenous collagen XIII in the muscle was extrasynaptic, in fibroblast-like cells, while some motor synapses were devoid of collagen XIII, possibly due to a dominant negative effect. Concomitantly, phenotypical changes in the NMJs of the Col13a1oe mice partly resembled those previously observed in Col13a1-/- mice. Namely, the overall increase in collagen XIII expression in the muscle produced both pre- and postsynaptic abnormalities at the NMJ, especially in the diaphragm. We discovered delayed and compromised acetylcholine receptor (AChR) clustering, axonal neurofilament aggregation, patchy acetylcholine vesicle (AChV) accumulation, disrupted adhesion of the nerve and muscle, Schwann cell invagination and altered evoked synaptic function. Furthermore, the patterns of the nerve trunks and AChR clusters in the diaphragm were broader in the adult muscles, and already prenatally in the Col13a1oe mice, suggesting collagen XIII involvement in the development of the neuromuscular system. Overall, these results confirm the role of collagen XIII at the neuromuscular synapses and highlight the importance of its correct expression and localization for motor synapse formation and function.

The Wnt/β-catenin signaling pathway affects the distribution of cytoskeletal proteins in Aβ treated PC12 cells.

Alzheimer's disease is pathologically characterized by the presence of senile plaques and neurofibrillary tangles in the central nervous system. Amyloid β-protein is toxic to neurons and induces phosphorylation of Tau protein, which accumulates in paired helical filaments and leads to the formation of neurofibrillary tangles. This study is focused on the Wnt/β-catenin pathway influence on Tau phosphorylation and the distribution of microtubules and neurofilaments in adrenal pheochromocytoma cells. It was found that neurofilament heavy polypeptide and microtubule-associated protein-2 aggregated after treatment with Aβ₁₋₄₂. Treatment with Wnt5a reduced this aggregation, while Dickkopf-1 treatment promoted microtubule and neurofilament aggregation. Furthermore, Tau phosphorylation at Ser396, Ser422, and Ser199 was significantly reduced after Wnt5a treatment, whereas Dickkopf-1 increased the level of phosphorylation. These results suggest that the Wnt/β-catenin pathway influences the distribution of microtubules and neurofilaments, possibly by modulating the phosphorylation of Tau protein in adrenal pheochromocytoma cells.

Neuronal apoptosis, axon damage and synapse loss occur synchronously in acute ocular hypertension

Retinal ganglion cells (RGCs) apoptosis and their axon degeneration are pivotal features in glaucoma. Previous studies suggest that the process of RGCs soma degeneration is distinct from axon degeneration and that both of them lead to vision loss but separately. However, since a normal visual function relies on the integrity of axon, synapse and soma in the retina, a comprehensive understanding of the changes of these neuron components in glaucoma is desired. Therefore, in an acute ocular hypertension (AOH) model in mice, we systematically evaluated retinal neuron soma, axon and synapse alteration at certain time points. We found that ocular hypertension led to a progressive apoptosis of retinal neural cells which proceeded from peripheral to central retina in the wholemount, meanwhile, started in the ganglion cell layer (GCL) and spread to the inner nuclear layer (INL) and then the outer nuclear layer (ONL) as time went on. The type of apoptotic cells was identified as RGCs in GCL, amacrine cells in INL and cone photoreceptor cells in ONL. Axon degeneration was observed at the same time as soma degenerated and also progressed from peripheral to central retina. More interestingly, accumulation of neurofilament in the soma caused by axon transport failure was detected synchronously. We also found that presynaptic and postsynaptic vesicle proteins were downregulated. Taken together, these data support a view that retinal neuronal apoptosis happens not only in RGCs, but also other neurons in laminar layers. Axon damage and synapse loss occur synchronously with soma loss in AOH. The combination of these three parameters might facilitate a systematic evaluation of the disease progression and treatment strategies in glaucoma.

Local Acceleration of Neurofilament Transport at Nodes of Ranvier.

Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.

Identification and immunophenotype of abnormal cells present in focal cortical dysplasia type IIb

BackgroundFocal cortical dysplasias (FCDs) are malformations of cortical development that present cortical dyslamination and abnormal cell morphology and are frequently associated with refractory epilepsy. FCD type IIb presents dysmorphic neurons (DNs) and balloon cells (BCs), which are the hallmarks of this dysplasia. Moreover, hypertrophic neurons (HyNs) may be present in FCD types I, II and III. The objective of this study was to perform a detailed morphology and immunophenotype study of BCs, DNs, and HyNs in a cohort of FCD IIb patients.MethodsCortices resected as a treatment for refractory epilepsy from 18 cases of FCD type IIb were analysed using Bielschowsky method and haematoxylin and eosin as routine stains. Immunophenotype was performed using specific antibodies to detect epitopes differentially expressed by abnormal cells.ResultsAll cases showed cortical dyslamination, BCs, DNs, and HyNs. No cell layer or column could be identified, except for cortical layer I. Lesions predominated in the frontal cortex (11 cases). DNs were large neurons and presented a clumped and or displaced Nissl substance towards the cell membrane, and a cytoplasm accumulation of neurofilament that displaced the nucleus to the cell periphery, as shown by Bielschowsky staining and immunohistochemistry. HyNs were as large as DNs, but without alterations of Nissl substance or dense neurofilament accumulation, with a central nucleus. BCs were identified as large, oval-shaped and pale eosinophilic cells, which lacked the Nissl substance, and presented an eccentric nucleus. BCs and DNs expressed epitopes of both undifferentiated and mature cells, detected using antibodies against nestin, vimentin, class III β-tubulin, pan-neuronal filaments, neurofilament proteins, β-tubulin and NeuN. Only BCs expressed GFAP.ConclusionFCDs present with disorganization of the cerebral cortex architecture, abnormal cell morphology, are frequently associated with refractory epilepsy, and their post-surgical prognosis depends on the type of FCD. The diagnosis of focal cortical dysplasia in a surgical specimen relies on the identification of the abnormal cells present in a dysplastic cortex specimen. The current report contributes to the identification of balloon cells, dysmorphic and hypertrophic neurons in the context of focal cortical dysplasia type IIb.

Alteration of O-GlcNAcylation affects assembly and axonal transport of neurofilament via phosphorylation

Neurofilaments (NFs), the most abundant cytoskeletal components in the mature neuron, are hyperphosphorylated and accumulated in the neuronal cell body of AD brain, and the abnormalities of NFs appear to contribute to neurodegeneration. Although previous studies have showed that O-GlcNAcylation and phosphorylation of NFs regulate each other reciprocally, the NFs O-GlcNAcylation and its effects on assembly and axonal transport are poorly explored. Here, we focus on the role of dysregulation of O-GlcNAcylation on structure and function of neurofilaments by corresponding phosphorylation.In the study, we found that decreased O-GlcNAcylation by intracerebroventricular administration of Alloxan, 6-diazo-5-oxonorleucine (Don) and okadaic acid (OA) in the rats resulted in increased phosphorylation with assembly of lower and shorter NFs. In contrast, in the sample of NAG-thiazoline (NAG-Ae) causing increased O-GlcNAcylation, NFs showed elongated filaments fibers and higher proportion of assembly. Furthermore, alloxan treatment induced abnormal accumulation of NFs bodies and delayed time of Fluorescence Recovery After Photobleaching (FRAP) in SK-N-SH cells, but the NAG-Ae treatment speeded up the axonal transport.Our experiments suggest that increased O-GlcNAcylation plays a key role in protecting the structure and function of NFs including filament assembly and axonal transport via decreased phosphorylation. These results expanded the function of O-GlcNAcylation in AD pathogenesis.

Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics.

Loss of sacsin, a large 520 kDa multidomain protein, causes autosomal recessive spastic ataxia of the Charlevoix-Saguenay, one of the most common childhood-onset recessive ataxias. A prominent feature is abnormal bundling of neurofilaments in many neuronal populations. This study shows the direct involvement of sacsin domains in regulating intermediate filament assembly and dynamics and identifies important domains for alleviating neurofilament bundles in neurons lacking sacsin. Peptides encoding sacsin internal repeat (SIRPT) 1, J-domains, and ubiquitin-like domain modified neurofilament assembly in vivo. The domains with chaperone homology, the SIRPT and the J-domain, had opposite effects, promoting and preventing filament assembly, respectively. In cultured Sacs-/- motor neurons, both the SIRPT1 and J-domain resolved preexisting neurofilament bundles. Increasing expression of heat shock proteins also resolved neurofilament bundles, indicating that this endogenous chaperone system can compensate to some extent for sacsin deficiency.-Gentil, B. J., Lai, G.-T., Menade, M., Larivière, R., Minotti, S., Gehring, K., Chapple, J.-P., Brais, B., Durham, H. D. Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics.

Phosphorylation, Dephosphorylation, and Multiprotein Assemblies Regulate Dynamic Behavior of Neuronal Cytoskeleton: A Mini-Review.

Cellular localization, assembly and abnormal aggregation of neurofilaments depend on phosphorylation. Pathological processes associated with neurodegeneration exhibit aberrant accumulation of microtubule associated aggregated forms of hyperphosphorylated neuronal protein tau in cell bodies. These processes are critical for the disease progression in patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. In healthy cells, tau is localized in axons. Topographic regulation suggests that whereas the sites of synthesis of kinases and neurofilaments are the cell bodies, and sites of their functional assemblies are axons, phosphorylation/dephosphorylation are the key processes that arrange the molecules at their precise locations. Phosphorylation sites in the dynamic developmental and degenerative processes differ. Not all these processes are well understood. New advancements identify epigenetic factors involved in AD which account for the influence of age-related environment/genome interactions leading to the disease. Progress in proteomics highlights previously found major proteins and adds more to the list of those involved in AD. New key elements of specificity provide determinants of molecular recognition important for the assembly of macromolecular complexes. In this review, we discuss aberrant spatial distribution of neuronal polypeptides observed in neuropathies: aggregation, association with proteins of the neuronal cytoskeleton, and phosphorylation dependent dynamics. Particularly, we emphasize recent advancements in understanding the function and determinants of specific association of molecules involved in Alzheimer’s disease with respect to the topographic regulation of phosphorylation in neuronal cytoskeleton and implications for the design of new therapies. Further, we address the role of various filament systems in maintenance of the shape, rigidity and dynamics of the cytoskeleton.

NFL in CSF and serum, a potential translational dynamic biomarker of neurodegeneration in preclinical models

Accumulation of Neurofilaments (NFs), the major constituents of the neuronal cytoskeleton, is a distinctive feature of neurological diseases and several studies have shown that soluble NFs can be detected in the cerebrospinal fluid (CSF) of patients with neurological diseases, such as multiple sclerosis and frontotemporal dementia. We have used an inducible transgenic mouse model of neurodegeneration, CamKII-TetOp25 mice (Tsai et al., 2003), to evaluate whether NF-L levels in CSF or blood can be used as a biochemical biomarker of neurodegeneration. Induction of p25 transgene brain expression led to increases in CSF and serum NF-L levels that correlated with increased neuronal stress and neurodegeneration. When p25 transgene expression was switched off this prevented further increases in both CSF and serum NF-L levels which correlated with the block of further increases in neuronal stress and neuronal loss. The levels of CSF NF-L detected in p25 mice are about 4–fold higher than the CSF levels detected in patients with chronic neurodegenerative diseases, such as symptomatic FTD (bvFTD). We also report increase with aging in CSF and plasma NFL in two other transgenic mouse models, ThyTau22 (Schindowski et al., 2006) and APPxPS1 mice (Balnchard et al., 2003). Finally, Western blot and NanoLC-MS/MS data indicate that the NF-L detected in CSF is most likely a cleaved form of NF-L. These results suggest that CSF and serum NF-L could be used as potential translational dynamic biomarkers of neurodegeneration in preclinical animal models and in clinical studies.

Vestibular findings in patients with schwannoma of the 8th cranial nerve: a survey of nine cases and review of the literature

Introduction: Some aspects of the natural history of vestibular schwannoma (VS) remain unsettled. A better understanding of the pathophysiology of the disease would improve its diagnostic workup and therapeutic management, as VS often requires surgery, which carries the risk of severe complications. This paper discusses the significance of ultrastructural findings in the vestibular organs removed during surgery.Material and methods: This study examined specimens from vestibular organs removed from patients who underwent VS resection through the translabyrinthine approach. The sample includes 9 patients, seven women and two men, with a mean age of 55 years (range 39–71 years).Results: The sensory epithelium showed a loss of stereocilia, numerous lipofuscin granules and vacuoles in the cytoplasm. The subepithelial myelinated nerve fibres showed signs of demyelination and sheath rearrangement. Some axons had markedly degenerative modifications, with neurofilament aggregation, more numerous mitochondria and vacuolization. As it lost its specialized structures, the epithelial covering of the semicircular canal walls progressively flattened out in the area farthest away from the sensorial area.Conclusions: There were no specific correlations between schwannoma size and instrumental or ultrastructural findings in the vestibular epithelium organs. The morphological features of the non-sensory epithelium suggest that the potassium circulation system is preserved in patients with VS even when they present prominent cochlear and/or vestibular symptoms. VS formation appears to be multifactorial; the vestibular nerve conduction block caused by tumour compression would not necessarily cause irreversible damage to the vestibular receptors at the outset.

Myelinated axons fail to develop properly in a genetically authentic mouse model of Charcot-Marie-Tooth disease type 2E

We have analyzed a mouse model of Charcot-Marie-Tooth disease 2E (CMT2E) harboring a heterozygous p.Asn98Ser (p.N98S) Nefl mutation, whose human counterpart results in a severe, early-onset neuropathy. Behavioral, electrophysiological, and pathological analyses were done on separate cohorts of NeflN98S/+ mutant mice and their wild type Nefl+/+ littermates between 8 and 48 weeks of age. The motor performance of NeflN98S/+ mice, as evidenced by altered balance and gait measures, was impaired at every age examined (from 6 to 25 weeks of age). At all times examined, myelinated axons were smaller and contained markedly fewer neurofilaments in NeflN98S/+ mice, in all examined aspects of the PNS, from the nerve roots to the distal ends of the sciatic and caudal nerves. Similarly, the myelinated axons in the various tracts of the spinal cord and in the optic nerves were smaller and contained fewer neurofilaments in mutant mice. The myelinated axons in both the PNS and the CNS of mutant mice had relatively thicker myelin sheaths. The amplitude and the nerve conduction velocity of the caudal nerves were reduced in proportion with the diminished sizes of myelinated axons. Conspicuous aggregations of neurofilaments were only seen in primary sensory and motor neurons, and were largely confined to the cell bodies and proximal axons. There was evidence of axonal degeneration and regeneration of myelinated axons, mostly in distal nerves. In summary, the p.N98S mutation causes a profound reduction of neurofilaments in the myelinated axons of the PNS and CNS, resulting in substantially reduced axonal diameters, particularly of large myelinated axons, and distal axon loss in the PNS.

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

ObjectiveWe 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.MethodsMotor 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.ResultsWe 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.ConclusionsThe 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.

Acrolein-mediated neurotoxicity in growing Wistar male rats

Acrolein is an environmental and food contaminant widely produced by thermal processed organic compounds. It can be used as a biocide and is known to be generated endogenously by lipid peroxidation. This study was designed for evaluating acrolein toxicity in the context of GFAP, MBP, neurofilaments and histological examination of brain tissues and also hematological paremeters in pubertal male rats. 3–4 weeks aged Wistar male rats were exposed to 0.5, 1 and 2 mg/kg/day acrolein for 90 days. According to acrolein exposure results, slight changes observed in hematological parameters, however no significant differences were observed in GFAP and MBP. Unlikely, acrolein caused several histopathological changes in brain tissues as well as neurofilament accumulation. It may be concluded that acrolein might contribute to formation of neurodegenerative disorders.

Schwann-Cell Autophagy, Functional Recovery, and Scar Reduction After Peripheral Nerve Repair.

The functional outcome after peripheral nerve repair is often unpredictable for many reasons, e.g., the severity of neuronal death and scarring. Axonal degeneration significantly affects outcomes. Post-injury axonal degeneration in peripheral nerves is accompanied by myelin degradation initiated by Schwann cells (SCs), which activate autophagy, a ubiquitous cytoprotective process essential for degrading and recycling cellular constituents. Scar formation occurs concomitantly with nerve insult and axonal degeneration. The association between SC autophagy and the mechanisms of nerve scar formation is still unknown. A rat model of peripheral nerve lesions induced by sciatic nerve transection injuries was used to examine the function of autophagy in fibrosis reduction during the early phase of nerve repair. Rats were treated with rapamycin (autophagy inducer) or 3-methyladenine (autophagy inhibitor). One week after the nerve damage, fibrosis was potently inhibited in rapamycin-treated rats and, based on gait analysis, yielded a better functional outcome. Immunohistochemistry showed that the autophagic activity of SCs and the accumulation of neurofilaments were upregulated in rapamycin-treated rats. A deficiency of SC autophagic activity might be an early event in nerve scar formation, and modulating autophagy might be a powerful pharmacological approach for improving functional outcomes.

Whole-Genome Linkage Analysis with Whole-Exome Sequencing Identifies a Novel Frameshift Variant in NEFH in a Chinese Family with Charcot-Marie-Tooth 2: A Novel Variant in NEFH for Charcot-Marie-Tooth 2

Background: Charcot-Marie-Tooth disease (CMT) is the most common neurodegenerative disorder of the peripheral nervous system. More than 50 genes/loci were found associated with the disease. We found a family with autosomal-dominant CMT2. Objective: To reveal the pathogenic gene of the family and further investigate the function of the variant. Methods: DNA underwent whole-genome linkage analysis for all family members and whole-exome sequencing for 2 affected members. Neurofilament light polypeptide and wild-type or mutant neurofilament heavy polypeptide (NEFH) were co-transfected into SW13 (vim–) cells. The nefh-knockdown zebrafish model was produced by using morpholino antisense oligonucleotides. Results: We identified a novel insertion variant (c.3057insG) in NEFH in the family. The variant led to the loss of a stop codon and an extended 41 amino acids in the protein. Immunofluorescence results revealed that mutant NEFH disrupted the neurofilament network and induced aggregation of NEFH protein. Knockdown of nefh in zebrafish caused a slightly or severely curled tail. The motor ability of nefh-knockdown embryos was impaired or even absent, and the embryos showed developmental defects of axons in motor neurons. The abnormal phenotype and axonal developmental defects could be rescued by injection of human wild-type but not human mutant NEFH mRNA. Conclusions: We identified a novel stop loss variant in NEFH that is likely pathogenic for CMT2, and the results provide further evidence for the role of an aberrant assembly of neurofilament in CMT.