Expression Of Neurofilament Light Research Articles

Downregulation of Neurofilament Light Chain Expression in Human Neuronal-Glial Cell Co-Cultures by a Microbiome-Derived Lipopolysaccharide-Induced miRNA-30b-5p.

Microbiome-derived Gram-negative bacterial lipopolysaccharide (LPS) has been shown by multiple laboratories to reside within Alzheimer's disease (AD)-affected neocortical and hippocampal neurons. LPS and other pro-inflammatory stressors strongly induce a defined set of NF-kB (p50/p65)-sensitive human microRNAs, including a brain-enriched Homo sapien microRNA-30b-5p (hsa-miRNA-30b-5p; miRNA-30b). Here we provide evidence that this neuropathology-associated miRNA, known to be upregulated in AD brain and LPS-stressed human neuronal-glial (HNG) cells in primary culture targets the neurofilament light (NF-L) chain mRNA 3'-untranslated region (3'-UTR), which is conducive to the post-transcriptional downregulation of NF-L expression observed within both AD and LPS-treated HNG cells. A deficiency of NF-L is associated with consequent atrophy of the neuronal cytoskeleton and the disruption of synaptic organization. Interestingly, miRNA-30b has previously been shown to be highly expressed in amyloid-beta (Aβ) peptide-treated animal and cell models, and Aβ peptides promote LPS entry into neurons. Increased miRNA-30b expression induces neuronal injury, neuron loss, neuronal inflammation, impairment of synaptic transmission, and synaptic failure in neurodegenerative disease and transgenic murine models. This gut microbiota-derived LPS-NF-kB-miRNA-30b-NF-L pathological signaling network: (i) underscores a positive pathological link between the LPS of gastrointestinal (GI)-tract microbes and the inflammatory neuropathology, disordered cytoskeleton, and disrupted synaptic signaling of the AD brain and stressed brain cells; and (ii) is the first example of a microbiome-derived neurotoxic glycolipid having significant detrimental miRNA-30b-mediated actions on the expression of NF-L, an abundant neuron-specific filament protein known to be important in the maintenance of neuronal cell shape, axonal caliber, and synaptic homeostasis.

NEFL is overexpressed and it modulates invasion and migration in neuroendocrine-like PC3-ML2 prostate cancer cells.

Prostate cancer clinical outcomes are varied, from non-aggressive asymptomatic to lethal aggressive neuroendocrine forms which represent a critical challenge in the management of the disease. The neurofilament light ( NEFL ) is proposed to be a tumor suppressor gene. Studies have shown that expression of the gene is decreased in various cancers. We have used quantitative RT-PCR, immunoblotting, methylation specific PCR, siRNA knockdown followed by migration/invasion assays to determine associations between NEFL expression and disease phenotype in a panel of prostate cells. We demonstrate that NEFL is overexpressed and it modulates invasion and migration in PC3-ML2 prostate cancers cells which have an aggressive neuroendocrine-like phenotype.

Neurofilament Light (NF-L) Chain Protein from a Highly Polymerized Structural Component of the Neuronal Cytoskeleton to a Neurodegenerative Disease Biomarker in the Periphery.

Neurofilaments (NFs) are critical scaffolding components of the axoskeleton of healthy neurons interacting directly with multiple synaptic-phosphoproteins to support and coordinate neuronal cell shape, cytoarchitecture, synaptogenesis and neurotransmission. While neuronal presynaptic proteins such as synapsin-2 (SYN II) degrade rapidly via the ubiquitin-proteasome pathway, a considerably more stable neurofilament light (NF-L) chain protein turns over much more slowly, and in several neurological diseases is accompanied by a pathological shift from an intracellular neuronal cytoplasmic location into various biofluid compartments. NF-L has been found to be significantly elevated in peripheral biofluids in multiple neurodegenerative disorders, however it is not as widely appreciated that NF-L expression within neurons undergoing inflammatory neurodegeneration exhibit a significant down-regulation in these neuron-specific intermediate-filament components. Down-regulated NF-L in neurons correlates well with the observed axonal and neuronal atrophy, neurite deterioration and synaptic disorganization in tissues affected by Alzheimer’s disease (AD) and other progressive, age-related neurological diseases. This Review paper: (i) will briefly assess the remarkably high number of neurological disorders that exhibit NF-L depolymerization, liberation from neuron-specific compartments, mobilization and enrichment into pathological biofluids; (ii) will evaluate how NF-L exhibits compartmentalization effects in age-related neurological disorders; (iii) will review how the shift of NF-L compartmentalization from within the neuronal cytoskeleton into peripheral biofluids may be a diagnostic biomarker for neuronal-decline in all cause dementia most useful in distinguishing between closely related neurological disorders; and (iv) will review emerging evidence that deficits in plasma membrane barrier integrity, pathological transport and/or vesicle-mediated trafficking dysfunction of NF-L may contribute to neuronal decline, with specific reference to AD wherever possible.

Expression of GSK3β, PICK1, NEFL, C4, NKCC1 and Synaptophysin in peripheral blood mononuclear cells of the first-episode schizophrenia patients.

Schizophrenia (SZ) is a severe neurodevelopmental disease with unknown pathogenic mechanisms characterized with impaired cognitive function. The disturbed synaptic plasticity and synaptic loss have been widely reported in SZ. In this study, 41 first-episode schizophrenia (FES) patients and 44 healthy controls (HC) were recruited and the expression of six genes commonly relevant to synaptic functions was examined in the peripheral blood mononuclear cells (PBMCs). These genes were glycogen synthase kinase 3β (GSK3β), protein interacting with C-kinase 1 (PICK1), synaptophysin (SYP), neurofilament light (NEFL), complement component 4 (C4) and Na+-K--2Cl- cotransporter 1 (NKCC1). Real-time quantitative polymerase chain reaction (qPCR) was performed to determine the quantity of individual mRNA template. Compared to HC, the expression of PICK1 and NKCC1 genes in FES patients was relatively lower whereas the expression of NEFL was higher. No difference for the mRNA expression of GSK3β, SYP and C4 genes was detected between FES patients and HC, nor was the gender difference; Interestingly, the mRNA expression of PICK1 in female FES patients was significantly decreased compared to female HC, but not in males; and the NEFL gene was up-regulated in male FES patients but not in females. Our findings support an abnormal expression profile of synapse-related genes in the PBMCs of FES patients.

Neurofilament light chain level in traumatic brain injury: A system review and meta-analysis.

Background:Neurofilament light (NfL) level was obviously increased in traumatic brain injury (TBI) individuals. But, no comprehensive meta-analysis has ever been conducted to assess the diagnostic performance of NfL. This study aims to evaluate the relationship between NfL level and TBI through a meta-analysis.Methods:Studies were selected from Pubmed, Web of science, Embase, Google Scholar, PMC and Chinese National Knowledge Infrastructure (CNKI), and the Chinese Biomedical Literature Database (CBM) through inclusion and exclusion criteria. The standard mean difference (SMD) and 95% confidence interval (CI) were calculated using the random-effect model or fixed-effect model to assess the association between NfL level and TBI. Subgroup analysis according to sample collection time, sample type and detection method was performed. The influence analysis and publication bias was also conducted. All analyses were performed using the RevMan 5.3 and Stata 12 software.Results:A total of 9 studies were included. Results indicated that TBI individuals had a higher NfL expression level compared with the non-TBI individuals (SMD = 2.48, 95% CI = 1.52–3.43, I2 = 96%, P < .01). Similar NfL increasing was also observed in Caucasian population, 0–48 hour and 6–10 days sample collection time, as well as cerebrospinal fluid (CSF), serum, plasma sample subgroup analysis. Moreover, the NfL increasing still existed no matter the NfL expression level was detected by ELISA or Simoa assay.Conclusion:NfL expression level was increased in TBI individuals, which indicated that NfL could be a potential biomarker in the diagnosis of TBI and other neurodegenerative diseases.

Japan realizes sharp rise in lead recovery from scrap batteries

Dioxin exposure is reported to affect nervous system development and increase the risk of neurodegenerative diseases. Generally, dioxin exerts its neurotoxicity via aryl hydrocarbon receptor (AhR). Neurofilament (NF) light (NFL) protein is a biomarker for both neuronal differentiation and neurodegeneration and its expression is controlled by the mitogen-activated protein kinase (MAPK) pathway. However, the effects of dioxin on NFL expression and involved mechanisms are incompletely understood. We aimed to investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on NFL expression and elucidate the underlining signaling pathways and their potential crosstalk, specifically between MAPK and AhR pathway. We employed primary cultured rat cortical neurons to evaluate the effect of TCDD exposure on NFL expression. We also used nerve growth factor (NGF)-treated PC12 cells with specific inhibitors to investigate the involvement of and potential crosstalk between the MAPK pathway and the AhR pathway in mediating the effects of TCDD on NFL expression. After TCDD exposure, NFL mRNA and protein levels were upregulated in cultured neurons. NFL protein was preferentially found in the cell body compared with neurites of the cultured neurons. In PC12 cells, TCDD enhanced both NGF-induced NFL expression and phosphorylation of ERK1/2 and p38. The addition of MAPK-pathway inhibitors (PD98059 and SB230580) partially blocked the TCDD-induced NFL upregulation. CH223191, an AhR antagonist, reversed the upregulation of NFL and phosphorylation of ERK1/2 and p38 induced by TCDD. This study demonstrated TCDD-induced upregulation of NFL in cultured neurons, with protein retained in the cell body. TCDD action was dependent on activation of AhR and MAPK, while crosstalk was found between these two signaling pathways.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and up-regulation of neurofilament expression in neuronal cells: Evaluation of AhR and MAPK pathways

Dioxin exposure is reported to affect nervous system development and increase the risk of neurodegenerative diseases. Generally, dioxin exerts its neurotoxicity via aryl hydrocarbon receptor (AhR). Neurofilament (NF) light (NFL) protein is a biomarker for both neuronal differentiation and neurodegeneration and its expression is controlled by the mitogen-activated protein kinase (MAPK) pathway. However, the effects of dioxin on NFL expression and involved mechanisms are incompletely understood. We aimed to investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on NFL expression and elucidate the underlining signaling pathways and their potential crosstalk, specifically between MAPK and AhR pathway. We employed primary cultured rat cortical neurons to evaluate the effect of TCDD exposure on NFL expression. We also used nerve growth factor (NGF)-treated PC12 cells with specific inhibitors to investigate the involvement of and potential crosstalk between the MAPK pathway and the AhR pathway in mediating the effects of TCDD on NFL expression. After TCDD exposure, NFL mRNA and protein levels were upregulated in cultured neurons. NFL protein was preferentially found in the cell body compared with neurites of the cultured neurons. In PC12 cells, TCDD enhanced both NGF-induced NFL expression and phosphorylation of ERK1/2 and p38. The addition of MAPK-pathway inhibitors (PD98059 and SB230580) partially blocked the TCDD-induced NFL upregulation. CH223191, an AhR antagonist, reversed the upregulation of NFL and phosphorylation of ERK1/2 and p38 induced by TCDD. This study demonstrated TCDD-induced upregulation of NFL in cultured neurons, with protein retained in the cell body. TCDD action was dependent on activation of AhR and MAPK, while crosstalk was found between these two signaling pathways.

Association of Acute Increase in Plasma Neurofilament Light with Repetitive Subconcussive Head Impacts: A Pilot Randomized Control Trial.

The purpose of the study was to examine an association of repetitive subconcussive head impacts with changes in plasma neurofilament light (NF-L) levels following 10 bouts of controlled soccer heading. In this randomized control trial, 37 healthy adult soccer players were randomly assigned into either a heading (n = 19) or kicking-control group (n = 18). The heading group executed 10 headers with soccer balls projected at a velocity of 25 mph over 10 min. Plasma samples were obtained at pre-heading baseline, 0 h, 2 h, and 24 h post-heading. The kicking-control group followed the same protocol with 10 kicks. Plasma NF-L was measured using ultrasensitive single-molecule array technology. Data from 34 subjects were eligible for analysis (heading n = 18 and kicking n = 16). Ten subconcussive head impacts induced a gradual increase in plasma NF-L expression for the heading group (β = 0.0297, standard error [SE] = 0.01, p = 0.0049), whereas there was no significant time effect for the kicking-control group. A follow-up analysis revealed that a significant difference appeared at 24 h post-heading (3.68 ± 0.30 pg/mL) compared with pre-heading (3.12 ± 0.29 pg/mL, p = 0.0013; Cohen's d = 1.898). At the 24 h post-heading time-point, the plasma NF-L level for the heading group was significantly higher than that of the kicking-control group with an estimated mean difference of 0.66 pg/mL (SE = 0.22, p = 0.0025). The data suggest that the increased level of plasma NF-L was driven by repetitive subconcussive head impacts and required longer than 2 h after the head impacts for the increase to be detected. Plasma NF-L levels may serve as an objective marker to monitor acute axonal burden from subconcussive head impacts.

Depressed neurofilament expression associates with apolipoprotein E3/E4 genotype in maturing human fetal neurons exposed to HIV-1

Exposure of differentiating human neural progenitor cells (NEP) to HIV-1 results in a neuronal“failure to thrive” phenotype characterized by a relative decrease in neurofilament-light (NF-L) expression. However,when NEP were segregated by their apolipoproteinE genotype, differentiating apolipoprotein E3/E4 cells showed reduced NF-L expression upon HIV-1 exposure,but differentiating apolipoprotein E3/E3 or apolipoproteinE4/E4 cells did not. These data suggest that apolipoproteinE genotype is a host factor that could affect the development of neurocognitive dysfunction in HIV-1 infected individuals.

Axotomy-induced cytoskeleton changes in unmyelinated mammalian central nervous system axons

Oligodendrocyte-derived myelin retards the ability of CNS axons to regenerate following transection. The intrinsic response of CNS axons to an axotomy insult may be vastly different in the absence of myelin. However, the paucity of adequate experimental models has limited detailed investigation of cellular behaviour following axon transection in an unmyelinated CNS environment. In this study we perform laser-induced axotomy of the porcine retinal ganglion cell axon, a physiologically unmyelinated, mature CNS axon that is structurally similar to humans to infer knowledge about axonal behaviour in the absence of myelin. Axotomy-induced changes to the neuronal cytoskeleton and supporting astrocytes during the early stages after transection are delineated by examining the sequence of neurofilament subunit, microtubule (TUB), microtubule associated protein (MAP), glial fibrillary acidic protein (GFAP) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) modification. Axonal transection induced an increase in the expression of neurofilament light at regions within, and immediately adjacent to, sites of axotomy. Other neurofilament subunits were not altered at sites of transection. Unlike myelinated axons where an increase in GFAP staining within hypertrophic glial scars have been shown to inhibit axonal repair we demonstrate a decrease in GFAP staining within regions of increased or preserved neurofilament expression. The behaviour of TUB and MAP proteins following transection of unmyelinated CNS axons are similar to what has previously been described in myelinated CNS axons. This study provides fundamental insights into astrocyte and axonal behaviour acutely after axotomy and demonstrates a series of degenerative events in unmyelinated CNS axons, which in comparison to prior reports are different to myelinated CNS axons. The findings of this report have relevance to understanding pathogenic mechanisms underlying neuro-degeneration in the CNS.

Lamina-Specific Abnormalities of NMDA Receptor-Associated Postsynaptic Protein Transcripts in the Prefrontal Cortex in Schizophrenia and Bipolar Disorder

The hypothesis of N-methyl-D-aspartate (NMDA) receptor hypofunction in schizophrenia was initially based on observations that blockade of the NMDA subtype of glutamate receptor by noncompetitive antagonists, such as phencyclidine and ketamine, can lead to clinical symptoms similar to those present in schizophrenia. Recently, glutamate has also been implicated in the pathophysiology of the mood disorders. As impaired NMDA receptor activity may be the result of a primary defect in the NMDA receptors themselves, or secondary to dysfunction in the protein complexes that mediate their signaling, we measured expression of both NMDA subunits and associated postsynaptic density (PSD) proteins (PSD95, neurofilament-light (NF-L), and SAP102) transcripts in the dorsolateral prefrontal cortex in subjects with schizophrenia, bipolar disorder, major depression, and a comparison group using tissue from the Stanley Foundation Neuropathology Consortium. We found decreased NR1 expression in all three illnesses, decreased NR2A in schizophrenia and major depression, and decreased NR2C in schizophrenia. We found no changes of NR2B or NR2D. Receptor autoradiography revealed no alterations in receptor binding in any of the illnesses, indicating no change in total receptor number, but taken with the subunit data suggests abnormal receptor stoichiometry. In the same subjects, PSD95 was unchanged in all three illnesses, while reduced NF-L expression was found in schizophrenia, especially in large cells of layer V. SAP102 expression was reduced in bipolar disorder restricted to small cells of layer II and large cells of layer III in bipolar disorder. These alterations likely reflect altered signaling cascades associated with glutamate-mediated neurotransmission within specific cortical circuits in these psychiatric illnesses.

Disruption of neurofilament network with aggregation of light neurofilament protein: a common pathway leading to motor neuron degeneration due to Charcot–Marie–Tooth disease-linked mutations in NFL and HSPB1

Mutations in neurofilament light (NFL) subunit and small heat-shock protein B1 (HSPB1) cause autosomal-dominant axonal Charcot-Marie-Tooth disease type 2E (CMT2E) and type 2F (CMT2F). Previous studies have shown that CMT mutations in NFL and HSPB1 disrupt NF assembly and cause aggregation of NFL protein. In this study, we investigate the role of aggregation of NFL protein in the neurotoxicity of CMT mutant NFL and CMT mutant HSPB1 in motor neurons. We find that expression of CMT mutant NFL leads to progressive degeneration and loss of neuronal viability of cultured motor neurons. Degenerating motor neurons show fragmentation and loss of neuritic processes associated with disruption of NF network and aggregation of NFL protein. Co-expression of wild-type HSPB1 diminishes aggregation of CMT mutant NFL, induces reversal of CMT mutant NFL aggregates and reduces CMT mutant NFL-induced loss of motor neuron viability. Like CMT mutant NFL, expression of S135F CMT mutant HSPB1 also leads to progressive degeneration of motor neurons with disruption of NF network and aggregation of NFL protein. Further studies show that wild-type and S135F mutant HSPB1 associate with wild-type and CMT mutant NFL and that S135F mutant HSPB1 has dominant effect on disruption of NF assembly and aggregation of NFL protein. Finally, we show that deletion of NFL markedly reduces degeneration and loss of motor neuron viability induced by S135F mutant HSPB1. Together, our data support the view that disruption of NF network with aggregation of NFL is a common triggering event of motor neuron degeneration in CMT2E and CMT2F disease.

Immunoautoradiographic analysis of NMDA receptor subunits and associated postsynaptic density proteins in the brain of dyskinetic MPTP‐treated common marmosets

l-3,4-dihydroxyphenylalanine methyl ester (l-DOPA)-induced dyskinesia in Parkinson's disease may result from aberrant glutamatergic stimulation of the striatum due to synaptic plasticity in the motor cortex or striatum as a consequence of adaptation of striatal output pathways. This might result from changes in NMDA receptor subunit or NMDA receptor associated postsynaptic density (PSD) scaffold protein expression. Using immunoautoradiography the expression levels of NR1 and NR2B subunits of the NMDA receptor and the postsynaptic density scaffold proteins, PSD-95, PSD-93, and neurofilament light (NFL) were examined in normal common marmosets (Callithrix jacchus) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned animals that exhibited high or low levels of l-DOPA-induced dyskinesia. Brains from MPTP-lesioned animals that were not primed for l-DOPA-induced dyskinesia were not included in this study. No alterations in the NR1 NMDA receptor subunit were observed. The NR2B NMDA receptor subunit was increased in caudal caudate nucleus and putamen, hippocampus, cingulate motor area (CMA), supplementary motor area (SMA) and dorsal primary motor cortex (dMI) of highly dyskinetic MPTP-lesioned marmosets, but not in animals with low levels of dyskinesia. PSD-93 was decreased in the globus pallidus of marmosets with high and low levels of dyskinesia and increased in the CMA, SMA and dMI of highly dyskinetic marmosets. PSD-95 was increased in the SMA of highly dyskinetic marmosets, but not in animals with low dyskinesia. NFL expression was elevated in the SMA and dorsal and ventral MI of highly dyskinetic marmosets. These results suggest that l-DOPA treatment of MPTP-lesioned marmosets can affect glutamatergic systems and indicate that altered NMDA receptor function may relate to dyskinesia.

Interleukin-1beta up-regulates expression of neurofilament light in human neuronal cells.

Elevated expression of interleukin-1 (IL-1beta), a pro-inflammatory cytokine secreted by activated microglia, is a pathogenic marker of numerous neurodegenerative processes including Alzheimer's disease (AD). We have characterized a link between IL-1beta and the 68-kDa neurofilament light (NF-L) protein, which is a major component of the neuronal cytoskeleton. Using human brain aggregate cultures, we found that IL-1beta treatment significantly increased NF-L expression in primary neurons. Analysis of mRNA levels demonstrated elevated NF-L expression within 72 h while imaging of neurons by immunofluorescent staining for NF-L confirmed IL-1beta-induced NF-L protein expression. These observations suggest a potential inflammatory-induced mechanism for deregulation of an important cytoskeletal protein, NF-L, possibly leading to neuronal dysfunction.

Different posttranscriptional controls for the human neurofilament light and heavy genes in transgenic mice

To investigate the mechanisms regulating neurofilament gene expression, we generated transgenic mice with high copy number of the intact human neurofilament light (NF-L) and heavy (NF-H) genes. Overexpression in transgenic mice of NF-L mRNA from 3- to 5-fold in different regions of the central nervous system (CNS) resulted only in a mild increase of 10–50% in the levels of NF-L proteins. The failure to enhance NF-L protein content was not due to interspecies differences in posttranscriptional NF-L regulation. For instance, based on specific immunodetection, it is estimated that human NF-L proteins composed 80% of total NF-L content in the spinal cord of transgenics. In contrast to the situation with NF-L, the CNS of transgenic mice bearing multiple copies of the human NF-H gene showed comparable increases in the levels of NF-H mRNA and proteins. These results suggest that the NF-L and NF-H genes are subject to different posttranscriptional regulation in the CNS. In vivo labeling of newly synthesized proteins by injection of [ 35S]methionine in the spinal cords of normal and transgenic mice provided evidence that the posttranscriptional regulation of NF-L expression in the CNS must occur, at least in part, at the level of translation.