Neurofilament markers in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

This study aims to determine the serum and cerebrospinal fluid (CSF) levels of neurofilament light chain (NFL) and phosphorylated neurofilament heavy chain (pNFH) in amyotrophic lateral sclerosis (ALS) patients, and to explore their feasibility as valid biomarkers for quantifying disease progression and predicting individual prognosis. 52 patients with ALS and 30 controls with noninflammatory neurological diseases were included. NFL and pNFH levels in serum and CSF were measured by enzyme-linked immunosorbent assay. Our findings showed that serum and CSF levels of NFL and pNFH in ALS patients were significantly increased. These values were negatively correlated with disease duration (except CSF NFL with disease duration) and ALSFRS-r score, and positively correlated with disease progression rate (DPR) and upper motor neuron (UMN) score, but did not correlate with bilateral median and ulnar nerve compound muscle action potential (cMAP) amplitudes (except a weak correlation between CSF NFL and cMAP amplitudes). The optimal cut-off values with high sensitivity and specificity were obtained in ROC curve analysis to discriminate ALS from controls. Kaplan-Meier survival curves illustrated that survival was significantly shorter for patients with higher neurofilament levels at diagnosis. The Cox proportional hazards regressions confirmed that NFL and pNFH were significant predictors of survival. Overall, NFL and pNFH in serum and CSF can be used as reliable biomarkers in ALS.

Neurofilament results for the phase II neuroprotection study of phenytoin in optic neuritis.

BACKGROUND: A randomized trial of phenytoin in acute optic neuritis (ON) demonstrated a 30% reduction in retinal nerve fiber layer (RNFL) loss with phenytoin versus placebo. Here we present the corresponding serum neurofilament analyses. METHODS: Eighty-six acute ON cases were randomized to receive phenytoin (4-6 mg/kg/day) or placebo for 3 months, and followed up for 6 months. Serum was collected at baseline, 3 and 6 months for analysis of neurofilament heavy chain (NfH) and neurofilament light chain (NfL). RESULTS: Sixty-four patients had blood sampling. Of these, 58 and 56 were available at 3 months, and 55 and 54 were available at 6 months for NfH and NfL, respectively. There was no significant correlation between serum NfH and NfL at the time points tested. For NfH, the difference in mean placebo - phenytoin was -44 pg/ml at 3 months (P = 0.019) and -27 pg/ml at 6 months (P = 0.234). For NfL, the difference was 1.4 pg/ml at 3 months (P = 0.726) and -1.6 pg/ml at 6 months (P = 0.766). CONCLUSIONS: At 3 months, there was a reduction in NfH, but not NFL, in the phenytoin versus placebo group, while differences at 6 months were not statistically significant. This suggests a potential neuroprotective role for phenytoin in acute ON, with the lower NfH at 3 months, when levels secondary to degeneration of the anterior visual pathway are still elevated, but not at 6 months, when levels have normalized.

Correlations between serum and CSF pNfH levels in ALS, FTD and controls: a comparison of three analytical approaches.

Background Phosphorylated neurofilament heavy (pNfH), a neuronal cytoskeleton protein, might provide a promising blood biomarker of neuronal damage in neurodegenerative diseases (NDDs). The best analytical approaches to measure pNfH levels and whether serum levels correlate with cerebrospinal fluid (CSF) levels in NDDs remain to be determined. Methods We here compared analytical sensitivity and reliability of three novel analytical approaches (homebrew Simoa, commercial Simoa and ELISA) for quantifying pNfH in both CSF and serum in samples of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and control subjects. Results While all three assays showed highly correlated CSF measurements, Simoa assays also yielded high between-assay correlations for serum measurements (ϱ = 0.95). Serum levels also correlated strongly with CSF levels for Simoa-based measurements (both ϱ = 0.62). All three assays allowed distinguishing ALS from controls by increased CSF pNfH levels, and Simoa assays also by increased serum pNfH levels. pNfH levels were also increased in FTD. Conclusions pNfH concentrations in CSF and, if measured by Simoa assays, in blood might provide a sensitive and reliable biomarker of neuronal damage, with good between-assay correlations. Serum pNfH levels measured by Simoa assays closely reflect CSF levels, rendering serum pNfH an easily accessible blood biomarker of neuronal damage in NDDs.

Serum biomarkers of neuronal injury in newborns evaluated for selective head cooling: a comparative pilot study.

Background Evaluation of newborns for hypoxic ischemic encephalopathy (HIE) includes laboratory and clinical parameters, as well as amplitude integrated electroencephalogram (aEEG). Based on qualifying criteria, selective head cooling (SHC) is initiated for infants with evidence of moderate to severe HIE. However, some newborns may not qualify for hypothermia therapy based on normal aEEG. Objective To compare levels of serum glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase-1 (UCHL-1) protein and phosphorylated axonal neurofilament heavy chain (pNF-H), in newborns who met initial screening criteria for HIE but did not qualify for head cooling, to the levels in healthy newborns. Study design Newborns ≥36 weeks of gestational age at risk for HIE, who were evaluated but did not qualify for SHC from July 2013 through June 2014 at NYU Langone Medical Center and Bellevue Hospital center were enrolled. A control group included healthy newborns from the newborn nursery (NBN). Serum samples were collected between 24 and 48 h of life from both groups. Results There was no significant difference in the serum levels of GFAP, UCHL-1 protein and pNF-H between the two groups of infants. Conclusion Newborns at risk for HIE who met the initial criteria for head cooling but who were excluded based on normal aEEG did not show significant elevation of biomarkers of brain injury compared to healthy newborns. These findings may help to validate using aEEG as an additional evaluation criteria in cooling.

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.

Plasma neurofilament heavy chain is not a useful biomarker in Charcot-Marie-Tooth disease.

INTRODUCTION: The negative results in trials of vitamin C in Charcot-Marie-Tooth disease (CMT) type 1A have highlighted the lack of sensitive outcome measures. Neurofilaments are abundant neuronal cytoskeletal proteins, and their concentration in blood is likely to reflect axonal breakdown. We therefore examined plasma neurofilament heavy-chain (NfH) concentration as a potential biomarker in CMT. METHODS: Blood samples were collected from healthy controls and patients with CMT over a 2-year period. Disease severity was measured using the CMT Examination Score. An in-house enzyme-linked immunoabsorbent assay was used to measure plasma NfH levels. RESULTS: There was no significant difference in plasma NfH concentrations between CMT patients and controls (P = 0.449). There was also no significant difference in plasma NfH levels in the CMT group over 1 year (mean difference = -0.02, SEM = 4.44, P = 0.98). CONCLUSIONS: Plasma NfH levels are not altered in patients with CMT and are not a suitable biomarker of disease activity. Muscle Nerve 53: 972-975, 2016.

Maternal exposure to 3,3'-iminodipropionitrile targets late-stage differentiation of hippocampal granule cell lineages to affect brain-derived neurotrophic factor signaling and interneuron subpopulations in rat offspring.

3,3'-Iminodipropionitrile (IDPN) causes neurofilament (NF)-filled swellings in the proximal segments of many large-caliber myelinated axons. This study investigated the effect of maternal exposure to IDPN on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 67 or 200 ppm IDPN in drinking water from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, female offspring subjected to analysis had decreased parvalbumin(+), reelin(+) and phospho-TrkB(+) interneurons in the dentate hilus at 200 ppm and increased granule cell populations expressing immediate-early gene products, Arc or c-Fos, at ≥ 67 ppm. mRNA expression in the dentate gyrus examined at 200 ppm decreased with brain-derived neurotrophic factor (Bdnf) and very low density lipoprotein receptor. Immunoreactivity for phosphorylated NF heavy polypeptide decreased in the molecular layer of the dentate gyrus and the stratum radiatum of the cornu ammonis (CA) 3, portions showing axonal projections from mossy cells and pyramidal neurons, at 200 ppm on PND 21, whereas immunoreactivity for synaptophysin was unchanged in the dentate gyrus. Observed changes all disappeared on PND 77. There were no fluctuations in the numbers of apoptotic cells, proliferating cells and subpopulations of granule cell lineage in the subgranular zone on PND 21 and PND 77. Thus, maternal IDPN exposure may reversibly affect late-stage differentiation of granule cell lineages involving neuronal plasticity as evident by immediate-early gene responses to cause BDNF downregulation resulting in a reduction in parvalbumin(+) or reelin(+) interneurons and suppression of axonal plasticity in the mossy cells and CA3 pyramidal neurons.

Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma.

BACKGROUND & AIMS: Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking. METHODS: We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking). RESULTS: Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 (SMPD3) and neurofilament, heavy polypeptide (NEFH), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort (P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6-6.5 in multivariate analysis). CONCLUSIONS: Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC.

Comparison of CSF and plasma NfL and pNfH for Alzheimer's disease diagnosis: a memory clinic study.

Plasma neurofilament light chain (NfL) is a promising biomarker of axonal damage for the diagnosis of neurodegenerative diseases. Phosphorylated neurofilament heavy chain (pNfH) has demonstrated its value in motor neuron diseases diagnosis, but has less been explored for dementia diagnosis. In a cross-sectional study, we compared cerebrospinal fluid (CSF) and plasma NfL and pNfH levels in n = 188 patients from Lariboisière Hospital, Paris, France, including AD patients at mild cognitive impairment stage (AD-MCI, n = 36) and dementia stage (n = 64), non-AD MCI (n = 38), non-AD dementia (n = 28) patients and control subjects (n = 22). Plasma NfL, plasma and CSF pNfH levels were measured using Simoa and CSF NfL using ELISA. The correlation between CSF and plasma levels was stronger for NfL than pNfH (rho = 0.77 and rho = 0.52, respectively). All neurofilament markers were increased in AD-MCI, AD dementia and non-AD dementia groups compared with controls. CSF NfL, CSF pNfH and plasma NfL showed high performance to discriminate AD at both MCI and dementia stages from control subjects [AUC (area under the curve) = 0.82-0.91]. Plasma pNfH displayed overall lower AUCs for discrimination between groups compared with CSF pNfH. Neurofilament markers showed similar moderate association with cognition. NfL levels displayed significant association with mediotemporal lobe atrophy and white matter lesions in the AD group. Our results suggest that CSF NfL and pNfH as well as plasma NfL levels display equivalent performance in both positive and differential AD diagnosis in memory clinic settings. In contrast to motoneuron disorders, plasma pNfH did not demonstrate added value as compared with plasma NfL.

Neurofilaments in Sporadic and Familial Amyotrophic Lateral Sclerosis: A Systematic Review and Meta-Analysis.

BACKGROUND: Neurofilament proteins have been implicated to be altered in amyotrophic lateral sclerosis (ALS). The objectives of this study were to assess the diagnostic and prognostic utility of neurofilaments in ALS. METHODS: Studies were conducted in electronic databases (PubMed/MEDLINE, Embase, Web of Science, and Cochrane CENTRAL) from inception to 17 August 2023, and investigated neurofilament light (NfL) or phosphorylated neurofilament heavy chain (pNfH) in ALS. The study design, enrolment criteria, neurofilament concentrations, test accuracy, relationship between neurofilaments in cerebrospinal fluid (CSF) and blood, and clinical outcome were recorded. The protocol was registered with PROSPERO, CRD42022376939. RESULTS: Sixty studies with 8801 participants were included. Both NfL and pNfH measured in CSF showed high sensitivity and specificity in distinguishing ALS from disease mimics. Both NfL and pNfH measured in CSF correlated with their corresponding levels in blood (plasma or serum); however, there were stronger correlations between CSF NfL and blood NfL. NfL measured in blood exhibited high sensitivity and specificity in distinguishing ALS from controls. Both higher levels of NfL and pNfH either measured in blood or CSF were correlated with more severe symptoms as assessed by the ALS Functional Rating Scale Revised score and with a faster disease progression rate; however, only blood NfL levels were associated with shorter survival. DISCUSSION: Both NfL and pNfH measured in CSF or blood show high diagnostic utility and association with ALS functional scores and disease progression, while CSF NfL correlates strongly with blood (either plasma or serum) and is also associated with survival, supporting its use in clinical diagnostics and prognosis. Future work must be conducted in a prospective manner with standardized bio-specimen collection methods and analytical platforms, further improvement in immunoassays for quantification of pNfH in blood, and the identification of cut-offs across the ALS spectrum and controls.

Phosphorylated neurofilament heavy chain in cerebrospinal fluid and plasma as a Nusinersen treatment response marker in childhood-onset SMA individuals from Serbia.

Introduction: Biomarkers capable of reflecting disease onset and short- and long-term therapeutic effects in individuals with spinal muscular atrophy (SMA) are still an unmet need and phosphorylated neurofilament heavy chain (pNF-H) holds significant promise. Methods: We conducted a longitudinal prospective study to evaluate pNF-H levels in the cerebrospinal fluid (CSF) and plasma of 29 individuals with childhood-onset SMA treated with Nuinersen (SMA type 1: n = 6, 2: n = 17, 3: n = 6). pNF-H levels before and during treatment were compared with the levels of controls (n = 22), patients with Duchenne muscular dystrophy (n = 17), myotonic dystrophy type 1 (n = 11), untreated SMA individuals with chronic type 3 disease (n = 8), and children with presymptomatic SMA (n = 3). Results: SMA type 1 showed the highest mean CSF pNF-H levels before treatment initiation. All Nusinersen-treated individuals (types 1, 2, and 3) showed significantly elevated mean baseline CSF pNF-H compared to controls, which inversely correlated with age at disease onset, age at first dose, disease duration and the initial CHOP INTEND result (SMA type 1 and 2). During 22 months of treatment, CSF pNF-H levels declined during loading doses, stabilizing at reduced levels from the initial maintenance dose in all individuals. Baseline plasma pNF-H levels in type 1 and 2 SMA were significantly increased compared to other cohorts and decreased notably in type 1 after 2 months of treatment and type 2 after 14 months. Conversely, SMA type 3, characterized by lower baseline pNF-H levels, did not show significant fluctuations in plasma pNF-H levels after 14 months of treatment. Conclusion: Our findings suggest that CSF pNF-H levels in untreated SMA individuals are significantly higher than in controls and that monitoring of CSF pNF-H levels may serve as an indicator of rapid short-term treatment response in childhood-onset SMA individuals, irrespective of the subtype of the disease, while also suggesting its potential for assessing long-term suppression of neurodegeneration. Plasma pNF-H may serve as an appropriate outcome measure for disease progression and/or response to treatment in types 1 and 2 but not in type 3. Presymptomatic infants with SMA may show elevated pNF-H levels, confirming early neuronal degeneration.

[Possibilities of magnetic resonance morphometry and laboratory biomarkers in studying the progression of multiple sclerosis]. / Vozmozhnosti magnitno-rezonansnoi morfometrii i laboratornykh biomarkerov v izuchenii progressirovaniya rasseyannogo skleroza.

OBJECTIVE: To show that magnetic resonance morphometry and laboratory biomarkers are promising methods for early detection of progressive forms of multiple sclerosis (MS). MATERIAL AND METHODS: Eighty-one patients with MS were examined, magnetic resonance morphometry was performed in all of them, 60 patients were analyzed for neurofilament light chains (sNFL), phosphorylated neurofilament heavy chains (spNFH) and glial fibrillary protein (sGFAP) in serum by enzyme-linked immunosorbent assay. RESULTS: Brain volumes were negatively correlated with disease duration, EDSS score, 25-foot walk test score and 9-ring test and positively correlated with the Symbol-Numeric Test and the Montreal Cognitive Assessment. Patients with progressive types of MS (PMS) had smaller volumes of brain gray matter, cerebellar white matter, occipital lobes, caudate nucleus, hippocampus, pallidum, thalamus, and contiguous nucleus. A CSF volume greater than 15.06% could suggest progression (CI 54.79-91%) with a sensitivity of 77.78% and specificity of 70.18%. When patients were on DMT, they had larger thalamic volumes (median 1.09% [1.6; 1.16] vs 1.04% [0.95; 1.14]; p=0.02) and smaller CSF volumes (13.86±2.87% vs. 15.55±3.49%; p=0.03). The levels of sNFL and spNFH were not increased in PMS and during exacerbations, and the low obtained values of sNFL suggest poor sensitivity of the method. There were trends (p=0.374) towards higher sGFAP in patients with PRS (median 3.2 ng/mL [1.85; 4.6] compared to remitting MS (2.05 ng/mL [1.29; 4.52]). CONCLUSION: The results demonstrate the differences in brain volumes in patients with different types of MS and emphasize the importance of long-term follow-up to better assess disease progression.

Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon.

Axonal injury and degeneration, whether primary or secondary, contribute to the morbidity and mortality seen in many acquired and inherited central nervous system (CNS) and peripheral nervous system (PNS) disorders, such as traumatic brain injury, spinal cord injury, cerebral ischemia, neurodegenerative diseases, and peripheral neuropathies. The calpain family of proteases has been mechanistically linked to the dysfunction and degeneration of axons. While the direct mechanisms by which transection, mechanical strain, ischemia, or complement activation trigger intra-axonal calpain activity are likely different, the downstream effects of unregulated calpain activity may be similar in seemingly disparate diseases. In this review, a brief examination of axonal structure is followed by a focused overview of the calpain family. Finally, the mechanisms by which calpains may disrupt the axonal cytoskeleton, transport, and specialized domains (axon initial segment, nodes, and terminals) are discussed.

A comparative study of CSF neurofilament light and heavy chain protein in MS.

BACKGROUND: There is a lack of reliable biomarkers of axonal degeneration. Neurofilaments are promising candidates to fulfil this task. We compared two highly sensitive assays to measure two subunits of the neurofilament protein (neurofilament light (NfL) and neurofilament heavy chain (NfH)). METHODS: We evaluated the analytical and clinical performance of the UmanDiagnostics NF-light(®) enzyme-linked immunosorbent assay (ELISA) in the cerebrospinal fluid (CSF) of a group of 148 patients with clinically isolated syndrome (CIS) or multiple sclerosis (MS), and 72 controls. We compared our results with referring levels of our previously-developed CSF NfH(SMI35) assay. RESULTS: Exposure to room temperature (up to 8 days) or repetitive thawing (up to 4 thaws) did not influence measurement of NfL concentrations. Values of NfL were higher in all disease stages of CIS/MS, in comparison to controls (p ≤ 0.001). NfL levels correlated with the Expanded Disability Status Scale (EDSS) score in patients with relapsing disease (r(s) = 0.31; p = 0.002), spinal cord relapses and with CSF markers of acute inflammation. The ability of NfL to distinguish patients from controls was greater than that of NfH(SMI35) in both CIS patients (p = 0.001) and all MS stages grouped together (p = 0.035). CONCLUSIONS: NfL proved to be a stable protein, an important prerequisite for a reliable biomarker, and the NF-light(®) ELISA performed better in discriminating patients from controls, compared with the ECL-NfH(SMI35) immunoassay. We confirmed and expanded upon previous findings regarding neurofilaments as quantitative markers of neurodegeneration. Our results further support the role of neurofilaments as a potential surrogate measure for neuroprotective treatment in MS studies.

Neurofilament light and heavy subunits compared as therapeutic biomarkers in multiple sclerosis.

BACKGROUND: Neurofilaments are promising biomarkers in multiple sclerosis (MS) and increased levels in cerebrospinal fluid (CSF) indicate axonal damage or degeneration. In a previous study, neurofilament light chain (NfL) levels in CSF of relapsing remitting (RR) patients with MS were normalized by natalizumab treatment. AIMS OF THE STUDY: We compared the coherence between NfL and neurofilament heavy chain (NfH(SMI) (35) ) levels in longitudinal CSF samples in a subset of these patients. METHODS: In 30 patients with RRMS, CSF was obtained prior to and following 12 months of natalizumab treatment. NfH(SMI) (35) was measured by an electrochemiluminescence-based immunoassay. NfL levels were determined previously by the UmanDiagnostics NF-light(®) assay. RESULTS: NfH(SMI) (35) decreased in 73.3% and NfL in 90% of the patients following natalizumab treatment (32.4 vs 27.4 pg/ml, P = 0.002 and 820 vs 375 pg/ml, P < 0.0001). Patients experiencing a relapse showed higher NfH(SMI) (35) levels compared with patients in remission (47.7 vs 27.6 pg/ml, n = 8, P = 0.001). This difference was less obvious for NfL (1055 vs 725 pg/ml, P = 0.256). In patients in remission, NfL levels were lower following natalizumab treatment (830 vs 365 pg/ml, n = 20, P = 0.0002), whereas the same comparison failed significance for NfH(SMI) (35) (28.3 vs 26.9 pg/ml, P = 0.086). CONCLUSIONS: We confirm previous findings, indicating reduced axonal damage under natalizumab treatment by measuring NfH(SMI) (35) , using an assay with independent methodology. In comparison with NfH(SMI) (35) , NfL changes were more pronounced and the treatment effect also included patients in remission. Our results suggest that NfL is superior over NfH(SMI) (35) as therapeutic biomarker and is a promising candidate to measure neuroaxonal damage in MS treatment trials.

Immunohistochemical characterization of stem cell, vascular, neural, and differentiation markers in the apical papilla and dental pulp of human teeth at various stages of root development.

This study aimed to evaluate the expression of several differentiation markers in the apical papilla (AP) and dental pulp (DP) of human permanent teeth. Twenty young human teeth were extracted and classified according to three Moorrees tooth development stages: initial root formation (Ri), root length ½ (R1/2), and root length complete (Rc). Immunohistochemical assays were performed using STRO-1, VEGF Receptor-2, Neurofilament heavy (NFH), and Nestin antibodies and analyzed under light microscopy. Decalcified, formalin fixed paraffin embedded tooth sections stained with hematoxylin and eosin showed an apical cell rich zone between the DP and AP. The AP revealed fewer vascular and cellular components than the DP. STRO-1 was expressed on vascular and neuronal elements beneath the odontoblast (OB) and in the sub-odontoblastic (SOB) zone, and VEGFR-2 positive cells were observed in the endothelium, arterioles, and blood vessels. Neuroepithelial stem cell protein (Nestin) was highly expressed in differentiated odontoblasts in the predentin odontotoblast and odontoblast cell processes. Neurofilament heavy (NFH) was expressed in mature axons throughout the DP. STRO-1 and VEGFR-2 microvascular expression was higher at the stages Ri and R1/2 while STRO-1 and NFH expression showed strong spatial distribution of Rc neuronal elements as compared to Ri and R1/2. Differentiated OB and SOB cells showed Nestin expression, indicating a reservoir of newly differentiated odontoblast-like cells.

Serum Neurofilaments in Motor Neuron Disease and Their Utility in Differentiating ALS, PMA and PLS.

Neurofilament levels are elevated in many neurodegenerative diseases and have shown promise as diagnostic and prognostic biomarkers in Amyotrophic Lateral Sclerosis (ALS), the most common form of Motor Neuron Disease (MND). This study assesses serum neurofilament light (NFL) and neurofilament heavy (NFH) chain concentrations in patients with ALS, other variants of motor neuron disease such as Progressive Muscular Atrophy (PMA) and Primary Lateral Sclerosis (PLS), and a range of other neurological diseases. It aims to evaluate the use of NFL and NFH to differentiate these conditions and for the prognosis of MND disease progression. NFL and NFH levels were quantified using electrochemiluminescence immunoassays (ECLIA). Both were elevated in 47 patients with MND compared to 34 patients with other neurological diseases and 33 healthy controls. NFL was able to differentiate patients with MND from the other groups with a Receiver Operating Characteristic (ROC) curve area under the curve (AUC) of 0.90 (p < 0.001). NFL correlated with the rate of disease progression in MND (rho 0.758, p < 0.001) and with the ALS Functional Rating Scale (rho -0.335, p = 0.021). NFL levels were higher in patients with ALS compared to both PMA (p = 0.032) and PLS (p = 0.012) and were able to distinguish ALS from both PMA and PLS with a ROC curve AUC of 0.767 (p = 0.005). These findings support the use of serum NFL to help diagnose and differentiate types of MND, in addition to providing prognostic information to patients and their families.

Biochemical analyses of tau and other neuronal markers in the submandibular gland and frontal cortex across stages of Alzheimer disease.

Hyperphosphorylation of the microtubule-associated protein tau is hypothesized to lead to the development of neurofibrillary tangles in select brain regions during normal aging and in Alzheimer disease (AD). The distribution of neurofibrillary tangles is staged by its involvement starting in the transentorhinal regions of the brain and in final stages progress to neocortices. However, it has also been determined neurofibrillary tangles can extend into the spinal cord and select tau species are found in peripheral tissues and this may be depended on AD disease stage. To further understand the relationships of peripheral tissues to AD, we utilized biochemical methods to evaluate protein levels of total tau and phosphorylated tau (p-tau) as well as other neuronal proteins (i.e., tyrosine hydroxylase (TH), neurofilament heavy chain (NF-H), and microtubule-associated protein 2 (MAP2)) in the submandibular gland and frontal cortex of human cases across different clinicopathological stages of AD (n = 3 criteria not met or low, n = 6 intermediate, and n = 9 high likelihood that dementia is due to AD based on National Institute on Aging-Reagan criteria). We report differential protein levels based on the stage of AD, anatomic specific tau species, as well as differences in TH and NF-H. In addition, exploratory findings were made of the high molecular weight tau species big tau that is unique to peripheral tissues. Although sample sizes were small, these findings are, to our knowledge, the first comparison of these specific protein changes in these tissues.

Alteration of neurofilament heavy chain and its phosphoforms reveals early subcellular damage beyond the optic nerve head in glaucoma.

Background: Retinal ganglion cells (RGCs) axon loss at the site of optic nerve head (ONH) is long believed as the common pathology in glaucoma since different types of glaucoma possessing different characteristic of intraocular pressure, and this damage was only detected at the later stage. Methods: To address these disputes and detect early initiating events underlying RGCs, we firstly detected somatic or axonal change and compared their difference in acute and chronic phase of primary angle-closed glaucoma (PACG) patient using optical coherence tomography (OCT), then an axonal-enriched cytoskeletal protein neurofilament heavy chain and its phosphoforms (NF-H, pNF-H) were utilized to reveal spatio-temporal undetectable damage insulted by acute and chronic ocular hypertension (AOH, COH) in two well characterized glaucoma mice models. Results: In clinic, we detected nonhomogeneous changes such as ONH and soma of RGCs presenting edema in acute phase but atrophy in chronic one by OCT. In AOH animal models, an increase expression of NF-H especially its phosphorylation modification was observed as early as 4 h before RGCs loss, which presented as somatic accumulation in the peripheral retina and at the sites of ONH. In contrast, in microbeads induced COH model, NF-H and pNF-H reduced significantly, these changes firstly occurred as NF-H or pNF-H disconnection at ONH and optic nerve after 2 weeks when the intraocular pressure reaching the peak; Meanwhile, we detected aqueous humor pNF-H elevation after AOH and slight reduction in the COH. Conclusion: Together, our data supports that early alteration of NF-H and its phosphoforms would reveal undetectable subcellular damage consisting of peripheral somatic neurofilament compaction, impaired axonal transport and distal axonal disorganization of cytoskeleton beyond the ONH, and identifies two distinct axonal degeneration which were Wallerian combination with retrograde degeneration in acute PACG and retrograde degeneration in the chronic one.