Neurofilament Heavy Chain Research Articles

Evaluating the Effect of a Bruton’s Tyrosine Kinase Inhibitor in a Murine Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis (P2-3.012)

<h3>Objective:</h3> To characterize a central nervous system (CNS)-penetrant, tolebrutinib-like Bruton’s tyrosine kinase inhibitor (BTKi) in murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) for its potential to modulate disease progression and expression of genes linked to disease-associated microglia (DAM). <h3>Background:</h3> BTK inhibition may slow disease progression in people with MS by targeting microglia-driven neuroinflammation in the CNS. We previously showed that a structural analogue of the BTKi tolebrutinib can reduce BTK-dependent inflammatory signalling in microglia in the mouse brain, using the cuprizone-mediated model of demyelination. <h3>Design/Methods:</h3> Female C57BL/6 mice were immunized with MOG_35–55 peptide (250 μg/mouse; two subcutaneous injections in the lower back) to induce EAE. Animals were randomized to receive vehicle (n=15) or oral BTKi (15 mg/kg; n=15), starting at clinical score of 1.0–1.5. Treatment groups were blinded until after final data analysis. Cohorts were monitored daily for development and severity of disease symptoms. After 10 days of treatment, spinal cord and plasma samples were taken for transcriptome analysis and neurofilament heavy chain (NfH) concentration measurement, respectively. <h3>Results:</h3> BTK inhibition significantly reduced the clinical score in the EAE mice, with significant differences between treatment groups starting after two days of treatment and continuing until end of study (Day 10). BTK inhibition significantly reduced plasma Nfh concentrations. A BTK-dependent transcriptional signature was identified, with BTK inhibition found to modulate pathways relevant to MS pathology. BTK inhibition modulated mRNA expression of genes that had previously been linked to DAM and/or are associated with BTK-signalling. <h3>Conclusions:</h3> We extended our previous <i>in vivo</i> findings from a demyelination model by demonstrating that dosing with a BTKi halted disease progression in an immune-mediated model of MS. This therapeutic benefit was accompanied by modulated expression of genes associated with activated microglia, which could abrogate microglia-driven neuroinflammation implicated in disease progression in MS. <b>Disclosure:</b> Dr. Gruber has received personal compensation for serving as an employee of Sanofi. Dr. Gruber has received stock or an ownership interest from Sanofi. Dr. Blazier has received personal compensation for serving as an employee of Sanofi. Lan Lee has nothing to disclose. Dr. Ryan has nothing to disclose. Dr. Cheong has received personal compensation for serving as an employee of Sanofi. Ms. Havari has received personal compensation for serving as an employee of sanofi. Timothy J. Turner has received personal compensation for serving as an employee of Sanofi. Timothy J. Turner has received stock or an ownership interest from Sanofi. Timothy J. Turner has received intellectual property interests from a discovery or technology relating to health care. Dimitry Ofengeim has received personal compensation for serving as an employee of Sanofi. Dimitry Ofengeim has received stock or an ownership interest from Sanofi. Dimitry Ofengeim has received personal compensation in the range of $500-$4,999 for serving as a Study Section Reveiwer with NIH.

Effect of RIPK1 Inhibitor, SAR443820, on Serum Neurofilament Light Levels in Patients with Multiple Sclerosis: A Phase 2 Trial Design (P6-3.011)

Effect of RIPK1 Inhibitor, SAR443820, on Serum Neurofilament Light Levels in Patients with Multiple Sclerosis: A Phase 2 Trial Design (P6-3.011)

Topiramate affords neuroprotection in diabetic neuropathy model via downregulating spinal GFAP/inflammatory burden and improving neurofilament production

The current study aimed to test the neuroprotective action of topiramate in mouse peripheral diabetic neuropathy (DN) and explored some mechanisms underlying this action. Mice were assigned as vehicle group, DN group, DN + topiramate 10-mg/kg and DN + topiramate 30-mg/kg. Mice were tested for allodynia and hyperalgesia and then spinal cord and sciatic nerves specimens were examined microscopically and neurofilament heavy chain (NEFH) immunostaining was performed. Results indicated that DN mice had lower the hotplate latency time (0.46-fold of latency to licking) and lower von-Frey test pain threshold (0.6-fold of filament size) while treatment with topiramate increased these values significantly. Sciatic nerves from DN control mice showed axonal degeneration while spinal cords showed elevated GFAP (5.6-fold) and inflammatory cytokines (∼3- to 4-fold) but lower plasticity as indicated by GAP-43 (0.25-fold). Topiramate produced neuroprotection and suppressed spinal cord GFAP/inflammation but enhanced GAP-43. This study reinforces topiramate as neuroprotection and explained some mechanisms included in alleviating neuropathy.

Comparison of neurofilament light and heavy chain in spinal muscular atrophy and amyotrophic lateral sclerosis: A pilot study.

Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) were two major motor neuron diseases with similar symptoms and poor outcomes. This study aimed to identify potential biomarkers in disease monitoring and differential diagnosis of adult SMA patients with sporadic ALS patients. This was a pilot study with ten adult SMA patients and ten ALS patients consecutively enrolled during hospitalization. Serum and cerebrospinal fluid (CSF) samples were collected for assessment of neurofilament light (NFL) and phosphorylated neurofilament heavy chain (pNFH). Serum creatine kinase (CK) and creatinine (Cr) were also compared between groups. The receiver operating characteristic (ROC) curves were used to identify differentiated values among ALS and SMA patients. Serum Cr, CSF NFL, and CSF pNFH levels of ALS patients were significantly higher than those of the adult SMA patients (p<.01). Serum CK and Cr were strongly correlated with baseline ALSFRS-R scores in SMA patients (p<.001). The ROC curves revealed an area under the curve (AUC) of 0.94 in serum Cr with a cut-off value of 44.5μmol/L (Sensitivity 90%, Specificity 90%). AUC from the ROC curve of CSF NFL and CSF pNFH were 1.0 and 0.84, with cut-off values of 1275 pg/mL and 0.395ng/mL, respectively (Sensitivity and Specificity of 100% and 100% in CSF NFL; Sensitivity and Specificity of 90% and 80% in CSF pNFH). CSF NFL and pNFH might be useful biomarkers for differential diagnosis of adult SMA and ALS.

The altered expression of cytoskeletal and synaptic remodeling proteins during epilepsy.

The cytoskeleton plays an important role in epilepsy; however, the mechanism is unknown. Therefore, this study aimed to reveal the mechanism of cytoskeletal proteins in epilepsy by investigating the expression of cytoskeletal proteins and synaptophysin (SYP) in mice at 0, 3, 6, and 24 h, 3 days, and 7 days in a kainic acid (KA)-induced epileptic model. Our results demonstrated that the expression of F-actin decreased significantly between 3 and 6 h, 6 and 24 h, and 24 h and 3 days (P < 0.05). Meanwhile, the expression of the neurofilament light chain, neurofilament medium chain, and neurofilament heavy chain subunits was significantly decreased (P < 0.001) at 3 h after the KA injection compared to the KA 0 h group, followed by an elevation at 6 h and a further decrease at 24 h compared to at 6 h. SYP expression was significantly decreased between 0 and 3 h as well as between 3 and 6 h (P < 0.05). At 24 h, the level was increased compared to at 6 h and continued to increase at 3 days after the KA injection. Thus, we propose that cytoskeletal proteins may be involved in the pathogenesis of epilepsy.

Infection of Mouse Neural Progenitor Cells by Toxoplasma gondii Reduces Proliferation, Migration, and Neuronal Differentiation in Vitro

Congenital toxoplasmosis constitutes a major cause of pre- and postnatal complications. Fetal infection with Toxoplasma gondii influences development and can lead to microcephaly, encephalitis, and neurologic abnormalities. Systematic studies concerning the effects of neural progenitor cell infection with T. gondii are unavailable. Cortical intermediate progenitor cells cultivated as neurospheres obtained from E16.5 Swiss Webster mice were infected with T. gondii (ME49 strain) tachyzoites to mimic the developing mouse cerebral cortex invitro. Infection was associated with decreased cell proliferation, detected by Ki-67 staining at 48 and 72 hours after infection in floating neurospheres, and reduced cellularity at 96 hours. Transient decreases in the expression of the neurogenesis-related transcription factors T-box brain protein 1, mouse atonal homolog protein 1, and hairy and enhancer of split protein 1 were found in infected cultures, while the level of transcription factor SOX-2 remained unaltered. Neurogenic potential, assessed in plated neurospheres, was impaired in infected cultures, as indicated by decreased late neuronal marker neurofilament heavy chain immunoreactivity. Infected cultures exhibited decreased overall migration rates at 48 and 120 hours. These findings indicate that T. gondii infection of neural progenitor cells may lead to reduced neurogenesis due to an imbalance in cell proliferation alongside an altered migratory profile. If translated to the invivo situation, these data could explain, in part, cortical malformations in congenitally infected individuals.

Abstract 3771: PiR-hsa-4447944 promotes the progression of prostate cancer through NEFH and induces androgen-independent growth

Abstract Introduction and Objective: Non-coding RNAs (ncRNAs) represent a diverse family of regulatory transcripts that drive tumorigenesis of prostate cancer (PCa) and various other types of cancers by their hyperactivity or diminished function. PIWI-interacting RNAs (piRNAs) are a class of small ncRNAs mechanistically similar to but much less studied than microRNAs. As the most diversified small ncRNAs, piRNAs are widely involved in the pathogenesis of different types of cancers. Our study aimed to investigate the role of piRNAs in the development of castration-resistant prostate cancer (CRPC). Methods: Small RNA sequencing was used to explore the CRPC associated piRNAs on a basis of 10 benign prostate tissues, 10 paired hormone-sensitive PCa tissues and CRPC tissues from the same patients. The top 12 upregulated piRNAs were validated through qRT-PCR in a bigger cohort. piR-hsa-4447944 was selected and investigated its biological function by gain of function in vitro. To study its loss of function role, an androgen-independent PCa cell (LNCaP-AI) was established. For mechanistic investigation, high-throughput RNA sequencing was conducted to identify dysregulated genes in PCa cells with control or piR-hsa-4447944 overexpression. Then, potential downstream targets were further predicted by miRanda program and validated by qRT-PCR, western-blot and dual-luciferase reporter assay. Results: piR-hsa-4447944 demonstrated a relatively higher expression level in both CRPC tissues and CRPC cell lines when compared with PCa tissues and cells. Functionally, overexpression of piR-hsa-4447944 could confer resistance to androgen deprivation and anti-androgen in PCa cells in vitro, whereas anti-sense RNA against the piR-hsa-4447944 could potentiate the sensitivity to androgen deprivation in LNCaP-AI cell. In addition, piR-hsa-4447944 showed the ability in promoting migration and invasion of PCa cells. Mechanistically, mRNA sequencing results identified a panel of downstream target-genes. Among them, neurofilament heavy chain (NEFH) was predicted and validated to have a direct binding relationship with piR-hsa-4447944, and was successfully verified to be negatively regulated by piR-hsa-4447944 in PCa cells. Conclusions: Our study showed, for the first time, that piR-hsa-4447944 can contribute to androgen insensitivity in CRPC. This sheds a new insight into the regulation of CRPC by a small ncRNA piRNA, which may form the basis of a novel target in the treatment of CRPC. Citation Format: Qiang Peng, Jingkai Sun, Peter Ka-Fung Chiu, Tingting Xie, Jeremy Yuen-Chun Teoh, Chi-Fai Ng. PiR-hsa-4447944 promotes the progression of prostate cancer through NEFH and induces androgen-independent growth. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3771.

Phosphorylated proteomics-based analysis of the effects of semaglutide on hippocampi of high-fat diet-induced-obese mice

The aim of this paper was to investigate the effects of semaglutide on phosphorylated protein expression, and its neuroprotective mechanism in hippocampi of high-fat-diet-induced obese mice. In total, 16 obese mice were randomly divided into model group (H group) and semaglutide group (S group), with 8 mice in each group. In addition, a control group (C group) was set up comprising 8 C57BL/6J male normal mice. The Morris water maze assay was conducted to detect cognitive function changes in the mice, and to observe and compare body weight and expression levels of serological indicators between groups after the intervention. Phosphorylated proteomic analysis was performed to detect the hippocampal protein profile in mice. Proteins up-regulated twofold or down-regulated 0.5-fold in each group and with t-test p < 0.05 were defined as differentially phosphorylated proteins and were analyzed bioinformatically. The results showed that the high-fat diet-induced obese mice had reduced body weight, improved oxidative stress indexes, significantly increased the percentage of water maze trips and the number of platform crossings, and significantly shortened the water maze platform latency after semaglutide intervention. The phosphorylated proteomics results identified that 44 overlapping proteins among the three experimental groups. Most of the phosphorylated proteins identified were closely associated with pathways of neurodegeneration-multiple diseases. In addition, we identified Huntington, Neurofilament light chain, Neurofilament heavy chain as drug targets. This study demonstrates for the first time that semaglutide exerts neuroprotective effects by reducing HTT Ser1843, NEFH Ser 661 phosphorylation and increasing NEFL Ser 473 phosphorylation in hippocampal tissue of obese mice.

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

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. 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. 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. 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.

Neuroserpin gene therapy inhibits retinal ganglion cell apoptosis and promotes functional preservation in glaucoma

Our research has proven that the inhibitory activity of the serine protease inhibitor neuroserpin (NS) is impaired because of its oxidation deactivation in glaucoma. Using genetic NS knockout (NS-/-) and NS overexpression (NS+/+ Tg) animal models and antibody-based neutralization approaches, we demonstrate that NS loss is detrimental to retinal structure and function. NS ablation was associated with perturbations in autophagy and microglial and synaptic markers, leading to significantly enhanced IBA1, PSD95, beclin-1, and LC3-II/LC3-I ratio and reduced phosphorylated neurofilament heavy chain (pNFH) levels. On the other hand, NS upregulation promoted retinal ganglion cell (RGC) survival in wild-type and NS-/- glaucomatous mice and increased pNFH expression. NS+/+Tg mice demonstrated decreased PSD95, beclin-1, LC3-II/LC3-I ratio, and IBA1 following glaucoma induction, highlighting its protective role. We generated a novel reactive site NS variant (M363R-NS) resistant to oxidative deactivation. Intravitreal administration of M363R-NS was observed to rescue the RGC degenerative phenotype in NS-/- mice. These findings demonstrate that NS dysfunction plays a key role in the glaucoma inner retinal degenerative phenotype and that modulating NS imparts significant protection to the retina. NS upregulation protected RGC function and restored biochemical networks associated with autophagy and microglial and synaptic function in glaucoma.

Clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations.

An expansion of the GGGGCC hexanucleotide in the non-coding region of C9orf72 represents the most common cause of familial amyotrophic lateral sclerosis. The objective was to describe and analyse the clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations in a large population. Between November 2011 and December 2020, clinical and genetic characteristics of n = 248 patients with amyotrophic lateral sclerosis carrying C9orf72 mutations were collected from the clinical and scientific network of German motoneuron disease centres. Clinical parameters included age of onset, diagnostic delay, family history, neuropsychological examination, progression rate, phosphorylated neurofilament heavy chain levels in CSF and survival. The number of repeats was correlated with the clinical phenotype. The clinical phenotype was compared to n = 84 patients with SOD1 mutations and n = 2178 sporadic patients without any known disease-related mutations. Patients with C9orf72 featured an almost balanced sex ratio with 48.4% (n = 120) women and 51.6% (n = 128) men. The rate of 33.9% patients (n = 63) with bulbar onset was significantly higher compared to sporadic (23.4%, P = 0.002) and SOD1 patients (3.1%, P < 0.001). Of note, 56.3% (n = 138) of C9orf72, but only 16.1% of SOD1 patients reported a negative family history (P < 0.001). The GGGGCC hexanucleotide repeat length did not influence the clinical phenotypes. Age of onset (58.0, interquartile range 52.0-63.8) was later compared to SOD1 (50.0, interquartile range 41.0-58.0; P < 0.001), but earlier compared to sporadic patients (61.0, interquartile range 52.0-69.0; P = 0.01). Median survival was shorter (38.0 months) compared to SOD1 (198.0 months, hazard ratio 1.97, 95% confidence interval 1.34-2.88; P < 0.001) and sporadic patients (76.0 months, hazard ratio 2.34, 95% confidence interval 1.64-3.34; P < 0.001). Phosphorylated neurofilament heavy chain levels in CSF (2880, interquartile range 1632-4638 pg/ml) were higher compared to sporadic patients (1382, interquartile range 458-2839 pg/ml; P < 0.001). In neuropsychological screening, C9orf72 patients displayed abnormal results in memory, verbal fluency and executive functions, showing generally worse performances compared to SOD1 and sporadic patients and a higher share with suspected frontotemporal dementia. In summary, clinical features of patients with C9orf72 mutations differ significantly from SOD1 and sporadic patients. Specifically, they feature a more frequent bulbar onset, a higher share of female patients and shorter survival. Interestingly, we found a high proportion of patients with negative family history and no evidence of a relationship between repeat lengths and disease severity.

Conformation of neurofilament heavy chain effect on neurofilament-neurofilament association and bundling.

Mammalian neurofilaments (NFs) are composed of Heavy (NF-H), Medium (NF-M), and Light (NF-L) subunits, named according to their molecular mass, along with α-internexin or peripherin (in central or peripheral nervous systems, respectively). Phosphorylated NF subunits assemble linearly, with C-terminal regions extending from the filament “backbone.” Phosphorylation of C-terminal “side arms” of NF-H promotes NF-NF interactions, providing stability to axons. Transport of NFs from the perikaryon into the axon and NF-NF interactions are dependent upon regulation of multiple kinases. The complete sequence of phosphorylation events does not occur until NFs have entered the axon. Sidearm phosphorylation fosters lateral extension away from the NF backbone. By contrast, sidearm conformation prior to phosphorylation remains unresolved. Scrutiny of the NF-H side-arm sequence revealed 4 proline-proline (PP) motifs; these motifs allow tremendous rotation of their peptide chains from a closed, folded-back hairpin (cis) to a fully extended, linear confirmation (trans). Notably, one PP motif is located within each of the consensus sequences for the NF kinases. We hypothesize that phosphorylation-induced alterations in the configuration of these PP motifs are critical for NF transport, NF-NF association within axons. Mutated NFs constructs with changed PP motifs were expressed (with or without PP motifs /Phosphorylated or unphosphorylated) and incubated with NFs medium chain then trapped using a his-tag column to demonstrate bundling. Our initial data were inconclusive. A different experimental approach, such as Localized Surface Plasma Resonance (LSPR) is necessary to analyze the role of PP motifs in NFs association. We predict that constructs lacking PP motifs will present lower association affinity to NF-M (even when phosphorylated. Understanding the mechanisms that allow NF-NF associations within neurites will help guide targeted treatments for NF-based neurodegenerative disorders.

An Exploratory Trial of EPI-589 in Amyotrophic Lateral Sclerosis (EPIC-ALS): Protocol for a Multicenter, Open-Labeled, 24-Week, Single-Group Study

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, with its currently approved drugs, including riluzole and edaravone, showing limited therapeutic effects. Therefore, safe and effective drugs are urgently necessary. EPI-589 is an orally available, small-molecule, novel redox-active agent characterized by highly potent protective effects against oxidative stress with high blood-brain barrier permeability. Given the apparent oxidative stress and mitochondrial dysfunction involvement in the pathogenesis of ALS, EPI-589 may hold promise as a therapeutic agent. This protocol aims to describe the design and rationale for the EPI-589 Early Phase 2 Investigator-Initiated Clinical Trial for ALS (EPIC-ALS). EPIC-ALS is an explorative, open-labeled, single-arm trial that evaluates the safety and tolerability of EPI-589 in patients with ALS. This trial consists of 12-week run-in, 24-week treatment, and 4-week follow-up periods. Patients will receive 500 mg of EPI-589 3 times daily over the 24-week treatment period. Clinical assessments include the mean monthly change of Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised total score. The biomarkers are selected to analyze the effect on oxidative stress and neuronal damage. The plasma biomarkers are 8-hydroxy-2'-deoxyguanosine (8-OHdG), 3-nitrotyrosine (3-NT), neurofilament light chain (NfL), phosphorylated neurofilament heavy chain (pNfH), homocysteine, and creatinine. The cerebrospinal fluid biomarkers are 8-OHdG, 3-NT, NfL, pNfH, and ornithine. The magnetic resonance biomarkers are fractional anisotropy in the corticospinal tract and N-acetylaspartate in the primary motor area. This trial began data collection in September 2021 and is expected to be completed in October 2023. This study can provide useful data to understand the characteristics of EPI-589. Japan Primary Registries Network jRCT2061210031; tinyurl.com/2p84emu6. DERR1-10.2196/42032.

A Dedicated 21-Plex Proximity Extension Assay Panel for High-Sensitivity Protein Biomarker Detection Using Microdialysis in Severe Traumatic Brain Injury: The Next Step in Precision Medicine?

Cerebral protein profiling in traumatic brain injury (TBI) is needed to better comprehend secondary injury pathways. Cerebral microdialysis (CMD), in combination with the proximity extension assay (PEA) technique, has great potential in this field. By using PEA, we have previously screened >500 proteins from CMD samples collected from TBI patients. In this study, we customized a PEA panel prototype of 21 selected candidate protein biomarkers, involved in inflammation (13), neuroplasticity/-repair (six), and axonal injury (two). The aim was to study their temporal dynamics and relation to age, structural injury, and clinical outcome. Ten patients with severe TBI and CMD monitoring, who were treated in the Neurointensive Care Unit, Uppsala University Hospital, Sweden, were included. Hourly CMD samples were collected for up to 7 days after trauma and analyzed with the 21-plex PEA panel. Seventeen of the 21 proteins from the CMD sample analyses showed significantly different mean levels between days. Early peaks (within 48 h) were noted with interleukin (IL)-1β, IL-6, IL-8, granulocyte colony-stimulating factor, transforming growth factor alpha, brevican, junctional adhesion molecule B, and neurocan. C-X-C motif chemokine ligand 10 peaked after 3 days. Late peaks (>5 days) were noted with interleukin-1 receptor antagonist (IL-1ra), monocyte chemoattractant protein (MCP)-2, MCP-3, urokinase-type plasminogen activator, Dickkopf-related protein 1, and DRAXIN. IL-8, neurofilament heavy chain, and TAU were biphasic. Age (above/below 22 years) interacted with the temporal dynamics of IL-6, IL-1ra, vascular endothelial growth factor, MCP-3, and TAU. There was no association between radiological injury (Marshall grade) or clinical outcome (Extended Glasgow Outcome Scale) with the protein expression pattern. The PEA method is a highly sensitive molecular tool for protein profiling from cerebral tissue in TBI. The novel TBI dedicated 21-plex panel showed marked regulation of proteins belonging to the inflammation, plasticity/repair, and axonal injury families. The method may enable important insights into complex injury processes on a molecular level that may be of value in future efforts to tailor pharmacological TBI trials to better address specific disease processes and optimize timing of treatments.

Two Cases of Isolated Neurofilament Heavy Chain Antibody Syndrome

Objective Novel clinical and laboratory findings in anti-neurofilament heavy chain encephalitis Background Antibodies to mature components of neuronal intermediate filament (NIF) have been implicated in several neurological disorders, including multiple sclerosis, amyotrophic lateral sclerosis, and more recently, various autoimmune encephalitides. The components include a-internexin, light chain and heavy chain. In the largest case series of anti-NIF syndromes (McKeon et al, 2021), patients' cell-based assays revealed antibodies to one, two or all three components. Heavy chain antibodies (anti-NfH) were present in most, including three out of four patients with encephalopathy and cerebellar involvement. One was due to a paraneoplastic phenomenon. Anti-NfH was also elevated in two cases of encephalopathy with spasticity. It was the lone autoantibody in one of the six aforementioned cases. Design/Methods N/A. Results Case 1: 37-year old female with a history of ovarian carcinoma, treated in 2016. Cognitive impairment started in fall 2019, with significant worsening to the point of catatonia and coma in October 2020. Though encephalopathy improved, severe ataxia and nystagmus persisted. Two MRI brain studies and an EEG were unremarkable, and no radiological evidence of cancer recurrence. Oligoclonal bands (in both CSF and serum) and serum anti-NfH were elevated. Case 2: 59 year-old female with gradual cognitive decline since March 2018, followed by rapid cognitive deterioration in Oct 2020. There was limb weakness, severe rigidity, clonus and a witnessed seizure. EEG showed intermittent rhythmic delta activity. MRI brain indicated severe bilateral hippocampal atrophy. CSF Protein and CSF anti-NfH were elevated. Conclusions This case series contains the first reported paraneoplastic encephalopathy with cerebellar involvement from isolated anti-NfH. Also presented is the first reported case of PERMS from any NIF antibody. Further research is needed on quantitative and qualitative factors of anti-NIF syndromes. Specifically, the clinical relevance of the number of antibodies, and associations between phenotype and specific antibody combination.

PD34 Neuron-specific Biomarkers Associated With Neurological Manifestations In COVID-19: An Evidence Mapping Systematic Review

IntroductionWe aimed to map and synthesize the available evidence on neuron-specific biomarkers related to COVID-19.MethodsA systematic review and qualitative evidence mapping synthesis was performed (PROSPERO-CRD42021266995). Searches were conducted in PubMed and Scopus, and complemented by manual search (July 2021). We included observational studies of any design assessing neurological biomarkers in adult patients (&gt;18 years; with or without neurological comorbidities) diagnosed with COVID-19. Methodological quality of nonrandomized studies (case-control, cohorts) was assessed using the Newcastle-Ottawa Scale.ResultsOverall, 14 studies (n=485 patients) conducted in Sweden (n=4 articles, 28.5%), Germany (n=3; 21.4%), USA (n=3; 21.4%), Canada, France, Italy and Norway (n=1 study each) were included. The most reported neurological symptoms (n=13 studies, 92.8%) were headache, confusion, general weakness, loss of smell/taste, cognitive impairments and behavioral changes. Prevalent neurological conditions included encephalopathies, neuropathies, myopathies, and delirium; most critical cases presented cerebrovascular events (n=4 studies, 28.5%). Hypertension, diabetes, obesity, dyslipidemia, and chronic lung disease were the most reported comorbidities. Eight different neuron-specific biomarkers were found in primary studies: neurofilament-light chain – NfL (n=10 studies; 71.4%), glial fibrillary acidic-protein – GFAp (n=5; 35.7%), tau protein (n=5; 35.7%), neurofilament-heavy chain – NfH, S100B protein, ubiquitin C-terminal hydrolase L1 - UCH-L1, neuron-specific enolase and beta protein-amyloid – Aβ (n=1 study each). These biomarkers were found both in cerebrospinal fluid and blood/plasma samples even without an evident cytokine storm. In patients with COVID-19, NfL and GFAp can act as sensitive indicators of neuroaxonal and astrocytic damages, respectively. Increased levels of NfL were significantly associated with severe COVID-19, unconsciousness and longer stay in the intensive care unit (p&lt;0.05). Studies had an overall poor to moderate methodological quality.ConclusionsWe identified eight neuron-specific biomarkers that should be further studied as prognostic factors of COVID-19. These findings can also guide the development of targeted therapies against SARS-CoV-2. Additional well-designed clinical trials are needed to strengthen this evidence and help understand the mechanisms of neurological symptoms and sequelae after COVID-19 infection.

An induced pluripotent stem cell-based model identifies molecular targets of vincristine neurotoxicity.

To model peripheral nerve degeneration and investigate molecular mechanisms of neurodegeneration, we established a cell system of induced pluripotent stem cell (iPSC)-derived sensory neurons exposed to vincristine, a drug that frequently causes chemotherapy-induced peripheral neuropathy. Sensory neurons differentiated from iPSCs exhibit distinct neurochemical patterns according to the immunocytochemical phenotypes, and gene expression of peripherin (PRPH, hereafter referred to as Peri) and neurofilament heavy chain (NEFH, hereafter referred to as NF). The majority of iPSC-derived sensory neurons were PRPH positive/NEFH negative, i.e. Peri(+)/NF(-) neurons, whose somata were smaller than those of Peri(+)/NF(+) neurons. On exposure to vincristine, projections from the cell body of a neuron, i.e. neurites, were degenerated quicker than somata, the lethal concentration to kill 50% (LC50) of neurites being below the LC50 for somata, consistent with the clinical pattern of length-dependent neuropathy. We then examined the molecular expression in the MAP kinase signaling pathways of, extracellular signal-regulated kinases 1/2 (MAPK1/3, hereafter referred to as ERK), p38 mitogen-activated protein kinases (MAPK11/12/13/14, hereafter referred to as p38) and c-Jun N-terminal kinases (MAPK8/9/10, hereafter referred to as JNK). Regarding these three cascades, only phosphorylation of JNK was upregulated but not that of p38 or ERK1/2. Furthermore, vincristine-treatment resulted in impaired autophagy and reduced autophagic flux. Rapamycin-treatment reversed the effect of impaired autophagy and JNK activation. These results not only established a platform to study peripheral degeneration of human neurons but also provide molecular mechanisms for neurodegeneration with the potential for therapeutic targets.

Diagnostic utility of neurofilament markers for MND is limited in restricted disease phenotype and for differentiation from compressive myeloradiculopathies.

Misdiagnosis is frequent in early motor neuron disease (MND), typically compressive radiculopathy, or in patients with restricted MND phenotype. In this retrospective, single tertiary centre study, we measured levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (p-NfH) chain in cerebrospinal fluid (CSF) and of p-NfH in serum with commercially available ELISA kits and assessed their respective diagnostic performance as a marker of MND. The entire study population (n = 164) comprised 71 MND patients, 30 patients with compressive myelo- or radiculopathy, and 63 disease controls (DC). Among MND patients, we specified subgroups with only lower motoneuron involvement (MND-LMN, n = 15) and with confounding nerve roots or spinal cord compression (MND-C, n = 18), representing clinical diagnostic pitfalls. MND-LMN displayed significantly lower CSF NfL (p = 0.003) and p-NFH (p = 0.017), but not serum p-NfH (p = 0.347) levels compared to other MND patients (n = 56). The discriminative ability (area under the curve-AUC) of both CSF Nfs towards all MND patients was comparable to each other but significantly higher than that of p-NfH in serum (ps < 0.001). AUC of both CSF Nfs between MND-LMN and DC and also between MND-C and myelo-/radiculopathies were reduced, as compared to AUC between other MND and DC or myelo-/radiculopathies, respectively. Our results suggest that both Nfs in CSF represent a reliable diagnostic marker in a general MND population, fulfilling Awaji criteria. As for diagnostic pitfalls, and also for p-NfH in serum, their discriminative ability and, therefore, clinical utility appears to be limited.

Differential Glial Chitotriosidase 1 and Chitinase 3-like Protein 1 Expression in the Human Primary Visual Cortex and Cerebellum after Global Hypoxia-Ischemia

Here, we studied the neuroinflammation- and ischemia-related glial markers chitotriosidase 1 (CHIT1) and chitinase-3-like protein 1 (CHI3L1, alias YKL-40) in the human striate cortex and cerebellum at different time points after global hypoxic-ischemic brain injury (HIBI). Both regions differ considerably in their glial cell population but are supplied by the posterior circulation. CHIT1 and CHI3L1 expression was compared to changes in microglial (IBA1, CD68), astrocytic (GFAP, S100β), and neuronal markers (H&E, neurofilament heavy chain, NfH; calretinin, CALR) using immunohistochemistry and multiple-label immunofluorescence. Initial striatal cortical and cerebellar Purkinje cell damage, detectable already 1/2 d after HIBI, led to delayed neuronal death, whereas loss of cerebellar NfH-positive stellate and CALR-positive granule cells was variable. During the first week post-HIBI, a transient reduction of IBA1-positive microglia was observed in both regions, and fragmented/clasmatodendritic cerebellar Bergmann glia appeared. In long-term survivors, both brain regions displayed high densities of activated IBA1-positive cells and CD68-positive macrophages, which showed CHIT1 co-localization in the striate cortex. Furthermore, enlarged GFAP- and S100β-positive astroglia emerged in both regions around 9–10 d post-HIBI, i.e., along with clearance of dead neurons from the neuropil, although GFAP-/S100β-positive gemistocytic astrocytes that co-expressed CHI3L1 were found only in the striate cortex. Thus, only GFAP-/S100β-positive astrocytes in the striate cortex, but not cerebellar Bergmann glia, differentiated into CHI3L1-positive gemistocytes. CHIT1 was co-expressed almost entirely in macrophages in the striate cortex and not cerebellum of long-term survivors, thereby indicating that CHIT1 and CHI3L1 could be valuable biomarkers for monitoring the outcome of global HIBI.

Different Profiles of the Triad of Lysophosphatidylcholine, Lysophosphatidic Acid, and Autotaxin in Patients with Neuropathic Pain Diseases: a Preliminary Observational Study.

The mechanisms underlying neuropathic pain remain unclear. Lysophosphatidic acid (LPA) is a bioactive phospholipid derived mainly from lysophosphatidylcholine (LPC) by extracellular autotaxin (ATX), and has attracted attention as a candidate biomarker of neuropathic pain. We aimed to investigate the levels of LPA, LPC, and ATX in patients with lumbar spinal canal stenosis (LSCS) or other neuropathic pain diseases, and to distinguish the underlying mechanism of LSCS from other neuropathic pain conditions. Furthermore, the levels of phosphorylated neurofilament heavy chain (pNF-H), an objective surrogate marker of axonal damage, were also measured. Cerebrospinal fluid (CSF) samples were obtained from 56 patients with LSCS (n = 31) and various etiologies other than LSCS (n = 25). Patients with LSCS complained of pain intensity comparable to that of patients without LSCS. The LPA levels were significantly higher in patients with LSCS than in non-LSCS patients, while the ATX levels were significantly lower. However, the differences in LPC and pNF-H levels between the two patient groups were not significant. The LPA/LPC ratio was significantly higher in the LSCS group. Notably, the difference in LPA between the two groups diminished in the analysis of covariance (ANCOVA) with ATX as a covariate. Thus, it helped to reveal that LPA synthesis in patients with LSCS depends more efficiently on ATX than in non-LSCS neuropathic pain patients with other etiologies. Our findings further suggest that the triad of LPA, LPC, and ATX in LSCS may contribute to the development and maintenance of neuropathic pain in a manner different from non-LSCS neuropathic conditions.