Association Between Serum Neurofilament Light Chain and Neurochemistry Deficits in Patients with Spinocerebellar Ataxia Type 3.

Extensive evidence supports the claim that the serum neurofilament light chain (sNfL) can be used as a biomarker to monitor disease severity in patients with spinocerebellar ataxia type 3 (SCA3). However, little is known about the associations between sNfL levels and neurochemical alterations in SCA3 patients. In this study, we performed a cross-sectional study to analyze the association between sNfL and brain metabolic changes in SCA3 patients. The severity of ataxia was assessed by using the Scale for the Assessment and Rating of Ataxia (SARA) and the International Cooperative Ataxia Rating Scale (ICARS). The sNfL levels and brain metabolic changes, represented by N-acetyl aspartate (NAA)/creatine (Cr) and choline complex (Cho)/Cr ratios, were measured by a single-molecule array and proton magnetic resonance spectroscopy, respectively. In this cohort, we observed consistently elevated sNfL levels and reduced brain metabolites in the cerebellar hemispheres, dentate nucleus, and cerebellar vermis. However, this correlation was further validated in the cerebellar cortex after analysis using pairwise comparisons and a Bonferroni correction. Taken together, our results further confirmed that sNfL levels were increased in SCA3 patients and were negatively correlated with metabolic changes in the cerebellar cortex. Our data also support the idea that sNfL levels are a promising potential complementary biomarker for patients with SCA3.

Astrocyte biomarkers GFAP and YKL-40 mediate early Alzheimer's disease progression.

INTRODUCTION: We studied how biomarkers of reactive astrogliosis mediate the pathogenic cascade in the earliest Alzheimer's disease (AD) stages. METHODS: We performed path analysis on data from 384 cognitively unimpaired individuals from the ALzheimer and FAmilies (ALFA)+ study using structural equation modeling to quantify the relationships between biomarkers of reactive astrogliosis and the AD pathological cascade. RESULTS: Cerebrospinal fluid (CSF) amyloid beta (Aß)42/40 was associated with Aß aggregation on positron emission tomography (PET) and with CSF p-tau181 , which was in turn directly associated with CSF neurofilament light (NfL). Plasma glial fibrillary acidic protein (GFAP) mediated the relationship between CSF Aß42/40 and Aß-PET, and CSF YKL-40 partly explained the association between Aß-PET, p-tau181 , and NfL. DISCUSSION: Our results suggest that reactive astrogliosis, as indicated by different fluid biomarkers, influences the pathogenic cascade during the preclinical stage of AD. While plasma GFAP mediates the early association between soluble and insoluble Aß, CSF YKL-40 mediates the latter association between Aß and downstream Aß-induced tau pathology and tau-induced neuronal injury. HIGHLIGHTS: Lower CSF Aß42/40 was directly linked to higher plasma GFAP concentrations. Plasma GFAP partially explained the relationship between soluble Aß and insoluble Aß. CSF YKL-40 mediated Aß-induced tau phosphorylation and tau-induced neuronal injury.

Stathmin-2 loss leads to neurofilament-dependent axonal collapse driving motor and sensory denervation.

The mRNA transcript of the human STMN2 gene, encoding for stathmin-2 protein (also called SCG10), is profoundly impacted by TAR DNA-binding protein 43 (TDP-43) loss of function. The latter is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Using a combination of approaches, including transient antisense oligonucleotide-mediated suppression, sustained shRNA-induced depletion in aging mice, and germline deletion, we show that stathmin-2 has an important role in the establishment and maintenance of neurofilament-dependent axoplasmic organization that is critical for preserving the caliber and conduction velocity of myelinated large-diameter axons. Persistent stathmin-2 loss in adult mice results in pathologies found in ALS, including reduced interneurofilament spacing, axonal caliber collapse that drives tearing within outer myelin layers, diminished conduction velocity, progressive motor and sensory deficits, and muscle denervation. These findings reinforce restoration of stathmin-2 as an attractive therapeutic approach for ALS and other TDP-43-dependent neurodegenerative diseases.

Plasma glial fibrillary acidic protein levels correlate with paramagnetic rim lesions in people with radiologically isolated syndrome.

BACKGROUND: There are no specific, evidence-based recommendations for the management of individuals with radiologically isolated syndrome. Imaging and blood biomarkers may have prognostic utility. OBJECTIVE: To determine whether plasma neurofilament light protein (NfL) or glial fibrillary acidic protein (GFAP) levels in people with radiologically isolated syndrome correlate with imaging measures that have been shown to be associated with negative clinical outcomes in people with multiple sclerosis. METHODS: Cross-sectional analysis of people with radiologically isolated syndrome. Participants underwent magnetic resonance imaging (MRI) of the brain and cervical spinal cord, and plasma was collected. Plasma NfL and GFAP levels were measured with a single-molecule array, and correlations with MRI measures were assessed, including the number of: T1-black holes, white-matter lesions demonstrating the central vein sign, paramagnetic rim lesions, cervical spinal cord lesions and infratentorial lesions. RESULTS: Plasma GFAP levels, but not NfL levels, showed correlations with the number of T1-black holes, white matter lesions demonstrating the central vein sign and paramagnetic rim lesions (all p < 0.05). CONCLUSION: We found correlations between plasma GFAP levels and imaging measures associated with poor clinical outcomes and chronic inflammation in individuals with radiologically isolated syndrome. Plasma GFAP may have prognostic utility in clinical trials and clinical practice.

Spatial transcriptomics and neurofilament light chain reveal changes in lesion patterns in murine autoimmune neuroinflammation.

OBJECTIVE: Ongoing neuroaxonal damage is a major contributor to disease progression and long-term disability in multiple sclerosis. However, spatio-temporal distribution and pathophysiological mechanisms of neuroaxonal damage during acute relapses and later chronic disease stages remain poorly understood. METHODS: Here, we applied immunohistochemistry, single-molecule array, spatial transcriptomics, and microglia/axon co-cultures to gain insight into spatio-temporal neuroaxonal damage in experimental autoimmune encephalomyelitis (EAE). RESULTS: Association of spinal cord white matter lesions and blood-based neurofilament light (sNfL) levels revealed a distinct, stage-dependent anatomical pattern of neuroaxonal damage: in chronic EAE, sNfL levels were predominately associated with anterolateral lumbar lesions, whereas in early EAE sNfL showed no correlation with lesions in any anatomical location. Furthermore, neuroaxonal damage in late EAE was largely confined to white matter lesions but showed a widespread distribution in early EAE. Following this pattern of neuroaxonal damage, spatial transcriptomics revealed a widespread cyto- and chemokine response at early disease stages, whereas late EAE was characterized by a prominent glial cell accumulation in white matter lesions. These findings were corroborated by immunohistochemistry and microglia/axon co-cultures, which further revealed a strong association between CNS myeloid cell activation and neuroaxonal damage both in vivo and in vitro. INTERPRETATION: Our findings indicate that CNS myeloid cells may play a crucial role in driving neuroaxonal damage in EAE. Moreover, neuroaxonal damage can progress in a stage-dependent centripetal manner, transitioning from normal-appearing white matter to focal white matter lesions. These insights may contribute to a better understanding of neurodegeneration and elevated sNfL levels observed in multiple sclerosis patients at different disease stages.

Upregulated expression of miR-4443 and miR-4488 in drug resistant melanomas promotes migratory and invasive phenotypes through downregulation of intermediate filament nestin.

BACKGROUND: BRAF-mutant melanoma patients benefit from the combinatorial treatments with BRAF and MEK inhibitors. However, acquired drug resistance strongly limits the efficacy of these targeted therapies in time. Recently, many findings have underscored the involvement of microRNAs as main drivers of drug resistance. In this context, we previously identified a subset of oncomiRs strongly up-regulated in drug-resistant melanomas. In this work, we shed light on the molecular role of two as yet poorly characterized oncomiRs, miR-4443 and miR-4488. METHODS: Invasion and migration have been determined by wound healing, transwell migration/invasion assays and Real Time Cell Analysis (RTCA) technology. miR-4488 and miR-4443 have been measured by qRT-PCR. Nestin levels have been tested by western blot, confocal immunofluorescence, immunohistochemical and flow cytometry analyses. RESULTS: We demonstrate that the two oncomiRs are responsible for the enhanced migratory and invasive phenotypes, that are a hallmark of drug resistant melanoma cells. Moreover, miR-4443 and miR-4488 promote an aberrant cytoskeletal reorganization witnessed by the increased number of stress fibers and cellular protrusions-like cancer cell invadopodia. Mechanistically, we identified the intermediate filament nestin as a molecular target of both oncomiRs. Finally, we have shown that nestin levels are able to predict response to treatments in melanoma patients. CONCLUSIONS: Altogether these findings have profound translational implications in the attempt i) to develop miRNA-targeting therapies to mitigate the metastatic phenotypes of BRAF-mutant melanomas and ii) to identify novel biomarkers able to guide clinical decisions.

Stabilizing vimentin phosphorylation inhibits stem-like cell properties and metastasis of hybrid epithelial/mesenchymal carcinomas.

Epithelial-mesenchymal transition (EMT) empowers epithelial cells with mesenchymal and stem-like attributes, facilitating metastasis, a leading cause of cancer-related mortality. Hybrid epithelial-mesenchymal (E/M) cells, retaining both epithelial and mesenchymal traits, exhibit heightened metastatic potential and stemness. The mesenchymal intermediate filament, vimentin, is upregulated during EMT, enhancing the resilience and invasiveness of carcinoma cells. The phosphorylation of vimentin is critical to its structure and function. Here, we identify that stabilizing vimentin phosphorylation at serine 56 induces multinucleation, specifically in hybrid E/M cells with stemness properties but not epithelial or mesenchymal cells. Cancer stem-like cells are especially susceptible to vimentin-induced multinucleation relative to differentiated cells, leading to a reduction in self-renewal and stemness. As a result, vimentin-induced multinucleation leads to sustained inhibition of stemness properties, tumor initiation, and metastasis. These observations indicate that a single, targetable phosphorylation event in vimentin is critical for stemness and metastasis in carcinomas with hybrid E/M properties.

Therapeutic targeting of vimentin by ALD-R491 impacts multiple pathogenic processes to attenuate acute and chronic colitis in mice.

BACKGROUND: Vimentin, an intermediate filament protein, crucially contributes to the pathogenesis of inflammatory bowel disease (IBD) by interacting with genetic risk factors, facilitating pathogen infection, and modulating both innate and adaptive immune responses. This study aimed to demonstrate preclinical proof-of-concept for targeting vimentin therapeutically in IBD across diverse etiologies. METHODS: The small molecule compound ALD-R491 was assessed for vimentin binding using microscale thermophoresis, off-target effects via Eurofins screening, and therapeutic effects in mice with dextran sulfate sodium (DSS)-induced acute colitis and in IL-10 KO with spontaneous colitis. Parameters measured included body weight, survival, disease activity, colon length, and histology. The study analyzed intestinal proinflammatory cytokines, Th17/Treg cells, and epithelial barrier molecules, along with gut microbiota profiling. RESULTS: ALD-R491 specifically bound vimentin with a dissociation constant (KD) of 328 ± 12.66 nM and no off-target effects. In the DSS model, orally administered ALD-R491 exhibited dose-dependent therapeutic effects, superior to 5-ASA and Tofacitinib. In the IL-10 KO model, ALD-R491 significantly delayed colitis onset and progression, with near-zero disease activity index scores over a 15-week treatment. ALD-R491 consistently showed in both models a reduced proinflammatory cytokine expression, including TNF-α, IL-1ß, IL-6, IL-17, IL-22, a rebalanced Th17/Treg axis by reducing RORγt while enhancing FoxP3 expression, and an improved epithelial barrier integrity by increasing intestinal expressions of Mucin-2, ZO-1 and Claudin5. The intestinal dysbiosis was restored with enriched presence of probiotics. CONCLUSIONS: Targeting vimentin exhibits significant therapeutic effects on various facets of IBD pathogenesis, representing a compelling approach for the development of highly effective treatments in IBD.

Increasing Neurofilament and Glial Fibrillary Acidic Protein After Treatment Discontinuation Predicts Multiple Sclerosis Disease Activity.

BACKGROUND AND OBJECTIVES: Stable patients with multiple sclerosis (MS) may discontinue treatment, but the risk of disease activity is unknown. Serum neurofilament light chain (sNfL) and serum glial fibrillary acidic protein (sGFAP) are biomarkers of subclinical disease activity and may help risk stratification. In this study, sNfL and sGFAP levels in stable patients were evaluated before and after treatment discontinuation to determine association with disease activity. METHODS: This observational study included patients enrolled in the Comprehensive Longitudinal Investigation in MS at the Brigham and Women's Hospital who discontinued treatment after >2 years disease activity-free. Two serum samples within 2 years, before and after treatment stop, were sent for sNfL and sGFAP measurements by single-molecule array. Biannual neurologic examinations and yearly MRI scans determined disease activity by 3 time-to-event outcomes: 6-month confirmed disability worsening (CDW), clinical attacks, and MRI activity (new T2 or contrast-enhancing lesions). Associations between each outcome and log-transformed sNfL and sGFAP levels pretreatment stop and posttreatment stop and the percent change were estimated using multivariable Cox regression analysis adjusting for age, disability, disease duration, and duration from attack before treatment stop. RESULTS: Seventy-eight patients (92% female) discontinued treatment at a median (interquartile range) age of 48.5 years (39.0-55.7) and disease duration of 12.3 years (7.5-18.8) and were followed up for 6.3 years (4.2-8.5). CDW occurred in 27 patients (35%), new attacks in 19 (24%), and new MRI activity in 26 (33%). Higher posttreatment stop sNfL level was associated with CDW (adjusted hazard ratio (aHR) 2.80, 95% CI 1.36-5.76, p = 0.005) and new MRI activity (aHR 3.09, 95% CI 1.42-6.70, p = 0.004). Patients who had >100% increase in sNfL level from pretreatment stop to posttreatment stop had greater risk of CDW (HR 3.87, 95% CI 1.4-10.7, p = 0.009) and developing new MRI activity (HR 4.02, 95% CI 1.51-10.7, p = 0.005). Patients who had >50% increase in sGFAP level also had greater risk of CDW (HR 5.34, 95% CI 1.4-19.9, p = 0.012) and developing new MRI activity (HR 5.16, 95% CI 1.71-15.6, p = 0.004). DISCUSSION: Stable patients who discontinue treatment may be risk stratified by sNfL and sGFAP levels measured before and after discontinuing treatment. Further studies are needed to validate findings and determine whether resuming treatment in patients with increasing biomarker levels reduces risk of subsequent disease activity.

Pretreatment Neurofilament Light Chain Serum Levels, Early Disease Severity, and Treatment Response in Pediatric Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: High disease activity and frequent therapy failure in pediatric multiple sclerosis (MS) make prognostic biomarkers urgently needed. We investigated whether serum neurofilament light chain (sNfL) levels in treatment-naive pediatric patients with MS are associated with early disease severity and indicate treatment outcomes. METHODS: A retrospective cohort study of patients seen in the Göttingen Center for MS in Childhood and Adolescence, Germany. Inclusion criteria were MS diagnosis according to the McDonald criteria, MS onset <18 years, and available pretreatment serum sample. sNfL levels were analyzed using a single-molecule array assay. Associations with clinical and MRI evidence of disease severity at sampling were evaluated using the Spearman correlations and nonparametric tests for group comparisons. Correlations between pretreatment sNfL and annualized relapse and new T2 lesion rate on first-line therapy, and odd ratios for switch to high-efficacy therapy were assessed. RESULTS: A total of 178 patients (116 women [65%]) with a mean sampling age of 14.3 years were included in the study. Pretreatment sNfL levels were above the ≥90th percentile reported for healthy controls in 80% of patients (median 21.1 pg/mL) and correlated negatively with age, but no correlation was seen with sex, oligoclonal band status, or body mass index. High pretreatment sNfL levels correlated significantly with a high number of preceding relapses, a shorter first interattack interval, a high T2 lesion count, and recent gadolinium-enhancing lesions. Of interest, sNfL levels reflected more strongly MRI activity rather than clinical activity. Pretreatment sNfL levels also correlated significantly with the relapse rate and occurrence of new/enlarging T2 lesions while on first-line injectable therapy. Odds of future therapy escalation increased from 0.14 for sNfL below 7.5 pg/mL to 6.38 for sNfL above 15 pg/mL. In patients with a recent relapse, higher sNfL levels were associated with poorer recovery 3 months after attack. DISCUSSION: The results of this study have 3 important implications: First, pretreatment sNfL levels are a valuable biomarker for underlying disease activity in pediatric patients with MS. Second, pretreatment sNfL levels in pediatric patients with MS have a predictive value for the response to first-line therapy and the necessity of future therapy escalation. Third, high sNfL levels during a relapse are associated with poor recovery in this age group.

Changes in stiffness of the optic nerve and involvement of neurofilament light chains in the course of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are often accompanied by optic neuritis associated with neurofilament disruption. In this study, the stiffness of the optic nerve was investigated by atomic force microscopy (AFM) in mice with induced EAE in the successive phases of the disease: onset, peak, and chronic. AFM results were compared with the intensity of the main pathological processes in the optic nerve: inflammation, demyelination, and axonal loss, as well as with the density of astrocytes, assessed by quantitative histology and immunohistochemistry. Optic nerve tissue and serum levels of neurofilament light chain protein (NEFL) were also examined by immunostaining and ELISA, respectively. The stiffness of the optic nerve in EAE mice was lower than that in control and naïve animals. It increased in the onset and peak phases and sharply decreased in the chronic phase. Serum NEFL level showed similar dynamics, while tissue NEFL level decreased in the onset and peak phases, indicating a leak of NEFL from the optic nerve to body fluids. Inflammation and demyelination gradually increased to reach the maximum in the peak phase of EAE, and inflammation slightly declined in the chronic phase, while demyelination did not. The axonal loss also gradually increased and had the highest level in the chronic phase. Among these processes, demyelination and especially axonal loss most effectively decrease the stiffness of the optic nerve. NEFL level in serum can be regarded as an early indicator of EAE, as it rapidly grows in the onset phase of the disease.

Diffuse argyrophilic grain disease with TDP-43 proteinopathy and neuronal intermediate filament inclusion disease: FTLD with mixed tau, TDP-43 and FUS pathologies.

Frontotemporal lobar degeneration (FTLD) is a group of disorders characterized by degeneration of the frontal and temporal lobes, leading to progressive decline in language, behavior, and motor function. FTLD can be further subdivided into three main subtypes, FTLD-tau, FTLD-TDP and FTLD-FUS based which of the three major proteins - tau, TDP-43 or FUS - forms pathological inclusions in neurons and glia. In this report, we describe an 87-year-old woman with a 7-year history of cognitive decline, hand tremor and gait problems, who was thought to have Alzheimer's disease. At autopsy, histopathological analysis revealed severe neuronal loss, gliosis and spongiosis in the medial temporal lobe, orbitofrontal cortex, cingulate gyrus, amygdala, basal forebrain, nucleus accumbens, caudate nucleus and anteromedial thalamus. Tau immunohistochemistry showed numerous argyrophilic grains, pretangles, thorn-shaped astrocytes, and ballooned neurons in the amygdala, hippocampus, parahippocampal gyrus, anteromedial thalamus, insular cortex, superior temporal gyrus and cingulate gyrus, consistent with diffuse argyrophilic grain disease (AGD). TDP-43 pathology in the form of small, dense, rounded neuronal cytoplasmic inclusion with few short dystrophic neurites was observed in the limbic regions, superior temporal gyrus, striatum and midbrain. No neuronal intranuclear inclusion was observed. Additionally, FUS-positive inclusions were observed in the dentate gyrus. Compact, eosinophilic intranuclear inclusions, so-called "cherry spots," that were visible on histologic stains were immunopositive for α-internexin. Taken together, the patient had a mixed neurodegenerative disease with features of diffuse AGD, TDP-43 proteinopathy and neuronal intermediate filament inclusion disease. She met criteria for three subtypes of FTLD: FTLD-tau, FTLD-TDP and FTLD-FUS. Her amnestic symptoms that were suggestive of Alzheimer's type dementia are best explained by diffuse AGD and medial temporal TDP-43 proteinopathy, and her motor symptoms were likely explained by neuronal loss and gliosis due to tau pathology in the substantia nigra. This case underscores the importance of considering multiple proteinopathies in the diagnosis of neurodegenerative diseases.

NfL and GFAP in serum are associated with microstructural brain damage in progressive multiple sclerosis.

BACKGROUND: The potential of neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) as biomarkers of disease activity and severity in progressive forms of multiple sclerosis (MS) is unclear. OBJECTIVE: To investigate the relationship between serum concentrations of NfL, GFAP, and magnetic resonance imaging (MRI) in progressive MS. METHODS: Serum concentrations of NfL and GFAP were measured in 32 healthy controls and 32 patients with progressive MS from whom clinical and MRI data including diffusion tensor imaging (DTI) were obtained during three years of follow-up. RESULTS: Serum concentrations of NfL and GFAP at follow-up were higher in progressive MS patients than in healthy controls and serum NfL correlated with the EDSS score. Decreasing fractional anisotropy (FA) in normal-appearing white matter (NAWM) correlated with worsening EDSS scores and higher serum NfL. Higher serum NfL and increasing T2 lesion volume correlated with worsening paced autitory serial addition test scores. In multivariable regression analyses with serum GFAP and NfL as independent factors and DTI measures of NAWM as dependent factors, we showed that high serum NfL at follow-up was independently associated with decreasing FA and increasing MD in NAWM. Moreover, we found that high serum GFAP was independently associated with decreasing MD in NAWM and with decreasing MD and increasing FA in cortical gray matter. CONCLUSION: Serum concentrations of NfL and GFAP are increased in progressive MS and are associated with distinct microstructural changes in NAWM and CGM.

Serum neurofilament light chain measurements following nerve trauma.

BACKGROUND: Optimal functional recovery following peripheral nerve injuries (PNIs) is dependent upon early recognition and prompt referral to specialist centres for appropriate surgical intervention. Technologies which facilitate the early detection of PNI would allow faster referral rates and encourage improvements in patient outcomes. Serum Neurofilament light chain (NfL) measurements are cheaper to perform, easier to access and interpret than many conventional methods used for nerve injury diagnosis, such as electromyography and/or magnetic resonance imaging assessments, but changes in serum NfL levels following traumatic PNI have not been investigated. This pre-clinical study aimed to determine whether serum NfL levels can: (1) detect the presence of a nerve trauma and (2) delineate between different severities of nerve trauma. METHODS: A rat sciatic nerve crush and common peroneal nerve crush were implemented as controlled animal models of nerve injury. At 1-, 3-, 7- and 21-days post-injury, serum samples were retrieved for analysis using the SIMOA® NfL analyser kit. Nerve samples were also retrieved for histological analysis. Static sciatic index (SSI) was measured at regular time intervals following injury. RESULTS: Significant 45-fold and 20-fold increases in NfL serum levels were seen 1-day post-injury following sciatic and common peroneal nerve injury, respectively. This corresponded with an eightfold higher volume of axons injured in the sciatic compared to the common peroneal nerve (p < .001). SSI measurements post-injury revealed greater reduction in function in the sciatic crush group compared with the common peroneal crush group. CONCLUSIONS: NfL serum measurements represent a promising method for detecting traumatic PNI and stratifying their severity. Clinical translation of these findings could provide a powerful tool to improve the surgical management of nerve-injured patients.

A New Mouse Model of Giant Axonal Neuropathy with Overt Phenotypes and Neurodegeneration Driven by Neurofilament Disorganization.

Research on pathogenic mechanisms underlying giant axonal neuropathy (GAN), a disease caused by a deficiency of gigaxonin, has been hindered by the lack of appropriate animal models exhibiting substantial symptoms and large neurofilament (NF) swellings, a hallmark of the human disease. It is well established that intermediate filament (IF) proteins are substrates for gigaxonin-mediated degradation. However, it has remained unknown to what extent NF accumulations contribute to GAN pathogenesis. Here, we report the generation of a new mouse model of GAN that is based on crossing transgenic mice overexpressing peripherin (Prph) with mice knockout for Gan The Gan-/-;TgPer mice developed early onset sensory-motor deficits along with IF accumulations made up of NF proteins and of Prph, causing swelling of spinal neurons at a young age. Abundant inclusion bodies composed of disorganized IFs were also detected in the brain of Gan-/-;TgPer mice. At 12 months of age, the Gan-/-;TgPer mice exhibited cognitive deficits as well as severe sensory and motor defects. The disease was associated with neuroinflammation and substantial loss of cortical neurons and spinal neurons. Giant axons (≥160 µm2) enlarged by disorganized IFs, a hallmark of GAN disease, were also detected in dorsal and ventral roots of the Gan-/-;TgPer mice. These results, obtained with both sexes, support the view that the disorganization of IFs can drive some neurodegenerative changes caused by gigaxonin deficiency. This new mouse model should be useful to investigate the pathogenic changes associated with GAN disease and for drug testing.SIGNIFICANCE STATEMENT Research on pathogenic mechanism and treatment of GAN has been hampered by the lack of animal models exhibiting overt phenotypes and substantial neurofilament disorganization, a hallmark of the disease. Moreover, it remains unknown whether neurologic defects associated with gigaxonin deficiency in GAN are because of neurofilament disorganization as gigaxonin may also act on other protein substrates to mediate their degradation. This study reports the generation of a new mouse model of GAN based on overexpression of Prph in the context of targeted disruption of gigaxonin gene. The results support the view that neurofilament disorganization may contribute to neurodegenerative changes in GAN disease. The Gan-/-;TgPer mice provide a unique animal model of GAN for drug testing.

Early neurofilament light and glial fibrillary acidic protein levels improve predictive models of multiple sclerosis outcomes.

BACKGROUND: Early risk-stratification in multiple sclerosis (MS) may impact treatment decisions. Current predictive models have identified that clinical and imaging characteristics of aggressive disease are associated with worse long-term outcomes. Serum biomarkers, neurofilament (sNfL) and glial fibrillary acidic protein (sGFAP), reflect subclinical disease activity through separate pathological processes and may contribute to predictive models of clinical and MRI outcomes. METHODS: We conducted a retrospective analysis of the Comprehensive Longitudinal Investigation of Multiple Sclerosis at the Brigham and Women's Hospital (CLIMB study), where patients with multiple sclerosis are seen every 6 months and undergo Expanded Disability Status Scale (EDSS) assessment, have annual brain MRI scans where volumetric analysis is conducted to calculate T2-lesion volume (T2LV) and brain parenchymal fraction (BPF), and donate a yearly blood sample for subsequent analysis. We included patients with newly diagnosed relapsing-remitting MS and serum samples obtained at baseline visit and 1-year follow-up (both within 3 years of onset), and were assessed at 10-year follow-up. We measured sNfL and sGFAP by single molecule array at baseline visit and at 1-year follow-up. A predictive clinical model was developed using age, sex, Expanded Disability Status Scale (EDSS), pyramidal signs, relapse rate, and spinal cord lesions at first visit. The main outcome was odds of developing of secondary progressive (SP)MS at year 10. Secondary outcomes included 10-year EDSS, brain T2LV and BPF. We compared the goodness-of-fit of the predictive clinical model with and without sNfL and sGFAP at baseline and 1-year follow-up, for each outcome by area under the receiver operating characteristic curve (AUC) or R-squared. RESULTS: A total 144 patients with median MS onset at age 37.4 years (interquartile range: 29.4-45.4), 64% female, were included. SPMS developed in 25 (17.4%) patients. The AUC for the predictive clinical model without biomarker data was 0.73, which improved to 0.77 when both sNfL and sGFAP were included in the model (P = 0.021). In this model, higher baseline sGFAP associated with developing SPMS (OR=3.3 [95%CI:1.1,10.6], P = 0.04). Adding 1-year follow-up biomarker levels further improved the model fit (AUC = 0.79) but this change was not statistically significant (P = 0.15). Adding baseline biomarker data also improved the R-squared of clinical models for 10-year EDSS from 0.24 to 0.28 (P = 0.032), while additional 1-year follow-up levels did not. Baseline sGFAP was associated with 10-year EDSS (ß=0.58 [95%CI:0.00,1.16], P = 0.05). For MRI outcomes, baseline biomarker levels improved R-squared for T2LV from 0.12 to 0.27 (P<0.001), and BPF from 0.15 to 0.20 (P = 0.042). Adding 1-year follow-up biomarker data further improved T2LV to 0.33 (P = 0.0065) and BPF to 0.23 (P = 0.048). Baseline sNfL was associated with T2LV (ß=0.34 [95%CI:0.21,0.48], P<0.001) and 1-year follow-up sNfL with BPF (ß=-2.53% [95%CI:-4.18,-0.89], P = 0.003). CONCLUSIONS: Early biomarker levels modestly improve predictive models containing clinical and MRI variables. Worse clinical outcomes, SPMS and EDSS, are associated with higher sGFAP levels and worse MRI outcomes, T2LV and BPF, are associated with higher sNfL levels. Prospective study implementing these predictive models into clinical practice are needed to determine if early biomarker levels meaningfully impact clinical practice.

Caspase-9-mediated cleavage of vimentin attenuates the aggressiveness of leukemic NB4 cells.

Vimentin is a main type 3 intermediate filament protein. It seems that abnormal expression of vimentin is contributed to the appearance of the aggressive feature of cancer cells. So that it has been reported that malignancy and epithelial-mesenchymal transition in solid tumors, and poor clinical outcomes in patients with lymphocytic leukemia and acute myelocytic leukemia have been associated with the high expression of vimentin. Vimentin is a non-caspase substrate of caspase-9 although its cleavage by caspase-9 in biological processes has not been reported. In the present study, we sought to understand whether vimentin cleavage mediated by caspase-9 could reverse the malignancy in leukemic cells. Herein, to address the issue, we investigated vimentin changes in differentiation and took advantage of the inducible caspase-9 (iC9)/AP1903 system in human leukemic NB4 cells. Following the transfection and treatment of the cells using the iC9/AP1903 system, vimentin expression, cleavage, and subsequently, the cell invasion and the relevant markers such as CD44 and MMP-9 were evaluated. Our results revealed the downregulation and cleavage of vimentin which attenuates the malignant phenotype of the NB4 cells. Considering the favorable effect of this strategy in keeping down the malignant features of the leukemic cells, the effect of the iC9/AP1903 system in combination with all-trans-retinoic acid (ATRA) treatment was evaluated. The obtained data prove that iC9/AP1903 significantly makes the leukemic cells more sensitive to ATRA.

Brain-derived neurotrophic factor, neurofilament light and glial fibrillary acidic protein do not change in response to aerobic training in people with MS-related fatigue - a secondary analysis of a randomized controlled trial.

BACKGROUND: Neuroinflammation and neurodegeneration are pathological hallmarks of multiple sclerosis (MS). Brain-derived neurotrophic factor (BDNF), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP) are blood-based biomarkers for neurogenesis, axonal damage and astrocyte reactivity, respectively. We hypothesize that exercise has a neuroprotective effect on MS reflected by normalization of BDNF, NfL and GFAP levels. OBJECTIVES: To investigate the neuroprotective effect of aerobic training (AT) compared to a control intervention on blood-based biomarkers (i.e. BDNF, NfL, GFAP) in people with MS (pwMS). METHODS: In the TREFAMS-AT (Treating Fatigue in Multiple Sclerosis - Aerobic Training) study, 89 pwMS were randomly allocated to either a 16-week AT intervention or a control intervention (3 visits to a MS nurse). In this secondary analysis, blood-based biomarker concentrations were measured in 55 patients using Simoa technology. Changes in pre- and post-intervention concentrations were compared and between-group differences were assessed using analysis of covariance (ANCOVA). Confounding effects of age, sex, MS-related disability assessed using the Expanded Disability Status Scale (EDSS), MS duration, use of disease-modifying medication, and Body Mass Index were considered. RESULTS: Blood samples were available for 30 AT and 25 control group participants (mean age 45.6 years, 71% female, median disease duration 8 years, median EDSS score 2.5). Within-group changes in both study groups were small and non-significant, with the exception of BDNF in the control group (median (interquartile range) -2.1 (-4.7; 0)). No between-group differences were found for any biomarker: BDNF (ß = 0.11, 95%CI (-3.78 to 4.00)), NfL (ß = -0.04, 95%CI (-0.26 to 0.18)), and GFAP (ß = -0.01, 95%CI (-0.16 to 0.15)), adjusted for confounders. CONCLUSION: Aerobic exercise therapy did not result in statistically significant changes in the tested neuro-specific blood-based biomarkers in people with MS. TRIAL REGISTRATION: this study is registered under number ISRCTN69520623 (https://www.isrctn.com/ISRCTN695206).

Neurofilament light chain is increased in the parahippocampal cortex and associates with pathological hallmarks in Parkinson's disease dementia.

BACKGROUND: Increased neurofilament levels in biofluids are commonly used as a proxy for neurodegeneration in several neurodegenerative disorders. In this study, we aimed to investigate the distribution of neurofilaments in the cerebral cortex of Parkinson's disease (PD), PD with dementia (PDD) and dementia with Lewy bodies (DLB) donors, and its association with pathology load and MRI measures of atrophy and diffusivity. METHODS: Using a within-subject post-mortem MRI-pathology approach, we included 9 PD, 12 PDD/DLB and 18 age-matched control donors. Cortical thickness and mean diffusivity (MD) metrics were extracted respectively from 3DT1 and DTI at 3T in-situ MRI. After autopsy, pathological hallmarks (pSer129-αSyn, p-tau and amyloid-ß load) together with neurofilament light-chain (NfL) and phosphorylated-neurofilament medium- and heavy-chain (p-NfM/H) immunoreactivity were quantified in seven cortical regions, and studied in detail with confocal-laser scanning microscopy. The correlations between MRI and pathological measures were studied using linear mixed models. RESULTS: Compared to controls, p-NfM/H immunoreactivity was increased in all cortical regions in PD and PDD/DLB, whereas NfL immunoreactivity was increased in the parahippocampal and entorhinal cortex in PDD/DLB. NfL-positive neurons showed degenerative morphological features and axonal fragmentation. The increased p-NfM/H correlated with p-tau load, and NfL correlated with pSer129-αSyn but more strongly with p-tau load in PDD/DLB. Lastly, neurofilament immunoreactivity correlated with cortical thinning in PD and with increased cortical MD in PDD/DLB. CONCLUSIONS: Taken together, increased neurofilament immunoreactivity suggests underlying axonal injury and neurofilament accumulation in morphologically altered neurons with increased pathological burden. Importantly, we demonstrate that such neurofilament markers at least partly explain MRI measures that are associated with the neurodegenerative process.

Vimentin affects inflammation and neutrophil recruitment in airway epithelium during Streptococcus suis serotype 2 infection.

Streptococcus suis serotype 2 (SS2) frequently colonizes the swine upper respiratory tract and can cause Streptococcal disease in swine with clinical manifestations of pneumonia, meningitis, and septicemia. Previously, we have shown that vimentin, a kind of intermediate filament protein, is involved in the penetration of SS2 through the tracheal epithelial barrier. The initiation of invasive disease is closely related to SS2-induced excessive local inflammation; however, the role of vimentin in airway epithelial inflammation remains unclear. Here, we show that vimentin deficient mice exhibit attenuated lung injury, diminished production of proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and the IL-8 homolog, keratinocyte-derived chemokine (KC), and substantially reduced neutrophils in the lungs following intranasal infection with SS2. We also found that swine tracheal epithelial cells (STEC) without vimentin show decreased transcription of IL-6, TNF-α, and IL-8. SS2 infection caused reassembly of vimentin in STEC, and pharmacological disruption of vimentin filaments prevented the transcription of those proinflammatory cytokines. Furthermore, deficiency of vimentin failed to increase the transcription of nucleotide oligomerization domain protein 2 (NOD2), which is known to interact with vimentin, and the phosphorylation of NF-κB protein p65. This study provides insights into how vimentin promotes excessive airway inflammation, thereby exacerbating airway injury and SS2-induced systemic infection.