Neurofilament Triplet Proteins Research Articles

Novel Approach for Detecting the Neurological or Behavioral Impact of Physiological Episodes (PEs) in Military Aircraft Crews.

Military and civil aviation have documented physiological episodes among aircrews. Therefore, continued efforts are being made to improve the internal environment. Studies have shown that exposures to many organic compounds present in emissions are known to cause a variety of physiological symptoms. We hypothesize that these compounds may reversibly inhibit acetylcholinesterase, which may disrupt synaptic signaling. As a result, neural proteins leak through the damaged blood-brain barrier into the blood and in some, elicit an autoimmune response. Neural-specific autoantibodies of immunoglobulin-G (IgG) class were estimated by the Western blotting technique in the sera of 26 aircrew members and compared with the sera of 19 normal healthy nonaircrew members, used as controls. We found significantly elevated levels of circulating IgG-class autoantibodies to neurofilament triplet proteins, tubulin, microtubule-associated tau proteins (Tau), microtubule-associated protein-2, myelin basic protein, and glial fibrillary acidic protein, but not S100 calcium-binding protein B compared to healthy controls. Repetitive physiological episodes may initiate cellular injury, leading to neuronal degeneration in selected individuals. Diagnosis and intervention should occur at early postinjury periods. Use of blood-based biomarkers to assess subclinical brain injury would help in both diagnosis and treatment.

De novo Blood Biomarkers in Autism: Autoantibodies against Neuronal and Glial Proteins.

Autism spectrum disorders (ASDs) are the most common neurodevelopmental disorders with unidentified etiology. The behavioral manifestations of ASD may be a consequence of genetic and/or environmental pathology in neurodevelopmental processes. In this limited study, we assayed autoantibodies to a panel of vital neuronal and glial proteins in the sera of 40 subjects (10 children with ASD and their mothers along with 10 healthy controls, age-matched children and their mothers). Serum samples were screened using Western Blot analysis to measure immunoglobulin (IgG) reactivity against a panel of 9 neuronal proteins commonly associated with neuronal degeneration: neurofilament triplet proteins (NFP), tubulin, microtubule-associated proteins (tau), microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), α-synuclein (SNCA) and astrocytes proteins such as glial fibrillary acidic protein (GFAP) and S100B protein. Our data show that the levels of circulating IgG class autoantibodies against the nine proteins were significantly elevated in ASD children. Mothers of ASD children exhibited increased levels of autoantibodies against all panel of tested proteins except for S100B and tubulin compared to age-matched healthy control children and their mothers. Control children and their mothers showed low and insignificant levels of autoantibodies to neuronal and glial proteins. These results strongly support the importance of anti-neuronal and glial protein autoantibodies biomarker in screening for ASD children and further confirm the importance of the involvement of the maternal immune system as an index that should be considered in fetal in utero environmental exposures. More studies are needed using larger cohort to verify these results and understand the importance of the presence of such autoantibodies in children with autism and their mothers, both as biomarkers and their role in the mechanism of action of autism and perhaps in its treatment.

A Systematic Review of the Use of Biochemical Markers in the Assessment of Spinal Cord Ischemia in Thoracoabdominal Aortic Aneurysm Repair.

Despite advances in perioperative critical care and surgical technique, spinal cord ischemia remains a devastating complication of thoracic and thoracoabdominal aortic aneurysm repair. Biochemical markers present in peripheral blood and cerebrospinal fluid (CSF) may be useful in assessing spinal cord injury. We systematically analyze and report the role of all reported biochemical markers that have been used in assessing and diagnosing spinal cord ischemia in thoracic and thoracoabdominal aortic aneurysm repair. Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines were used for this review. Published literature was searched to identify all studies reporting on the use of biochemical markers in thoracoabdominal aortic aneurysm repair in the assessment of spinal cord ischemia. Marker-specific and patient-specific data were extracted from all studies and where possible, subgroup analysis was performed on marker-specific data sets. Fourteen studies of 321 patients undergoing thoracic and thoracoabdominal aortic aneurysm repair were eligible for further analysis. Seven distinct biochemical markers were used in both CSF and blood samples: S100B proteins (S100B), neurone-specific enolase, lactate dehydrogenase, glial fibrillary acidic protein (GFAp), neurofilament triplet protein (NFL) and Tau protein, and glucose. There was substantial evidence demonstrating the heightened levels of S100, NFL, and GFAp in CSF in patients with spinal cord ischemia. There is however, wide variability in the correlation of the same 6 biochemical markers in peripheral blood and spinal cord ischemia. In patients with spinal cord injury, dramatic rises occur with S100B, NFL, and GFAp in CSF. However, further work is needed if biochemical markers are to impact on the future of thoracoabdominal aortic aneurysm repair.

A Panel of Autoantibodies Against Neural Proteins as Peripheral Biomarker for Pesticide-Induced Neurotoxicity.

In the present study, we screened the sera of subjects chronically exposed to mixtures of pesticides (composed mainly of organophosphorus compounds (OPs) and others) and developed neurological symptoms for the presence of autoantibodies against cytoskeletal neural proteins. OPs have a well-characterized clinical profile resulting from acute cholinergic crisis. However, some of these compounds cause neuronal degeneration and demyelination known as organophosphorus compound-induced delayed neurotoxicity (OPIDN) and/or organophosphorus compound-induced chronic neurotoxicity (OPICN). Studies from our group have demonstrated the presence of autoantibodies to essential neuronal and glial proteins against cytoskeletal neural proteins in patients with chemical-induced brain injury. In this study, we screened the serum of 50 pesticide-exposed subjects and 25 non-exposed controls, using Western blot analysis against the following proteins: neurofilament triplet proteins (NFPs), tubulin, microtubule-associated tau proteins (Tau), microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), glial fibrillary acidic protein (GFAP), calcium-calmodulin kinase II (CaMKII), glial S100-B protein, and alpha-synuclein (SNCA). Serum reactivity was measured as arbitrary chemiluminescence units. As a group, exposed subjects had significantly higher levels of autoantibody reactivity in all cases examined. The folds of increase in of autoantibodies against neural proteins of the subjects compared to healthy humans in descending order were as follows: MBP, 7.67, MAG 5.89, CaMKII 5.50, GFAP 5.1, TAU 4.96, MAP2 4.83, SNCA 4.55, NFP 4.55, S-100B 2.43, and tubulin 1.78. This study has demonstrated the presence of serum autoantibodies to central nervous system-specific proteins in a group of farmers chronically exposed to pesticides who developed neurological signs and symptoms of neural injury. These autoantibodies can be used as future diagnostic/therapeutic target for OP-induced neurotoxicity.

The third wave: Intermediate filaments in the maturing nervous system.

Intermediate filaments are critical for the extreme structural specialisations of neurons, providing integrity in dynamic environments and efficient communication along axons a metre or more in length. As neurons mature, an initial expression of nestin and vimentin gives way to the neurofilament triplet proteins and α-internexin, substituted by peripherin in axons outside the CNS, which physically consolidate axons as they elongate and find their targets. Once connection is established, these proteins are transported, assembled, stabilised and modified, structurally transforming axons and dendrites as they acquire their full function. The interaction between these neurons and myelinating glial cells optimises the structure of axons for peak functional efficiency, a property retained across their lifespan. This finely calibrated structural regulation allows the nervous system to maintain timing precision and efficient control across large distances throughout somatic growth and, in maturity, as a plasticity mechanism allowing functional adaptation.

Screening for novel central nervous system biomarkers in veterans with Gulf War Illness

Gulf War illness (GWI) is primarily diagnosed by symptom report; objective biomarkers are needed that distinguish those with GWI. Prior chemical exposures during deployment have been associated in epidemiologic studies with altered central nervous system functioning in veterans with GWI. Previous studies from our group have demonstrated the presence of autoantibodies to essential neuronal and glial proteins in patients with brain injury and autoantibodies have been identified as candidate objective markers that may distinguish GWI. Here, we screened the serum of 20 veterans with GWI and 10 non-veteran symptomatic (low back pain) controls for the presence of such autoantibodies using Western blot analysis against the following proteins: neurofilament triplet proteins (NFP), tubulin, microtubule associated tau proteins (Tau), microtubule associated protein-2 (MAP-2), myelin basic protein (MBP), myelin associated glycoprotein (MAG), glial fibrillary acidic protein (GFAP), calcium-calmodulin kinase II (CaMKII) and glial S-100B protein. Serum reactivity was measured as arbitrary chemiluminescence units. As a group, veterans with GWI had statistically significantly higher levels of autoantibody reactivity in all proteins examined except S-100B. Fold increase of the cases relative to controls in descending order were: CaMKII 9.27, GFAP 6.60, Tau 4.83, Tubulin 4.41, MAG 3.60, MBP 2.50, NFP 2.45, MAP-2 2.30, S-100B 1.03. These results confirm the continuing presence of neuronal injury/gliosis in these veterans and are in agreement with the recent reports indicating that 25years after the war, the health of veterans with GWI is not improving and may be getting worse. Such serum autoantibodies may prove useful as biomarkers of GWI, upon validation of the findings using larger cohorts.

Developmental pattern of the neuronal intermediate filament inaa in the zebrafish retina.

α-Internexin is a member of the neuronal intermediate filament (nIF) protein family, which also includes peripherin and neurofilament (NF) triplet proteins. Previous studies found that expression of α-internexin precedes that of the NF triplet proteins in mammals and suggested that α-internexin plays a key role in the neuronal cytoskeleton network during development. In this study, we aimed to analyze the expression patterns and function of internexin neuronal intermediate filament protein-alpha a (inaa), the encoding gene of which is a homolog of the mammalian α-internexin, during retinal development in zebrafish. Via in vitro and in vivo studies, we demonstrated that zebrafish inaa is an α-internexin homolog that shares characteristics with nIFs. An immunohistochemical analysis of zebrafish revealed that inaa was distributed dynamically in the developing retina. It was widely localized in retinal neuroepithelial cells at 1 day postfertilization (dpf), and was mainly found in the ganglion cell layer (GCL) and inner part of the inner nuclear layer (INL) from 3-9 dpf; after 14 dpf, it was restricted to the outer nuclear layer (ONL). Moreover, we demonstrated for the first time that inaa acted distinctively from the cytoskeletal scaffold of zebrafish cone photoreceptors during development. In conclusion, we demonstrated the morphological features of a novel nIF, inaa, and illustrated its developmental expression pattern in the zebrafish retina. J. Comp. Neurol. 524:3810-3826, 2016. © 2016 Wiley Periodicals, Inc.

α-Internexin and Peripherin: Expression, Assembly, Functions, and Roles in Disease.

α-Internexin and peripherin are neuronal-specific intermediate filament (IF) proteins. α-Internexin is a type IV IF protein like the neurofilament triplet proteins (NFTPs, which include neurofilament light chain, neurofilament medium chain, and neurofilament high chain) that are generally considered to be the primary components of the neuronal IFs. However, α-internexin is often expressed together with the NFTPs and has been proposed as the fourth subunit of the neurofilaments in the central nervous system. α-Internexin is also expressed earlier in the development than the NFTPs and is a maker for neuronal IF inclusion disease. α-Internexin can self-polymerize in vitro and in transfected cells and it is present in the absence of the NFTP in development and in granule cells in the cerebellum. In contrast, peripherin is a type III IF protein. Like α-internexin, peripherin is specific to the nervous system, but it is expressed predominantly in the peripheral nervous system (PNS). Peripherin can also self-assemble both in vitro and in transfected cells. It is as abundant as the NFTPs in the sciatic nerve and can be considered a fourth subunit of the neurofilaments in the PNS. Peripherin has multiple isoforms that arise from intron retention, cryptic intron receptor site or alternative translation initiation. The functional significance of these isoforms is not clear. Peripherin is a major component found in inclusions of patients with amyotrophic lateral sclerosis (ALS) and peripherin expression is upregulated in ALS patients.

Tumors displaying hybrid schwannoma and neurofibroma features in patients with neurofibromatosis type 2.

Although schwannoma and neurofibroma tumors are generally reported as distinct pathologic diagnoses, sporadic schwannoma/neurofibroma hybrid nerve sheath tumors have been reported in the general population with components of both entities. We report the clinicopathological features of these hybrid nerve sheath tumors in patients with neurofibromatosis type 2 (NF2). A retrospective review of nerve sheath tumor surgical specimens from patients with NF2 enrolled at the National Institutes of Health was performed. Those specimens reported to have schwannoma-like and neurofibroma-like features were selected for further characterization by morphology, immunohistochemical panel (CD34, S100, neurofilament triplet protein (immunostain) (NFTP), epithelial membrane antigen (EMA)), and confirmation as hybrid tumors. Of 43 total NF2 patients undergoing resection of nerve sheath tumors, 11 specimens from 11 (26%) patients were found to be benign nerve sheath tumors exhibiting hybrid features of both neurofibroma and schwannoma. Immunohistochemical studies showed the schwannoma component to be S100+, CD 34- while the neurofibroma component was CD34+, variable S100+. Our experience emphasizes the importance of including this distinct tumor subtype, the schwannoma/neurofibroma hybrid tumor, in the differential diagnosis of nerve sheath tumors in NF2 patients and suggests that the relationship between neurofibroma and schwannoma tumors is closer than previously suspected.

Neurofilament light gene deletion exacerbates amyloid, dystrophic neurite, and synaptic pathology in the APP/PS1 transgenic model of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with the loss of cognitive function. Neurofilament (NF) triplet proteins, the major structural (intermediate filament) proteins of neurons, are expressed in a subset of pyramidal cells that show a high degree of vulnerability to degeneration in AD. Alterations in the NF triplet proteins in amyloid-beta (Aβ) plaque-associated dystrophic neurites (DNs) represent the first cytoskeletal aberration to occur in the neocortex in the earliest stages of AD. We generated transgenic APP/PS1 (APPswe/PSEN1dE9) mice on the neurofilament light knockout (NFL KO) background to explore the role of NFL deletion in the context of DN formation, synaptic changes, and other neuropathologic features. Our analysis demonstrated that NFL deficiency significantly increased neocortical DN pathology, Aβ deposition, synapse vulnerability, and microgliosis in APP/PS1 mice. Thus, NFs may have a role in protecting neurites from dystrophy and in regulating cellular pathways related to the generation of Aβ plaques.

Diffuse axonal injury in brain trauma: insights from alterations in neurofilaments.

Traumatic brain injury (TBI) from penetrating or closed forces to the cranium can result in a range of forms of neural damage, which culminate in mortality or impart mild to significant neurological disability. In this regard, diffuse axonal injury (DAI) is a major neuronal pathophenotype of TBI and is associated with a complex set of cytoskeletal changes. The neurofilament triplet proteins are key structural cytoskeletal elements, which may also be important contributors to the tensile strength of axons. This has significant implications with respect to how axons may respond to TBI. It is not known, however, whether neurofilament compaction and the cytoskeletal changes that evolve following axonal injury represent a component of a protective mechanism following damage, or whether they serve to augment degeneration and progression to secondary axotomy. Here we review the structure and role of neurofilament proteins in normal neuronal function. We also discuss the processes that characterize DAI and the resultant alterations in neurofilaments, highlighting potential clues to a possible protective or degenerative influence of specific neurofilament alterations within injured neurons. The potential utility of neurofilament assays as biomarkers for axonal injury is also discussed. Insights into the complex alterations in neurofilaments will contribute to future efforts in developing therapeutic strategies to prevent, ameliorate or reverse neuronal degeneration in the central nervous system (CNS) following traumatic injury.

Autoantibodies against cytoskeletal neuronal proteins in sera of arsenic-exposed subjects correlate with neurological symptoms

This descriptive study correlates the presence of serum autoantibodies to five neuronal cytoskeletal proteins with neurologic deficits in subjects exposed to arsenic (As). Arsenic exposure remains a serious problem in many countries producing skin lesions and hair loss. More serious manifestations include: liver and kidney damage, encephalopathy, peripheral neuropathy, gangrene of extremities, and cancer. Bangladesh represents one of the countries where high percentage of population is exposed to As through highly contaminated drinking groundwater. Autoantibodies were determined against the following proteins: neurofilament triplet proteins (NFP), microtubule associated protein-2 (MAP2), and microtubule associated protein tau (tau). Sera were obtained from 14 subjects in the severely affected village of Mianpur, Charghat Thana, Rajshahi. Individuals were further divided into group A with objective complaints of organic neurologic damage and group B without neurologic deficits manifestations and eight healthy controls from Rajshahi city. High levels of autoantibodies were detected in five subjects against low molecular weight neurofilaments (NFL) and in two subjects against high molecular weight neurofilament (NFH). Two subjects displayed significantly increased levels of MAP-2 autoantibodies and two others high levels of tau autoantibodies. All elevated autoantibodies were detected in group A subjects who were diagnosed with neurological disorders ranging from sleep disorders to paralysis. The results demonstrate a good correlation between neurologic defects in As-exposed subjects and presence of sera neuronal autoantibodies to cytoskeletal proteins.

Molecular cloning and characterization of chicken neuronal intermediate filament protein α‐internexin

α-Internexin is one of the neuronal intermediate filament (IF) proteins, which also include low-, middle-, and high-molecular-weight neurofilament (NF) triplet proteins, designated NFL, NFM, and NFH, respectively. The expression of α-internexin occurs in most neurons as they begin differentiation and precedes the expression of the NF triplet proteins in mammals. However, little is known about the gene sequence and physiological function of α-internexin in avians. In this study we describe the molecular cloning of the mRNA sequence encoding the chicken α-internexin (chkINA) protein from embryonic brains. The gene structure and predicted amino acid sequence of chkINA exhibited high similarity to those of its zebrafish, mouse, rat, bovine, and human homologs. Data from transient-transfection experiments show that the filamentous pattern of chkINA was found in transfected cells and colocalized with other endogenous IFs, as demonstrated via immunocytochemistry using a chicken-specific antibody. The expression of chkINA was detected at the early stage of development and increased during the developmental process of the chicken. chkINA was expressed widely in chicken brains and colocalized with NF triplet proteins in neuronal processes, as assessed using immunohistochemistry. We also found that chkINA was expressed abundantly in the developing cerebellum and was the major IF protein in the parallel processes of granule neurons. Thus, we suggest that chkINA is a neuron-specific IF protein that may be a useful marker for studies of chicken brain development.

Cytoskeletal changes during development and aging in the cortex of neurofilament light protein knockout mice

The neurofilament light (NFL) subunit is considered as an obligate subunit polymer for neuronal intermediate filaments comprising the neurofilament (NF) triplet proteins. We examined cytoskeletal protein levels in the cerebral cortex of NFL knockout (KO) mice at postnatal day 4 (P4), 5 months, and 12 months of age compared with age-matched wild-type (WT) mice of a similar genetic background (C57BL/6). The absence of NFL protein resulted in a significant reduction of phosphorylated and dephosphorylated NFs (NF-P, NF-DP), the medium NF subunit (NFM), and the intermediate filament α-internexin (INT) at P4. At 5 months, NF-DP, NFM, and INT remained significantly lower in knockouts. At 12 months, NF-P was again significantly decreased, and INT significantly increased, in KOs compared with wild type. In addition, protein levels of class III neuron-specific β-tubulin and microtubule-associated protein 2 were significantly increased in NFL KO mice at P4, 5 months, and 12 months, whereas β-actin levels were significantly decreased at P4. Immunocytochemical studies demonstrated that NF-DP accumulated abnormally in the perikarya of cortical neurons by 5 months of age in NFL KO mice. Neurons that lacked NF triplet proteins, such as calretinin-immunolabeled nonpyramidal cells, showed no alterations in density or cytoarchitectural distribution in NFL KO mice at 5 months relative to WT mice, although calretinin protein levels were decreased significantly after 12 months in NFL KO mice. These findings suggest that a lack of NFL protein alters the expression of cytoskeletal proteins and disrupts other NF subunits, causing intracellular aggregation but not gross structural changes in cortical neurons or cytoarchitecture. The data also indicate that changes in expression of other cytoskeletal proteins may compensate for decreased NFs.

Neuronal intermediate filament α-internexin is expressed by neuronal lineages in the developing chicken retina

α-Internexin, a neuronal intermediate filament (IF) protein that is expressed in most neurons at the beginning of differentiation, has been studied in the developing vertebrate retina. It has been suggested that α-internexin plays a role in neuroplasticity during development. However, the expression of α-internexin in the chicken retina has not been investigated. In this study, we identified the expression patterns of the chicken α-internexin (chkINA) and neurofilament (NF) proteins in the developing chicken retina. These proteins exhibited dynamic patterns of expression with the progression of retinal development. Expression of chkINA was detected in the processes of ganglion, amacrine, and horizontal cells throughout development. chkINA was also transiently expressed in photoreceptor and bipolar cell lineages. NF triplet proteins exhibited more restricted expression patterns than did chkINA during development. Our observations suggest the coexpression of chkINA and NF triplet proteins in ganglion and amacrine cells in the developing retina, as well as in horizontal cell processes in the adult chicken retina. Our results indicate that chkINA is transiently expressed in all neuronal lineages in the developing chicken retina. These findings provide novel information on chicken retinal cell development and suggest that chkINA is a good neuronal marker for the study of retinal differentiation.

Autoantibodies to Nervous System-Specific Proteins Are Elevated in Sera of Flight Crew Members: Biomarkers for Nervous System Injury

This descriptive study reports the results of assays performed to detect circulating autoantibodies in a panel of 7 proteins associated with the nervous system (NS) in sera of 12 healthy controls and a group of 34 flight crew members including both pilots and attendants who experienced adverse effects after exposure to air emissions sourced to the ventilation system in their aircrafts and subsequently sought medical attention. The proteins selected represent various types of proteins present in nerve cells that are affected by neuronal degeneration. In the sera samples from flight crew members and healthy controls, immunoglobin (IgG) was measured using Western blotting against neurofilament triplet proteins (NFP), tubulin, microtubule-associated tau proteins (tau), microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and glial S100B protein. Significant elevation in levels of circulating IgG-class autoantibodies in flight crew members was found. A symptom-free pilot was sampled before symptoms and then again afterward. This pilot developed clinical problems after flying for 45 h in 10 d. Significant increases in autoantibodies were noted to most of the tested proteins in the serum of this pilot after exposure to air emissions. The levels of autoantibodies rose with worsening of his condition compared to the serum sample collected prior to exposure. After cessation of flying for a year, this pilot's clinical condition improved, and eventually he recovered and his serum autoantibodies against nervous system proteins decreased. The case study with this pilot demonstrates a temporal relationship between exposure to air emissions, clinical condition, and level of serum autoantibodies to nervous system-specific proteins. Overall, these results suggest the possible development of neuronal injury and gliosis in flight crew members anecdotally exposed to cabin air emissions containing organophosphates. Thus, increased circulating serum autoantibodies resulting from neuronal damage may be used as biomarkers for chemical-induced CNS injury. The authors thank all of the participants who volunteered to take part in this case study. The technical work of Dr. Hagir B. Suliman and the art work of Sheref M. Abou-Donia are appreciated. This study was supported in part by the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.

Neurites containing the neurofilament-triplet proteins are selectively vulnerable to cytoskeletal pathology in Alzheimer’s disease and transgenic mouse models

Amyloid-β plaque accumulation in Alzheimer’s disease (AD) is associated with dystrophic neurite (DN) formation and synapse loss in principal neurons, but interneuron pathology is less clearly characterized. We compared the responses of neuronal processes immunoreactive for either neurofilament triplet (NF+) or calretinin (CR+) to fibrillar amyloid (Aβ) plaques in human end-stage and preclinical AD, as well as in APP/PS1 and Tg2576 transgenic mouse AD models. Neurites traversing the Aβ plaque core, edge, or periphery, defined as 50, 100, and 150% of the plaque diameter, respectively, in human AD and transgenic mouse tissue were compared to age-matched human and wild-type mouse controls. The proportion of NF+ neurites exhibiting dystrophic morphology (DN) was significantly larger than the proportion of dystrophic CR+ neurites in both human AD and transgenic mice (p < 0.01). Additionally, the number of NF+, but not CR+, DNs, correlated with Aβ plaque size. We conclude that CR+ interneurons appear to be more resistant than NF+ neurons to AD-mediated cytoskeletal pathology.

Nervous system autoantibodies and vitamin D receptor gene polymorphisms in hemodialysis patients

Cognitive deficits are prevalent in hemodialysis (HD) patients. Vitamin D deficiency and vitamin D receptor (VDR) gene single nucleotide polymorphism (SNPs) have been linked to both neurodegeneration (ND) and neuroprotection, respectively. Autoantibodies (Ab) to myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and neurofilament (NF) triplet proteins arise secondary to nervous system (NS) damage providing a means to assess neurological injury. Characterization of Ab biomarkers of NS damage in HD patients, their association with VDR SNPs, and nutritional vitamin D (NVD) therapy was performed. VDR genotypes, cytokines, and Ab biomarkers to NS proteins in HD subjects receiving ergocalciferol (n = 40) were compared with nonusers (n = 71). Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and immunoglobulin G (IgG) titers against NFs, GFAP, and MBP were measured by immunoassay. Subjects were genotyped for VDR SNPs BsmI (rs1544410) and FokI (rs2228570). Subjects (age 63.3 ± 16.1 years, 66% male) who were C allele carriers of BsmI had higher values of NF-68 antibody titers (p = 0.027). Ergocalciferol users (n = 40) compared with nonusers (n = 71) had lower Ab titers to NS proteins; however, only anti-NF-160 and anti-MBP titers were significantly (p < 0.05) higher. IgG against NS proteins in HD patients suggests neuronal and glial insult and a relationship with VDR alleles. NVD may provide some neuroprotection, indicated by anti-NF-160 and anti-MBP, which was markedly lowered in ergocalciferol patients. This preliminary study suggests that Ab detection may be useful in monitoring ND and the potential of NVD for neuroprotection in HD patients.

Peripherin Is a Subunit of Peripheral Nerve Neurofilaments: Implications for Differential Vulnerability of CNS and Peripheral Nervous System Axons

Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament triplet proteins [neurofilament light (NFL), medium (NFM), and heavy (NFH) chain] but has an unknown function. The earlier peak expression of peripherin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that peripherin and neurofilament triplets form separate filament systems. However, here, we demonstrate that, despite a postnatal decline in expression, peripherin is as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry with these subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins in sciatic axons and colocalizes with NFL on single neurofilaments by immunogold electron microscopy. Peripherin also coassembles into a single network of filaments containing NFL, NFM, and NFH with and without α-internexin in quadruple- or quintuple-transfected SW13vim(-) cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons. Moreover, peripherin mutations has been shown to disrupt the neurofilament network in transfected SW13vim(-) cells. These data show that peripherin and the neurofilament proteins are functionally interdependent. The results strongly support the view that, rather than forming an independent structure, peripherin is a subunit of neurofilaments in the adult PNS. Our findings provide a basis for its close relationship with neurofilaments in PNS diseases associated with neurofilament accumulation.

Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior

Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.