Uman-type neurofilament light antibodies are effective reagents for the imaging of neurodegeneration.

Recent work shows that certain antibody-based assays for the neurofilament light chain detect informative signals in the CSF and blood of human and animals affected by a variety of CNS injury and disease states. Much of this work has been performed using two mouse monoclonal antibodies to neurofilament light, UD1 and UD2, also known as Clones 2.1 and 47.3, respectively. These are the essential components of the Uman Diagnostics Neurofilament-Light™ ELISA kit, the Quanterix Simoa™ bead-based assay and others. We show that both antibodies bind to neighbouring epitopes in a short, conserved and unusual peptide in the centre of the neurofilament light Coil 2 segment of the 'rod' domain. We also describe a surprising and useful feature of Uman and similar reagents. While other well-characterized neurofilament antibodies generally show robust staining of countless cells and processes in CNS sections from healthy rats, both Uman antibodies reveal only a minor subset of profiles, presumably spontaneously degenerating or degenerated neurons and their processes. However, following experimental mid-cervical spinal cord injuries to rats, both Uman antibodies recognize numerous profiles in fibre tracts damaged by the injury administered. These profiles were typically swollen, beaded, discontinuous or sinusoidal as expected for degenerating and degenerated processes. We also found that several antibodies to the C-terminal 'tail' region of the neurofilament light protein bind undamaged axonal profiles but fail to recognize the Uman-positive material. The unmasking of the Uman epitopes and the loss of the neurofilament light tail epitopes can be mimicked by treating sections from healthy animals with proteases suggesting that the immunostaining changes we discovered are due to neurodegeneration-induced proteolysis. We have also generated a novel panel of monoclonal and polyclonal antibodies directed against the Uman epitopes that have degeneration-specific staining properties identical to the Uman reagents. Using these, we show that the region to which the Uman reagents bind contains further hidden epitopes distinct from those recognized by the two Uman reagents. We speculate that the Uman-type epitopes are part of a binding region important for higher order neurofilament assembly. The work provides important insights into the properties of the Uman assay, describes novel and useful properties of Uman-type and neurofilament light tail-binding antibodies and provides a hypothesis relevant to further understanding of neurofilament assembly.

DAT and TH expression marks human Parkinson's disease in peripheral immune cells.

Parkinson's disease (PD) is marked by a loss of dopamine neurons, decreased dopamine transporter (DAT) and tyrosine hydroxylase (TH) expression. However, this validation approach cannot be used for diagnostic, drug effectiveness or investigational purposes in human patients because midbrain tissue is accessible postmortem. PD pathology affects both the central nervous and peripheral immune systems. Therefore, we immunophenotyped blood samples of PD patients for the presence of myeloid derived suppressor cells (MDSCs) and discovered that DAT+/TH+ monocytic MDSCs, but not granulocytic MDSCs are increased, suggesting a targeted immune response to PD. Because in peripheral immune cells DAT activity underlies an immune suppressive mechanism, we investigated whether expression levels of DAT and TH in the peripheral immune cells marks PD. We found drug naïve PD patients exhibit differential DAT+/TH+ expression in peripheral blood mononuclear cells (PBMCs) compared to aged/sex matched healthy subjects. While total PBMCs are not different between the groups, the percentage of DAT+/TH+ PBMCs was significantly higher in drug naïve PD patients compared to healthy controls irrespective of age, gender, disease duration, disease severity or treatment type. Importantly, treatment for PD negatively modulates DAT+/TH+ expressing PBMCs. Neither total nor the percentage of DAT+/TH+ PBMCs were altered in the Alzheimer's disease cohort. The mechanistic underpinning of this discovery in human PD was revealed when these findings were recapitulated in animal models of PD. The reverse translational experimental strategy revealed that alterations in dopaminergic markers in peripheral immune cells are due to the disease associated changes in the CNS. Our study demonstrates that the dopaminergic machinery on peripheral immune cells displays an association with human PD, with exciting implications in facilitating diagnosis and investigation of human PD pathophysiology.

TNFα increases tyrosine hydroxylase expression in human monocytes.

Most, if not all, peripheral immune cells in humans and animals express tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Since TH is typically studied in the context of brain catecholamine signaling, little is known about changes in TH production and function in peripheral immune cells. This knowledge gap is due, in part, to the lack of an adequately sensitive assay to measure TH in immune cells expressing lower TH levels compared to other TH expressing cells. Here, we report the development of a highly sensitive and reproducible Bio-ELISA to quantify picogram levels of TH in multiple model systems. We have applied this assay to monocytes isolated from blood of persons with Parkinson's disease (PD) and to age-matched, healthy controls. Our study unexpectedly revealed that PD patients' monocytes express significantly higher levels of TH protein in peripheral monocytes relative to healthy controls. Tumor necrosis factor (TNFα), a pro-inflammatory cytokine, has also been shown to be increased in the brains and peripheral circulation in human PD, as well as in animal models of PD. Therefore, we investigated a possible connection between higher levels of TH protein and the known increase in circulating TNFα in PD. Monocytes isolated from healthy donors were treated with TNFα or with TNFα in the presence of an inhibitor. Tissue plasminogen activator (TPA) was used as a positive control. We observed that TNFα stimulation increased both the number of TH+ monocytes and the quantity of TH per monocyte, without increasing the total numbers of monocytes. These results revealed that TNFα could potentially modify monocytic TH production and serve a regulatory role in peripheral immune function. The development and application of a highly sensitive assay to quantify TH in both human and animal cells will provide a novel tool for further investigating possible PD immune regulatory pathways between brain and periphery.

Cerebrospinal fluid levels of GFAP and pNF-H are elevated in patients with chronic spinal cord injury and neurological deterioration.

BACKGROUND: Years after a traumatic spinal cord injury (SCI), a subset of patients may develop progressive clinical deterioration due to intradural scar formation and spinal cord tethering, with or without an associated syringomyelia. Meningitis, intradural hemorrhages, or intradural tumor surgery may also trigger glial scar formation and spinal cord tethering, leading to neurological worsening. Surgery is the treatment of choice in these chronic SCI patients. OBJECTIVE: We hypothesized that cerebrospinal fluid (CSF) and plasma biomarkers could track ongoing neuronal loss and scar formation in patients with spinal cord tethering and are associated with clinical symptoms. METHODS: We prospectively enrolled 12 patients with spinal cord tethering and measured glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase L1 (UCH-L1), and phosphorylated Neurofilament-heavy (pNF-H) in CSF and blood. Seven patients with benign lumbar intradural tumors and 7 patients with cervical radiculopathy without spinal cord involvement served as controls. RESULTS: All evaluated biomarker levels were markedly higher in CSF than in plasma, without any correlation between the two compartments. When compared with radiculopathy controls, CSF GFAP and pNF-H levels were higher in patients with spinal cord tethering (p ≤ 0.05). In contrast, CSF UCH-L1 levels were not altered in chronic SCI patients when compared with either control groups. CONCLUSIONS: The present findings suggest that in patients with spinal cord tethering, CSF GFAP and pNF-H levels might reflect ongoing scar formation and neuronal injury potentially responsible for progressive neurological deterioration.

Protein aggregate formation permits millennium-old brain preservation.

Human proteins have not been reported to survive in free nature, at ambient temperature, for long periods. Particularly, the human brain rapidly dissolves after death due to auto-proteolysis and putrefaction. The here presented discovery of 2600-year-old brain proteins from a radiocarbon dated human brain provides new evidence for extraordinary long-term stability of non-amyloid protein aggregates. Immunoelectron microscopy confirmed the preservation of neurocytoarchitecture in the ancient brain, which appeared shrunken and compact compared to a modern brain. Resolution of intermediate filaments (IFs) from protein aggregates took 2-12 months. Immunoassays on micro-dissected brain tissue homogenates revealed the preservation of the known protein topography for grey and white matter for type III (glial fibrillary acidic protein, GFAP) and IV (neurofilaments, Nfs) IFs. Mass spectrometry data could be matched to a number of peptide sequences, notably for GFAP and Nfs. Preserved immunogenicity of the prehistoric human brain proteins was demonstrated by antibody generation (GFAP, Nfs, myelin basic protein). Unlike brain proteins, DNA was of poor quality preventing reliable sequencing. These long-term data from a unique ancient human brain demonstrate that aggregate formation permits for the preservation of brain proteins for millennia.

A novel approach to study markers of dopamine signaling in peripheral immune cells.

Human monocytes express known markers of dopamine synthesis, storage and clearance, including dopamine transporter (DAT), tyrosine hydroxylase (TH), all subtypes of dopamine receptors and vesicular monoamine transporter 2 (VMAT2). Immunohistochemical and immunofluorescent methodologies have traditionally been employed to determine DAT and TH expression in the CNS, their detection in the blood and specifically in the peripheral monocytes has not been studied by flow cytometry. Flow cytometry assays are widely used in medicine and in basic, preclinical or clinical research to quantify physical and chemical characteristics of target cell populations. Here, we have established a highly sensitive and reproducible flow cytometry panel to detect and quantify DAT and TH expression in freshly isolated or cryopreserved human peripheral monocytes. In healthy humans (n = 41 biological replicates), we show baseline DAT and TH expressing monocytes constitute ~12% of the peripheral blood mononuclear cell (PBMC) fraction when examined in fresh isolation from whole blood. Using an identical flow cytometry panel, we found that cryopreservation of PBMCs using multiple techniques resulted in altered PBMC populations as compared to fresh isolation and relative to one another. Among these, we identified an optimum cryopreservation method for detecting TH and DAT in cryopreserved PBMCs. Our data provide a sensitive and reproducible approach to examine dopamine signaling in peripheral human immune cells. This approach can be applied to study peripheral dopamine signaling under healthy and potentially under disease conditions. The use of dopamine signaling could also be explored as a technique to monitor therapeutic interventions particularly those targeting DAT and TH in the periphery.

Ubiquitin C-Terminal Hydrolase 1 and Phosphorylated Axonal Neurofilament Heavy Chain in Infants Undergoing Cardiac Surgery: Preliminary Assessment as Potential Biomarkers of Brain Injury.

BACKGROUND: There are no reliable markers to assess brain injury in neonates following cardiac surgery. We examine ubiquitin C-terminal hydrolase 1 (UCHL1) and phosphorylated axonal neurofilament heavy chain (pNF-H), neuronal-specific biomarkers released following axonal and cortical injury, in neonates undergoing cardiac surgery involving cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). METHODS: Twenty-six patients younger than three months were prospectively enrolled (CPB only, n = 12 and DHCA, n = 14). Healthy newborns (n = 22) served as the control. Blood samples were collected preoperatively and postoperatively upon intensive care unit admission (hour 0) and subsequently at 12, 24, 36, and 48 hours. Serum was tested for UCHL1 and pNF-H using enzyme-linked immunosorbent assay. Concomitant arterial blood gas, lactate, and cerebral near-infrared spectroscopy (NIRS) monitoring were performed. RESULTS: Ubiquitin C-terminal hydrolase 1 showed a significant rise at 0 hours in the DHCA group compared to baseline (74.9 ± 13.7 pg/mL vs 33.9 ± 37.3 pg/mL, P < .0001). Levels returned to baseline at 12 hours. There was an early rise in UCHL1 at 0 hours in the CPB group, P = .09. Phosphorylated axonal neurofilament heavy chain was decreased at 0 hours in both the CPB and DHCA groups compared to baseline, P = .06. There was no difference between control and baseline levels of UCHL1 ( P = .9) or pNF-H ( P = .77). Decreased NIRS was observed in the DHCA group at 0 hours (57.3 ± 10.5) versus baseline (64.2 ± 12.3), but not significant ( P = .21). There was no correlation between biomarkers and NIRS at 0 hours. CONCLUSION: A rapid rise in UCHL1 levels was observed in the DHCA group, suggesting that it may be a marker for acute brain injury. Follow-up with neurodevelopmental studies is ongoing.

Extracellular vesicles: pathogenetic, diagnostic and therapeutic value in traumatic brain injury.

INTRODUCTION: Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Accurate classification according to injury-specific and patient-specific characteristics is critical to help informed clinical decision-making and to the pursuit of precision medicine in TBI. Reliable biomarker signatures for improved TBI diagnostics are required but still an unmet need. Areas covered: Extracellular vesicles (EVs) represent a new class of biomarker candidates in TBI. These nano-sized vesicles have key roles in cell signaling profoundly impacting pathogenic pathways, progression and long-term sequelae of TBI. As such EVs might provide novel neurobiological insights, enhance our understanding of the molecular mechanisms underlying TBI pathophysiology and recovery, and serve as biomarker signatures and therapeutic targets and delivery systems. Expert commentary: EVs are fast gaining momentum in TBI research, paving the way for new transformative diagnostic and treatment approaches. Their potential to sort out TBI variability and active involvement in the mechanisms underpinning different clinical phenotypes point out unique opportunities for improved classification, risk-stratification ad intervention, harboring promise of predictive, personalized, and even preemptive therapeutic strategies. Although a great deal of progress has been made, substantial efforts are still required to ensure the needed rigorous validation and reproducibility for clinical implementation of EVs.

Erythropoietin Does Not Alter Serum Profiles of Neuronal and Axonal Biomarkers After Traumatic Brain Injury: Findings From the Australian EPO-TBI Clinical Trial.

OBJECTIVE: To determine profiles of serum ubiquitin carboxy-terminal hydrolase L1 and phosphorylated neurofilament heavy-chain, examine whether erythropoietin administration reduce their concentrations, and whether biomarkers discriminate between erythropoietin and placebo treatment groups. DESIGN: Single-center, prospective observational study. SETTING: A sub-study of the erythropoietin-traumatic brain injury clinical trial, conducted at the Alfred Hospital, Melbourne, Australia. PATIENTS: Forty-four patients with moderate-to-severe traumatic brain injury. INTERVENTIONS: Epoetin alfa 40,000 IU or 1 mL sodium chloride 0.9 as subcutaneous injection within 24 hours of traumatic brain injury. MEASUREMENTS AND MAIN RESULTS: Ubiquitin carboxy-terminal hydrolase L1, phosphorylated neurofilament heavy-chain, and erythropoietin concentrations were measured in serum by enzyme-linked immunosorbent assay from D0 (within 24 hr of injury, prior to erythropoietin/vehicle administration) to D5. Biomarker concentrations were compared between injury severities, diffuse versus focal traumatic brain injury and erythropoietin or placebo treatment groups. Ubiquitin carboxy-terminal hydrolase L1 peaked at 146.0 ng/mL on D0, significantly decreased to 84.30 ng/mL on D1, and declined thereafter. Phosphorylated neurofilament heavy-chain levels were lowest at D0 and peaked on D5 at 157.9 ng/mL. D0 ubiquitin carboxy-terminal hydrolase L1 concentrations were higher in diffuse traumatic brain injury. Peak phosphorylated neurofilament heavy-chain levels on D3 and D4 correlated with Glasgow Outcome Score-Extended, predicting poor outcome. Erythropoietin did not reduce concentrations of ubiquitin carboxy-terminal hydrolase L1 or phosphorylated neurofilament heavy-chain. CONCLUSIONS: Serum ubiquitin carboxy-terminal hydrolase L1 and phosphorylated neurofilament heavy-chain increase after traumatic brain injury reflecting early neuronal and progressive axonal injury. Consistent with lack of improved outcome in traumatic brain injury patients treated with erythropoietin, biomarker concentrations and profiles were not affected by erythropoietin. Pharmacokinetics of erythropoietin suggest that the dose given was possibly too low to exert neuroprotection.

Type 3 Adenylyl Cyclase and Somatostatin Receptor 3 Expression Persists in Aged Rat Neocortical and Hippocampal Neuronal Cilia.

The primary cilia of forebrain neurons assemble around birth and become enriched with neuromodulatory receptors. Our understanding of the permanence of these structures and their associated signaling pathways in the aging brain is poor, but they are worthy of investigation because disruptions in neuronal cilia signaling have been implicated in changes in learning and memory, depression-like symptoms, and sleep anomalies. Here, we asked whether neurons in aged forebrain retain primary cilia and whether the staining characteristics of aged cilia for type 3 adenylyl cyclase (ACIII), somatostatin receptor 3 (SSTR3), and pericentrin resemble those of cilia in younger forebrain. To test this, we analyzed immunostained sections of forebrain tissues taken from young and aged male Fischer 344 (F344) and F344 × Brown Norway (F344 × BN) rats. Analyses of ACIII and SSTR3 in young and aged cortices of both strains of rats revealed that the staining patterns in the neocortex and hippocampus were comparable. Virtually every NeuN positive cell examined possessed an ACIII positive cilium. The lengths of ACIII positive cilia in neocortex were similar between young and aged for both strains, whereas in F344 × BN hippocampus, the cilia lengths increased with age in CA1 and CA3, but not in dentate gyrus (DG). Additionally, the percentages of ACIII positive cilia that were also SSTR3 positive did not differ between young and aged tissues in either strain. We also found that pericentrin, a protein that localizes to the basal bodies of neuronal cilia and functions in primary cilia assembly, persisted in aged cortical neurons of both rat strains. Collectively, our data show that neurons in aged rat forebrain possess primary cilia and that these cilia, like those present in younger brain, continue to localize ACIII, SSTR3, and pericentrin. Further studies will be required to determine if the function and signaling pathways regulated by cilia are similar in aged compared to young brain.

Physiologically relevant factors influence tau phosphorylation by leucine-rich repeat kinase 2.

Hyperphosphorylation and aggregation of tau are observed in multiple neurodegenerative diseases termed tauopathies. Tau has also been implicated in the pathogenesis of Parkinson's disease (PD) and parkinsonisms. Some PD patients with mutations in the leucine-rich repeat kinase 2 (LRRK2) gene exhibit tau pathology. Mutations in LRRK2 are a major risk factor for PD, but LRRK2 protein function remains unclear. The most common mutation, G2019S, is located in the kinase domain of LRRK2 and enhances kinase activity in vitro. This suggests that the kinase activity of LRRK2 may underlie its cellular toxicity. Recently, in vitro studies have suggested a direct interaction between tubulin-bound tau and LRRK2 that results in tau phosphorylation at one identified site. Here we present data suggesting that microtubules (MTs) enhance LRRK2-mediated tau phosphorylation at three different epitopes. We also explore the effect of divalent cations as catalytic cofactors for G2019S LRRK2-mediated tau phosphorylation and show that manganese does not support kinase activity but inhibits the efficient ability of magnesium to catalyze LRRK2-mediated phosphorylation of tau. These results suggest that cofactors such as MTs and cations in the cellular milieu have an important impact on LRRK2-tau interactions and resultant tau phosphorylation.

Combination of serum phosphorylated neurofilament heavy subunit and hyperintensity of intramedullary T2W on magnetic resonance imaging provides better prognostic value of canine thoracolumbar intervertebral disc herniation.

The aim of this study was to evaluate the prognostic value of concurrent measurement of serum phosphorylated neurofilament heavy subunit (pNF-H) concentration and intramedullary T2W hyperintensity in paraplegic to paraplegic dogs. Our hypothesis was that concurrent measurement of these would provide a more accurate prediction of functional outcome in dogs with thoracolumbar intervertebral disc herniation (IVDH). A prospective case-control clinical study was designed using 94 dogs with acute onset of thoracolumbar IVDH. The association of serum pNF-H concentration, T2W hyperintensity on sagittal MRI (T2H/L2), deep pain perception and surgical outcome were evaluated with logistic regression analysis after three months for all 94 surgically treated dogs. Sensitivity to predict non-ambulatory outcome was compared among pNF-H and T2H/L2 and combination of both. Logistic regression analysis indicated that serum pNF-H concentration and T2H/L2 were significantly correlated with surgical outcome (P<0.05); however, deep pain perception was not (P=0.41). The results of logistic regression analysis indicated that the odds ratios of unsuccessful long-term outcome were 2.6 for serum pNF-H concentration, 1.9 for T2H/L2 and 2.3 for deep pain sensation. The sensitivity and specificity to predict non-ambulatory outcome for using serum parameter pNF-H>2.6 ng/ml, using T2H/L2 value of>0.84 and using both serum pNF-H and T2H/L2, were 95% and 75.7%, 65% and 86.5%, and 90.0% and 97.5%, respectively. Therefore, combined measurements of serum pNF-H and T2H/L2 might be useful for predicting long-term outcome in dogs with thoracolumbar IVDH.

Measurement of novel biomarkers of neuronal injury and cerebral oxygenation after routine vaginal delivery versus cesarean section in term infants.

AIMS: It remains unclear if mode of delivery can have any impact on the neonatal brain. Our aim was to determine in term newborns any differences based on mode of delivery in either neuronal injury biomarkers, phosphorylated axonal neurofilament heavy chain (pNF-H) and ubiquitin C-terminal hydrolase (UCHL1), or brain oxygenation values, regional cerebral tissue oxygen saturation (CrSO2) and cerebral fractional tissue oxygen extraction (CFOE). METHODS: An Institutional Review Board approved prospective observational pilot study of well newborns. Serum pNF-H and UCHL1 levels were measured on the day following delivery. CrSO2 values along with CFOE values were also measured using near-infrared spectroscopy (NIRS) and pulse oximetry. RESULTS: There were 22 subjects, 15 born vaginally and seven born by cesarean section. No difference was found in mean pNF-H (107.9±54.3 pg/mL vs. 120.2±43.3 pg/mL, P=0.66) or mean UCHL1 (4.0±3.5 pg/mL vs. 3.0±2.2 pg/mL, P=0.68). No difference was found in mean CrSO2 (80.8±5.3% vs. 80.8±5.6%, P=0.99) or mean CFOE (0.17±0.06 vs. 0.15±0.08, P=0.51). CONCLUSIONS: We found no difference in neuronal injury markers between term neonates born vaginally compared to those born by cesarean section. From a neurologic standpoint, this supports current obstetric practice guidelines that emphasize vaginal birth as the preferred delivery method whenever possible.

Brain injection of α-synuclein induces multiple proteinopathies, gliosis, and a neuronal injury marker.

Intracerebral injection of amyloidogenic α-synuclein (αS) has been shown to induce αS pathology in the CNS of nontransgenic mice and αS transgenic mice, albeit with varying efficiencies. In this study, using wild-type human αS transgenic mice (line M20), we demonstrate that intracerebral injection of recombinant amyloidogenic or soluble αS induces extensive αS intracellular inclusion pathology that is associated with robust gliosis. Near the injection site, a significant portion of αS inclusions are detected in neurons but also in astrocytes and microglia. Aberrant induction of expression of the intermediate filament protein peripherin, which is associated with CNS neuronal injury, was also observed predominantly near the site of injection. In addition, many pSer129 αS-induced inclusions colocalize with the low-molecular-mass neurofilament subunit (NFL) or peripherin staining. αS inclusion pathology was also induced in brain regions distal from the injection site, predominantly in neurons. Unexpectedly, we also find prominent p62-immunoreactive, αS-, NFL-, and peripherin-negative inclusions. These findings provide evidence that exogenous αS challenge induces αS pathology but also results in the following: (1) a broader disruption of proteostasis; (2) glial activation; and (3) a marker of a neuronal injury response. Such data suggest that induction of αS pathology after exogenous seeding may involve multiple interdependent mechanisms.

Endogenously regulated Dab2 worsens inflammatory injury in experimental autoimmune encephalomyelitis.

BACKGROUND: Neuroinflammation regulates both disease pathogenesis and repair in multiple sclerosis. In early multiple sclerosis lesion development, neuroinflammation causes demyelination and axonal injury, the likely final common determinant of disability. Here we report the identification of a novel neuroinflammatory mediator, Disabled-2 (Dab2). Dab2 is an intracellular adaptor protein with previously unknown function in the central nervous system. RESULTS: We report that Dab2 is up-regulated in lesional macrophages/microglia in the spinal cord in murine experimental autoimmune encephalomyelitis, a model of multiple sclerosis. We demonstrate that dab2 expression is positively correlated with experimental autoimmune encephalomyelitis disease severity during the acute disease phase. Furthermore, dab2-deficient mice have a less severe experimental autoimmune encephalomyelitis disease course and suffer less neuroinflammation and less axonal injury than their wild-type littermates. We demonstrate that dab2 expression is strongly associated with the expression of inducible nitric oxide synthase. We further demonstrate that Dab2 is expressed at the protein level by macrophages in early acute human multiple sclerosis lesions and that this correlates with axonal injury. CONCLUSIONS: Together, these results suggest that endogenous Dab2 exacerbates central nervous system inflammation, potentially acting to up-regulate reactive oxygen species expression in macrophages and microglia, and that it is of potential pathogenic relevance in Multiple Sclerosis.

Unbiased screen reveals ubiquilin-1 and -2 highly associated with huntingtin inclusions.

Recently mutations in ubiquilin-2 were identified in patients with amyotrophic lateral sclerosis (ALS) and ALS/dementia providing direct evidence for the importance of this protein in neurodegenerative diseases. Histological studies have suggested that ubiquilin-1/-2 are associated with various pathological inclusions including Lewy bodies in Parkinson's disease, neurofibrillary tangles in Alzheimer's disease, polyQ inclusions in expansion repeat diseases and various proteinopathies associated with ALS and frontotemporal dementia. Using specific ubiquilin-2 antibodies and a series of transgenic mouse models of proteinopathies associated with neurodegenerative disease, we show that ubiquilin-2 preferentially associates with huntingtin polyQ expansion aggregates compared to α-synuclein, tau and several other types of protein inclusions. These results were confirmed by similar findings for ubiquilin-1 and -2 in human brain tissue sections, where accumulation was observed in huntingtin inclusions, but only infrequently in other types of protein inclusions. In cultured cells, ubiquilin-2 associates with huntingtin/polyQ aggregates, but this is not compromised by disease-causing mutations. Although ubiquilin proteins can function as chaperones to shuttle proteins for degradation, there is persistent co-localization between ubiquilin-2 and polyQ aggregated proteins during disease progression in the N586-82Q-C63 Huntington's disease mouse model. Thus, the co-localization of ubiquilin-2 with the huntingtin aggregates does not appear to facilitate aggregate removal.

Noninvasive assessments of optic nerve neurodegeneration in transgenic mice with isolated optic neuritis.

PURPOSE: To determine if phosphorylated neurofilament heavy chain (pNF-H) released into the bloodstream and the pattern ERG are noninvasive indicators of neurodegeneration in experimental optic neuritis. METHODS: Serum from Myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor-positive (TCR+) transgenic mice that develop isolated optic neuritis usually without any other characteristic lesions of inflammation or demyelination in the spinal cord and littermates negative for the transgene were assayed for the presence of serum phosphorylated neurofilament H (pNF-H). In vivo measurements of optic nerve and retinal ganglion cell injury were assessed by magnetic resonance imaging (MRI), optical coherence tomography (OCT), and pattern electroretinogram (PERG). Automated two dimensional fluorescence differential in-gel electrophoresis (2D-DIGE) of pooled optic nerve samples, light, and transmission electron micrographs were used to evaluate optic atrophy postmortem. RESULTS: We found an almost 3-fold elevation in serum pNF-H levels in MOG+ mice relative to MOG-littermates (P = 0.02). 2D-DIGE revealed a 3-fold reduction in optic nerve neurofilaments. Visual function assessed by the PERG was reduced by one-quarter (P = 0.033) and latencies increased by 38% (P = 0.036). MOG+ mice with the lowest PERG amplitudes had optic nerve atrophy visualized by MRI. Optic nerve diameters were reduced by one-third (P = 0.0001) and axon counts reduced by more than two-thirds. Histopathology of the spinal cords was normal. CONCLUSIONS: Elevated serum pNF-H levels and the PERG are useful markers of neurodegeneration of the optic nerve in isolated experimental optic neuritis. Our findings suggest that elevations of this axonal protein in patients with optic neuritis who had a poor visual outcome are likely also due to demise of optic nerve axons.

Phosphorylated neurofilament heavy subunit (pNF-H) in peripheral blood and CSF as a potential prognostic biomarker in amyotrophic lateral sclerosis.

BACKGROUND: The phosphorylated neurofilament heavy subunit (pNF-H), a major structural component of motor axons, is a promising putative biomarker in amyotrophic lateral sclerosis (ALS) but has been studied mainly in CSF. We examined pNF-H concentrations in plasma, serum and CSF as a potential biomarker for disease progression and survival in ALS. METHODOLOGY: We measured pNF-H concentration by monoclonal sandwich ELISA in plasma (n=43), serum and CSF (n=20) in ALS patients collected at the Mayo Clinic Florida and Emory University. We included plasma from an ALS cohort (n=20) from an earlier pilot study in order to evaluate baseline pNF-H levels in relation to disease progression using the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R), survival and anatomical region of ALS onset. RESULTS: Higher pNF-H levels in plasma, serum and CSF showed evidence of association with faster decline in ALSFRS-R. There was evidence for a relationship of higher serum and plasma pNF-H levels with shorter survival, although evidence was weaker for CSF. pNF-H concentration in plasma (n=62) may be higher in patients with bulbar onset than in patients with spinal onset. CONCLUSIONS: In ALS, increased pNF-H concentration in plasma, serum and CSF appears to be associated with faster disease progression. Factors affecting pNF-H levels or their detection in serum and plasma in relation to disease course may differ from those in CSF. Data raising the possibility that site of ALS onset (bulbar vs spinal) may influence pNF-H levels in peripheral blood seems noteworthy but requires confirmation. These data support further study of pNF-H in CSF, serum and plasma as a potential ALS biomarker.

Leukemia inhibitory factor protects axons in experimental autoimmune encephalomyelitis via an oligodendrocyte-independent mechanism.

Leukemia inhibitory factor (LIF) and Ciliary Neurotrophic factor (CNTF) are members of the interleukin-6 family of cytokines, defined by use of the gp130 molecule as an obligate receptor. In the murine experimental autoimmune encephalomyelitis (EAE) model, antagonism of LIF and genetic deletion of CNTF worsen disease. The potential mechanism of action of these cytokines in EAE is complex, as gp130 is expressed by all neural cells, and could involve immuno-modulation, reduction of oligodendrocyte injury, neuronal protection, or a combination of these actions. In this study we aim to investigate whether the beneficial effects of CNTF/LIF signalling in EAE are associated with axonal protection; and whether this requires signalling through oligodendrocytes. We induced MOG35₋55 EAE in CNTF, LIF and double knockout mice. On a CNTF null background, LIF knockout was associated with increased EAE severity (EAE grade 2.1±0.14 vs 2.6±0.19; P<0.05). These mice also showed increased axonal damage relative to LIF heterozygous mice, as indicated by decreased optic nerve parallel diffusivity on MRI (1540±207 µm²-/s vs 1310±175 µm²-/s; P<0.05), and optic nerve (-12.5%) and spinal cord (-16%) axon densities; and increased serum neurofilament-H levels (2.5 fold increase). No differences in inflammatory cell numbers or peripheral auto-immune T-cell priming were evident. Oligodendrocyte-targeted gp130 knockout mice showed that disruption of CNTF/LIF signalling in these cells has no effect on acute EAE severity. These studies demonstrate that endogenous CNTF and LIF act centrally to protect axons from acute inflammatory destruction via an oligodendrocyte-independent mechanism.