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.

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.

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.

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.

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.

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.

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.

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.

Combination of neurofilament heavy chain and complement C3 as CSF biomarkers for ALS.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease with an average survival of 3 years from symptom onset. Rapid and conclusive early diagnosis is essential if interventions with disease-modifying therapies are to be successful. Cytoskeletal modification and inflammation are known to occur during the pathogenesis of ALS. We measured levels of cytoskeletal proteins and inflammatory markers in the CSF of ALS, disease controls and healthy subjects. We determined threshold values for each protein that provided the optimal sensitivity and specificity for ALS within a training set, as determined by receiver operating characteristic analysis. Interestingly, the optimal assay was a ratio of the levels for phosphorylated neurofilament heavy chain and complement C3 (pNFH/C3). We next applied this assay to a separate test set of CSF samples to verify our results. Overall, the predictive pNFH/C3 ratio identified ALS with 87.3% sensitivity and 94.6% specificity in a total of 71 ALS subjects, 52 disease control subjects and 40 healthy subjects. In addition, the level of CSF pNFH correlated with survival of ALS patients. We also detected increased pNFH in the plasma of ALS patients and observed a correlation between CSF and plasma pNFH levels within the same subjects. These findings support large-scale prospective biomarker studies to determine the clinical utility of diagnostic and prognostic signatures in ALS.

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.

Identification and preliminary characterization of ubiquitin C terminal hydrolase 1 (UCHL1) as a biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage.

By using two different approaches, ubiquitin C-terminal hydrolase 1 (UCHL1) was identified as a potential cerebrospinal fluid (CSF) biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage (ASAH) and presumably other CNS damage and disease states. Appropriate antibodies and a sensitive ELISA were generated, and the release of UCHL1 into CSF was compared with that of pNF-H and S100beta in a cohort of 30 ASAH patients. Both UCHL1 and pNF-H showed persistent release into CSF in almost all patients in the second week postaneurysmal rupture (AR), and S100beta levels rapidly declined to baseline levels in 23 of 30 patients. Seven of thirty patients showed persistently elevated S100beta levels over the first 5 days post-AR and also had relatively higher levels of pNF-H and UCHL1 higher compared with the rest. These patients proved to have very poor outcomes, with 6 of 7 expiring. Patients who did reduce S100beta levels tended to have a better outcome if pNF-H and UCHL1 levels were also lower, and elevated UCHL1 levels in the second week post-AR were particularly predictive of poor outcome. Acute coordinated releases of large amounts of UCHL1, pNF-H, and S100beta in 16 of 30 patients were observed, suggesting sudden loss of brain tissues associated with secondary events. We conclude that measurement of the CSF levels of these proteins reveals details of ASAH progression and recovery and predicts patient outcome.

A pilot study of novel biomarkers in neonates with hypoxic-ischemic encephalopathy.

Severe hypoxic-ischemic encephalopathy (HIE) is a devastating condition that can lead to mortality and long-term disabilities in term newborns. No rapid and reliable laboratory test exists to assess the degree of neuronal injury in these patients. We propose two possible biomarkers: 1) phosphorylated axonal neurofilament heavy chain (pNF-H) protein, one of the major subunits of neurofilaments, found only in axonal cytoskeleton of neurons and 2) Ubiquitin C-terminal hydrolase 1 (UCHL1 protein) that is heavily and specifically concentrated in neuronal perikarya and dendrites. High-serum pNF-H and UCHL1 levels are reported in subarachnoid hemorrhage and traumatic brain injury, suggesting that they are released into blood following neuronal injury. We hypothesized that serum pNF-H and UCHL1 were higher in neonates with moderate-to-severe HIE than in healthy neonates. A time-limited enrollment of 14 consecutive patients with HIE and 14 healthy controls was performed. UCHL1 and pNF-H were correlated with clinical data and brain MRI. UCHL1 and pNF-H serum levels were higher in HIE versus controls. UCHL1 showed correlation with the 10-min Apgar score, and pNF-H showed correlation with abnormal brain MRI. Our findings suggest that serum UCHL1 and pNF-H could be explored as diagnostic and prognostic tools in neonatal HIE.

Hematopoietic- and neurologic-expressed sequence 1 (Hn1) depletion in B16.F10 melanoma cells promotes a differentiated phenotype that includes increased melanogenesis and cell cycle arrest.

The Hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a small protein that is highly conserved among species. Hn1 expression is upregulated in regenerating neural tissues, including the axotomized adult rodent facial motor nerve and dedifferentiating retinal pigment epithelial cells of the Japanese newt. It is also expressed in numerous tissues during embryonic development as well as in regions of the adult brain that exhibit high plasticity. Hn1 has also been reported as a marker for human ovarian carcinoma and it is expressed in high-grade human gliomas. This study was directed toward understanding the function of Hn1 in a murine melanoma cell line. Hn1 mRNA and protein were identified in B16.F10 cells and in tumors formed from these cells. Inhibition of Hn1 protein expression with siRNA increased melanogenesis. Hn1-depleted cells expressed higher levels of the melanogenic proteins tyrosinase and Trp2 and an increased interaction between actin and Rab27a. The in vitro cell growth rate of Hn1-depleted cells was significantly reduced due to G1/S cell cycle arrest. This was consistent with a reduction in the phosphorylation of retinoblastoma protein as well as lower levels of p27 and increased expression of p21. Decreased expression of c-Met, the receptor for hepatocyte growth factor, was also detected in the Hn1-depleted cells, however HGF-dependent stimulation of phosphorylated-ERK was unaffected. Hn1 depletion also led to increased basal levels of phosphorylated p38 MAPK, while basal ERK phosphorylation was reduced. Moreover, Hn1-depleted cells had reduced expression of transcription factors MITF and USF-1, and increased expression of TFE3. These data, coupled with reports on Hn1 expression in regeneration and development, suggest that Hn1 functions as a suppressor of differentiation in cells undergoing repair or proliferation.

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.

Immunoreactivity of the phosphorylated axonal neurofilament H subunit (pNF-H) in blood of ALS model rodents and ALS patients: evaluation of blood pNF-H as a potential ALS biomarker.

Levels of neurofilament subunits, potential biomarkers of motor axon breakdown, are increased in amyotrophic lateral sclerosis (ALS) patient's CSF but data on blood are not available. We measured blood levels of the phosphorylated axonal form of neurofilament H (pNF-H) by ELISA in transgenic rodent models of superoxide dismutase 1 (SOD1) ALS, and in 20 ALS patients and 20 similar aged controls monthly for 4 months. All symptomatic rodent ALS models showed robust levels of blood pNF-H, while control rodents or mice transgenic for unmutated SOD1 showed no detectable blood pNF-H. Average pNF-H levels in the G93A SOD1 mouse progressively increased from day 74 through death (day approximately 130). Median blood pNF-H level in ALS patients was 2.8-fold higher than controls (p < 0.001). Median ALSFRS-R declined a median of 0.8 pt/month (p < 0.001); higher baseline pNF-H level appeared to be associated with faster ALSFRS-R decline over 4 months (p = 0.087). The median rate of decline in ALSFRS-R was 1.9 pt/month in patients with baseline pNF-H levels above the median pNF-H value of 0.53 ng/mL; ALSFRS-R declined at a median of 0.6 pt/month in patients below this level. The pNF-H levels were relatively stable month to month in individual patients, raising questions regarding the molecular pathogenesis of ALS. Baseline control human pNF-H levels were higher in men than women and increased minimally over time. These data suggest that blood pNF-H can be used to monitor axonal degeneration in ALS model rodents and support further study of this protein as a potential biomarker of disease prognosis in ALS patients.

A neurotoxic peripherin splice variant in a mouse model of ALS.

Peripherin, a neuronal intermediate filament (nIF) protein found associated with pathological aggregates in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and of transgenic mice overexpressing mutant superoxide dismutase-1 (SOD1G37R), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. Mouse peripherin is unique compared with other nIF proteins in that three peripherin isoforms are generated by alternative splicing. Here, the properties of the peripherin splice variants Per 58, Per 56, and Per 61 have been investigated in transfected cell lines, in primary motor neurons, and in transgenic mice overexpressing peripherin or overexpressing SOD1G37R. Of the three isoforms, Per 61 proved to be distinctly neurotoxic, being assembly incompetent and inducing degeneration of motor neurons in culture. Using isoform-specific antibodies, Per 61 expression was detected in motor neurons of SOD1G37R transgenic mice but not of control or peripherin transgenic mice. The Per 61 antibody also selectively labeled motor neurons and axonal spheroids in two cases of familial ALS and immunoprecipitated a higher molecular mass peripherin species from disease tissue. This evidence suggests that expression of neurotoxic splice variants of peripherin may contribute to the neurodegenerative mechanism in ALS.

Immunostaining evidence for PI(4,5)P2 localization at the leading edge of chemoattractant-stimulated HL-60 cells.

It is well known that in fMLP-stimulated neutrophils, phosphatidyl inositol 3,4,5-trisphosphate [PI(3,4,5)P3] localizes at the leading edge of the cells. However, no effort has been made to study the PI 4,5-bisphosphate [PI(4,5)P2] distribution in these cells. In fact, it has been suggested that PI(4,5)P2 is unlikely to localize, as its basal level is orders of magnitude higher than that of PI(3,4,5)P3. We developed an optimized immunostaining protocol for studying the endogenous distribution of PI(4,5)P2 in neutrophil-like HL-60 cells. We show that PI(4,5)P2 localizes sharply at the leading edge with an intensity gradient similar to that for PI(3,4,5)P3. The enzymes for the production of PI(4,5)P2, namely, PI5KIalpha and PI5KIgamma, were also found to localize at the leading edge, further supporting our finding that PI(4,5)P2 localizes at the leading edge. These results imply that complementary regulation of PI3K and phosphate and tensin homolog (PTEN) is not the sole or dominant mechanism of PI(3,4,5)P3 polarization in HL-60 cells.

Detection of phosphorylated NF-H in the cerebrospinal fluid and blood of aneurysmal subarachnoid hemorrhage patients.

Blood and cerebrospinal fluid (CSF) of 30 Fisher grade 3 aneurysmal subarachnoid hemorrhage (ASAH) patients were analyzed for the presence of the phosphorylated axonal form of the major neurofilament subunit NF-H (pNF-H), a promising biomarker of axonal injury. Patient demographic data including development of vasospasm and outcome scores at 6 months after aneurysmal rupture (AR) were evaluated. Higher pNF-H blood levels in the first few days after AR were strongly predictive of a negative outcome. Blood pNF-H levels in most recovering patients showed a steady increase into the second week after AR, presumably reflecting axonal degeneration secondary to the original insult. Almost half of the patients studied showed sudden dramatic peaks of pNF-H protein release into CSF in the 3- to 14-day time period after AR, which must reflect profound, coordinated, and secondary loss of axons. Patients in whom vasospasm was detected had significantly more pNF-H in both blood and CSF compared with those in whom vasospasm was not detected. We conclude that the analysis of pNF-H levels in blood and CSF differentiates between patients with poor and favorable outcomes and also reveals several novel features of ASAH progression and recovery.