pNF-H Concentrations Research Articles

Phosphorylated neurofilament H (pNF-H) as a potential diagnostic marker for neurological disorders in horses

The current study aimed at the investigating the potential use of phosphorylated neurofilament H (pNF-H) as a diagnostic biomarker for neurologic disorders in the horse. Paired serum and cerebrospinal fluid (CSF) samples (n=88) and serum only (n=30) were obtained from horses diagnosed with neurologic disorders and clinically healthy horses as control. The neurologic horses consisted of equine protozoal myeloencephalitis (EPM) (38 cases) and cervical vertebral malformation (CVM) (23 cases). Levels of pNF-H were determined using an ELISA. The correlation between CSF and serum concentrations of pNF-H was evaluated using Spearman's Rank test and the significance of the difference among the groups was assessed using a nonparametric test. Horses had higher pNF-H levels in the CSF than serum. Horses afflicted with EPM had significantly higher serum pNF-H levels in comparison to controls or CVM cases. The correlation between CSF and serum pNF-H levels was poor in both the whole study population and among subgroups of horses included in the study. There was significant association between the likelihood of EPM and the concentrations of pNF-H in either the serum or CSF. These data suggest that pNF-H could be detected in serum and CSF samples from neurologic and control horses. This study demonstrated that pNF-H levels in serum and CSF have the potential to provide objective information to help in the early diagnosis of horses afflicted with neurologic disorders.

Brain injury with systemic inflammation in newborns with congenital heart disease undergoing heart surgery.

The potential role of systemic inflammation on brain injury in newborns with congenital heart disease (CHD) was assessed by measuring levels of central nervous system (CNS)-derived proteins in serum prior to and following cardiac surgery. A total of 23 newborns (gestational age, 39±1 weeks) with a diagnosis of CHD that required cardiac surgery with cardiopulmonary bypass (CPB) were enrolled in the current study. Serum samples were collected immediately prior to surgery and 2, 24 and 48 h following CPB, and serum levels of phosphorylated neurofilament-heavy subunit (pNF-H), neuron-specific enolase (NSE) and S100B were analyzed. Systemic inflammation was assessed by measuring serum concentrations of complement C5a and complement sC5b9, and the following cytokines: Interleukin (IL)-1β, IL-6, IL-8, IL-10, IL12p70, interferon γ and tumor necrosis factor (TNF)-α. Analysis of cord blood from normal term deliveries (n=26) provided surrogate normative values for newborns. pNF-H and S100B were 2.4- to 2.8-fold higher (P<0.0001) in patient sera than in cord blood prior to surgery and remained elevated following CPB. Pre-surgical serum pNF-H and S100B levels directly correlated with interleukin (IL)-12p70 (ρ=0.442, P<0.05). pNF-H was inversely correlated with arterial pO2 prior to surgery (ρ=−0.493, P=0.01) and directly correlated with arterial pCO2 post-CPB (ρ=0.426, P<0.05), suggesting that tissue hypoxia and inflammation contribute to blood brain barrier (BBB) dysfunction and neuronal injury. Serum IL12p70, IL-6, IL-8, IL-10 and TNF-α levels were significantly higher in patients than in normal cord blood and levels of these cytokines increased following CPB (P<0.001). Activation of complement was observed in all patients prior to surgery, and serum C5a and sC5b9 remained elevated up to 48 h post-surgery. Furthermore, they were correlated (P<0.05) with low arterial pO2, high pCO2 and elevated arterial pressure in the postoperative period. Length of mechanical ventilation was associated directly with post-surgery serum IL-12p70 and IL-8 concentrations (P<0.05). Elevated serum concentrations of pNF-H and S100B in neonates with CHD suggest BBB dysfunction and CNS injury, with concurrent hypoxemia and an activated inflammatory response potentiating this effect.

Neurofilament markers for ALS correlate with extent of upper and lower motor neuron disease.

To determine the diagnostic performance and prognostic value of phosphorylated neurofilament heavy chain (pNfH) and neurofilament light chain (NfL) in CSF as possible biomarkers for amyotrophic lateral sclerosis (ALS) at the diagnostic phase. We measured CSF pNfH and NfL concentrations in 220 patients with ALS, 316 neurologic disease controls (DC), and 50 genuine disease mimics (DM) to determine and assess the accuracy of the diagnostic cutoff value for pNfH and NfL and to correlate with other clinical parameters. pNfH was most specific for motor neuron disease (specificity 88.2% [confidence interval (CI) 83.0%-92.3%]). pNfH had the best performance to differentially diagnose patients with ALS from DM with a sensitivity of 90.7% (CI 84.9%-94.8%), a specificity of 88.0% (CI 75.7%-95.5%) and a likelihood ratio of 7.6 (CI 3.6-16.0) at a cutoff of 768 pg/mL. CSF pNfH and NfL levels were significantly lower in slow disease progressors, however, with a poor prognostic performance with respect to the disease progression rate. CSF pNfH and NfL levels increased significantly as function of the number of regions with both upper and lower motor involvement. In particular, CSF pNfH concentrations show an added value as diagnostic biomarkers for ALS, whereas the prognostic value of pNfH and NfL warrants further investigation. Both pNfH and NfL correlated with the extent of motor neuron degeneration. This study provides Class II evidence that elevated concentrations of CSF pNfH and NfL can accurately identify patients with ALS.

Cerebrospinal Fluid Levels of Phosphorylated Neurofilament Heavy as a Diagnostic Marker of Canine Degenerative Myelopathy.

BackgroundNo definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF‐H) is a promising biomarker for nervous system diseases.Hypothesis/ObjectiveCerebrospinal fluid (CSF) and serum pNF‐H is a detectable biological marker for diagnosis of canine DM.AnimalsFifty‐three DM‐affected, 27 neurologically normal, 7 asymptomatic at‐risk, and 12 DM mimic dogs.MethodsArchived CSF and serum pNF‐H concentrations were determined by a commercially available ELISA. A receiver‐operating characteristic (ROC) curve was generated with CSF values.ResultsCompared with old control dogs, median CSF pNF‐H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4–9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8–29.6; P < .05) versus DM stage 2 36.8 ng/mL (IQR 22.9–51.2; P < .0001) versus DM stage 3 25.2 ng/mL (IQR 20.2–61.8; P < .001) versus DM stage 4 38.0 ng/mL (IQR 11.6–59.9; P < .01). Degenerative myelopathy stage 1 dogs had increased median CSF pNF‐H concentrations compared with asymptomatic, at‐risk dogs (3.4 ng/mL [IQR 1.5–10.9; P < .01]) and DM mimics (6.6 ng/mL [IQR 3.0–12.3; P < .01]). CSF pNF‐H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09–90.64%) and 93.6% specific (CI 78.58–99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92–0.9974). No differences in serum pNF‐H concentration were found between control and DM‐affected dogs.Conclusions and Clinical Importance pNF‐H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies.

New Surgical Attitude in Complete Acute Traumatic Spinal Cord Injury

Introduction Studies have shown the values pattern of the phosphorylated form of the high-molecular-weight neurofilament subunit (pNF-H) in CSF is a predictive biomarker for acute traumatic spinal cord injury (SCI). Materials and Methods A previous study of pNF-H in CSF of patients with SCI showed it is a specific biomarker for SCI, it can distinguish the severity of SCI and it is a predictive biomarker because of its values pattern can show the reducing or stopping of the secondary lesion and a favorable result. At 22 subjects with acute traumatic SCI we correlated the pNF-H concentration in CSF with the MRI, with the diffusion tensor tractography on the SCI site and with the clinical evolution. All patients underwent surgery during the first 24 hours (decompression, stabilization). In two cases with unfavorable pattern of pNF-H after 4 and 7 days, MRI identified the SCI site with an extensive spinal cord edema and a necrotic cavity. A second microneurosurgery was done: opening the dura 5 levels, duraplasty and opening the spinal cord in the midline and debridement of the necrotic tissue. Results Complete SCI cases had a specific pattern of values of pNF-H correlated with the MRI and the clinical evolution. A second microneurosurgery solved the cord edema and the spinal cord laceration. One case has improved progressively after 6 months and the other case - no neurological improvement after one year of rehabilitation. Conclusions Patients must undergo surgery during the first 24 hours: a large decompression on 5 – 7 levels with duraplasty and stabilization. After these we can use the predictive unfavorable pattern of pNF-H correlated with MRI and a second microneurosurgery in SCI site must be done: opening the spinal cord in the midline and microsurgical debridement of the necrotic tissue. This surgical attitude can create favorable conditions for functional recovery of the remaining spinal cord.

Serum Phosphorylated Neurofilament-Heavy Chain, a Potential Biomarker, is Associated With Peripheral Neuropathy in Patients With Type 2 Diabetes.

Neurofilament (NF), one of the major axonal cytoskeletal proteins, plays a critical role in degenerative diseases in both the central and the peripheral nervous systems. The aim of this study is to explore the relationship between serum phosphorylated neurofilament-heavy chain (pNF-H) and diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes.Serum pNF-H concentrations were measured by ELISA in hospitalized patients with and without DPN (n = 118). DPN was assessed by clinical symptoms, signs, and electromyography.Compared with the non-DPN group (311.98 [189.59–634.12] pg/mL), the confirmed group (605.99 [281.17–1332.78] pg/mL) patients had the higher serum pNF-H levels (P = 0.007). DPN was significantly correlated with C-peptide (r = −0.269), total cholesterol (TC) (r = 0.185), and pNF-H (r = 0.258). Serum pNF-H levels were independently associated with DPN (P = 0.004), even after adjusting for age, sex, duration of diabetes, fasting plasma glucose, glycosylated hemoglobin A1c, TC, C-peptide, urinary albuminto/creatinine ratio, and estimated glomerular filtration rate. Compared with pNF-H quartile 1 (referent), patients in quartile 3 (odds ratio [OR], 3.977; 95% confidence interval [CI], 1.243–12.728; P = 0.021) and quartile 4 (OR, 10.488; 95% CI, 3.020–34.429; P = 0.000) had the higher risk of DPN after adjusting for the confounders.Serum pNF-H levels might be associated with the DPN, and the correlationship between serum pNF-H and DPN should be further studied.

Cerebrospinal Fluid Phosphorylated Neurofilament Subunit NF-H (pNF-H) is a Specific Biomarker and Its Pattern is Predictive for Spinal Cord Injury

Introduction In this study, we measured the phosphorylated neurofilament subunit NF-H (pNF-H) in the cerebrospinal fluid (CSF) of patients with spinal cord injury (SCI) and we set the correlation between the pNF-H levels and the severity of the injury. Patients and Methods The study included 20 patients with acute traumatic SCI: 12 patients with complete SCI and 8 patients with incomplete SCI. All patients were classified according to the American Spinal Injury Association Impairment Scale and all patients underwent surgery during the first 24 hours (decompression, stabilization). We measured daily the heavy phosphorylated neurofilament subunit (pNF-H) concentration by sandwich ELISA test in CSF of all patients and we correlated the values of pNF-H with the clinical evolution. Results For all patients with SCI, pNF-H was detectable in CSF samples and the values were different in the cases of complete SCI compared with the cases of incomplete SCI and the cerebrospinal pNF-H level was more elevated in cases of complete SCI. The level of CSF pNF-H was 10 to 100 times higher in complete SCI than in incomplete SCI, where the level was close to normal. The patients with a favorable neurological evolution after treatment had a specific pattern of daily values of NFP-H: a sudden increase up to a maximum value then a progressive decrease until normal. The maximum values were different in each case, from 10 times up to 200 times higher than the normal. Conclusions The phosphorylated form of the high molecular weight neurofilament subunit NF-H (pNF-H) in CSF is a specific biomarker for SCI and it can distinguish the severity of SCI. pNF-H is a predictive biomarker because its value pattern can show the reducing or stopping of the secondary lesion and the favorable result.

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.

Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients

ObjectivesWe evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS).MethodsSerum pNF-H concentrations were...

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Study objective. The objective of this study was to measure the phosphorylated neurofilament subunit NF-H (pNF-H) in cerebro-spinal fluid of patients with spinal cord injury and to determine the correlation between the pNF-H levels and the severity of the injury. Materials and methods. The study included 15 subjects with acute traumatic spinal cord injury: eight patients with complete spinal cord injury (SCI) and seven patients with incomplete SCI. All patients were classified according to the American Spinal Injury Association impairment scale (ASIA) and all patients underwent surgery during the first 24 hours (decompression, stabilization). We measured daily the heavy phosphorylated neurofilament subunit (pNF-H) concentration by sandwich ELISA test in CSF in all patients and we correlated the values of pNF-H with the clinical evolution. Results. For all patients with SCI pNF-H was detectable in CSF samples and the values were different in the cases of complete SCI toward the cases of incomplete SCI and the cerebro-spinal pNF-H level was more elevated in cases of complete SCI. The level of CSF pNF-H was ten to a hundred times higher in complete SCI than the level of CSF pNF-H in cases with incomplete SCI, where the level of this biomarker was close to normal. The patients with a favorable neurological evolution after treatment had a specific pattern of daily values of pNF-H: a sudden increase up to a maximum value then a progressive decrease until normal. The maximum values were different in each case, from 10 times up to 170 times higher than the normal. Conclusion. The phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H) in cerebro-spinal fluid can be a specific biomarker for spinal cord injury and it can distinguish the severity of SCI. pNF-H is a predictive biomarker because of its values pattern can show the reducing or stopping of the secondary lesion and the favorable result.

Increased CSF Levels of Phosphorylated Neurofilament Heavy Protein following Bout in Amateur Boxers

IntroductionDiagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau) and glial (GFAP and S-100B) damage in cerebrospinal fluid (CSF) after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of APOE ε4 allele genotype which has been associated with poor outcome after TBI.Materials and MethodsThirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up). The controls were tested once. CSF levels of neurofilament heavy (pNFH), amyloid precursor proteins (sAPPα and sAPPβ), ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed.ResultsCSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001). The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018). CSF pNFH concentrations correlated with NFL (r = 0.57 after bout and 0.64 at follow up, p<0.001). No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele had similar biomarker concentrations as non-carriers.ConclusionsSubconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the APOE ε4 allele was not found to influence biomarker levels after acute TBI.

Predictive value of phosphorylated axonal neurofilament subunit H for clinical outcome in patients with acute intracerebral hemorrhage

BackgroundPhosphorylated axonal neurofilament subunit H (pNF-H) is a biomarker of axonal injury. We investigated whether plasma pNF-H concentrations were associated with 6-month clinical outcomes and early neurological deterioration (END) of patients with acute intracerebral hemorrhage. MethodsPlasma pNF-H concentrations of 112 patients and 112 healthy individuals were quantified by ELISA. Unfavorable outcome was defined as modified Rankin Scale score >2. Associations of plasma pNF-H concentrations with END, 6-month mortality and unfavorable outcome were evaluated. ResultsPlasma pNF-H concentrations were increased in patients than in healthy individuals [700.2 (430.8) pg/ml vs. 25.5 (32.4) pg/ml, P<0.001]. A logistic regression analysis selected plasma pNF-H concentration as an independent predictor for 6-month mortality [OR: 1.287, 95% CI: 1.140–1.524, P<0.001], 6-month unfavorable outcome (OR 1.265, 95% CI 1.121–1.517, P<0.001) and END (OR 1.246, 95% CI 1.109–1.498, P<0.001). A receiver operating characteristic curve analysis showed that plasma pNF-H concentration predicted 6-month clinical outcomes and END with high area under curves (all P<0.001). The predictive value of pNF-H was similar to that of the National Institutes of Health Stroke Scale score (all P>0.05). In a combined logistic-regression model, pNF-H did not improve the predictive value of National Institutes of Health Stroke Scale score (all P>0.05). ConclusionsIncreased plasma pNF-H concentration was highly associated with 6-month clinical outcomes and END of patients with intracerebral hemorrhage.

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

BackgroundThe 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....

Plasma neurofilament pNF‐H concentration is not increased in acute equine grass sickness

Although a presumptive diagnosis of acute grass sickness (AGS) can be made on the basis of clinical signs, a definitive ante mortem diagnosis currently requires histological examination of enteric ganglia. Development of an accurate noninvasive ante mortem diagnostic test is therefore warranted. The objective of this study was to determine whether quantification of the plasma concentrations of the heavily phosphorylated form of major neurofilament subunit NF-H (pNF-H), which mirror the degree of axonal degeneration in some human and animal neurodegenerative disorders, could distinguish AGS-affected and control horses. The pNF-H was quantified in plasma from 20 AGS cases and 20 control horses using a commercial enzyme-linked immunosorbent assay kit. Five AGS and 4 control samples had detectable pNF-H concentrations (>0.0759 ng/ml). There was no significant intergroup difference in pNF-H concentrations. It was concluded that plasma pNF-H is not a useful biomarker for the diagnosis of AGS.

Folate is related to phosphorylated neurofilament‐H and P‐tau (Ser396) in rat brain

Protein phosphatase PP2A dephosphorylates phosphorylated tau (P-tau) and neurofilaments (pNFs). PP2A is S-adenosylmethionine (SAM)-dependent and might thus link methylation with neurodegeneration. Low SAM and increased S-adenosylhomocysteine (SAH) can enhance the risk of dementia. We studied the effect of hyperhomocysteinemia on P-tau (Ser396), pNF-H (heavy chain), and PP2A-activity and level (the C subunit) in rat brain. Wistar rats (total n=55) were fed either on a standard, a homocystine 1.7% or a methionine 2.4%-rich diet for 5 months. P-tau was tested in 21 frontal cortex tissue slices using immuno-fluorescence. Concentrations of pNF-H and the activity and level of PP2A were measured in brain extracts. Concentrations of homocysteine, SAM and SAH strongly increased in plasma of rats on the modified diets. The diets caused lowering of plasma folate and vitamin B12 and a significant increase in P-tau (Ser396) in brain tissues but PP2A activity and level were unchanged. Plasma folate correlated to brain tissue PP2A activity (r=0.28), pNF-H (r=-0.30), and P-tau (Ser396) staining (r=-0.57) all p<0.05. Phosphorylation of brain functional proteins was related to folate. The effect of the diet on P-tau and pNF-H seemed not to be explained by a lower activity or protein level of PP2A. Folate might prove protective against multiple steps in the process of neurodegeneration.

Biomarkers of Brain Injury in Foals with Hypoxic‐Ischemic Encephalopathy

Neonatal hypoxic-ischemic encephalopathy (NHIE) is a disease affecting newborn foals for which there is no antemortem diagnostic test. Ubiquitin C-terminal hydrolase 1 (UCHL1) and the phosphorylated axonal forms of neurofilament H (pNF-H) are markers of brain injury in foals with NHIE. Thirty-three foals with a clinical diagnosis consistent with NHIE and 17 healthy foals. Retrospective study. Concentrations of UCHL1 and pNF-H in plasma were measured by ELISA. The performance of the assays for the diagnosis of NHIE was assessed by receiver operating characteristic curve analysis. Concentrations of UCHL1 and pNF-H were measured throughout the brains of 2 healthy foals. The diagnostic performance of UCHL1 (AUC = 0.86) was significantly higher (P = .001) than that of pNF-H (0.52) for the diagnosis of NHIE. Median concentrations of UCHL1 (6.57 ng/mL; 2.35-11.90 ng/mL) in foals with a clinical diagnosis of NHIE were significantly (P < .001) higher than those of healthy controls (2.52 ng/mL; 1.4-4.01 ng/mL). The right sided reference interval for UCHL1 concentrations in healthy foals was 0-4.01 ng/mL. The sensitivity and specificity of UCHL1 (>4.01 ng/mL) for diagnosis of NHIE were 70% (51-84%) and 94% (72-99%), respectively. UCHL1 concentrations were higher in gray than white matter, while pNF-H concentrations were higher in white than gray matter. UCHL1 has potential as a marker of brain injury in foals with NHIE.

Comparative Study of CSF Neurofilaments in HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis and Other Neurological Disorders

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive CNS disease leading to corticospinal tract degeneration. Various degenerative diseases have increased neurofilament subunit concentration in cerebrospinal fluid (CSF), frequently showing hyperphosphorylation in neurofilaments. The aim of this study was to determine if there were elevated concentrations of neurofilament light subunit (NFL) and phosphorylated forms of neurofilament heavy subunit (PNFH) in HAM/TSP CSF. NF concentrations were compared with those of controls and patients with neurodegenerative diseases associated with other retroviruses (HIV-associated dementia, HAD) and a form of prion disease (familiar Creutzfeldt-Jakob, FCJD). Western blotting of CSF with antibodies against NFL showed two immunoreactive bands of 66 and 59 kDa, the latter probably corresponding to a partially degraded NFL form. The concentration of the 59-kDa form was not different in HAM/TSP compared with controls, but it was significantly increased in HAD and FCJD groups. ELISA assay for PNFH did not show differences among HAM/TSP, HAD, and control groups, while PNFH concentration was significantly elevated in FCJD. Our results show that CSF NFL and PNFH are not molecular markers of axonal damage for HAM/TSP probably due to the slow progression of this disease. NFL phosphorylation studies required previous immunoprecipitation from CSF for mass spectrometric analysis. This preliminary analysis indicated phosphorylation at S472 and at some other residues.