Evaluating the utility of serum NfL, GFAP, UCHL1 and tTAU as estimates of CSF levels and diagnostic instrument in neuroinflammation and multiple sclerosis.

BACKGROUND: This study aimed to evaluate the utility of neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase L1 (UCHL1) and total tau (tTAU) serum concentrations as approximation for cerebrospinal fluid (CSF) concentrations of the respective biomarkers in the context of neuroinflammation and multiple sclerosis (MS). METHODS: NfL, GFAP, UCHL1 and tTAU concentrations in serum and CSF were measured in 183 patients (122 with neuroinflammatory disease and 61 neurological or somatoform disease controls) using the single molecule array HD-1 analyzer (Quanterix, Boston, MA). Spearman's rank correlations were computed between serum and CSF concentrations. In a second step, the effects of age, BMI, gadolinium-enhancing lesions in MRI, integrity of the blood-brain barrier (BBB) and presence of acute relapse were accounted for by computing partial correlations. The analyses were repeated for a subsample consisting of MS phenotype patients only (n = 118). EDSS, MS disease activity and acute relapse were considered as additional covariates. Receiver operating characteristic (ROC) analysis was performed for each serum/CSF biomarker concentration to assess how well the particular biomarker concentration differentiates MS patients from somatoform disease controls. Correlations between serum and CSF levels as well as area under the curve (AUC) values were compared for the different biomarkers using z-test statistics. RESULTS: Serum concentrations correlated positively with CSF levels for NfL (r = 0.705, p < 0.01) as well as for GFAP (r = 0.259, p < 0.01). Correlation coefficients were significantly higher for NfL than for GFAP (z = 5.492, p < 0.01). We found no significant serum-CSF correlations for UCHL1 or tTAU. After adjusting for covariates, the results remained unchanged. In the analysis focusing only on MS patients, the results were replicated. ROC analysis demonstrated similarly acceptable performance of serum and CSF NfL values in differentiating MS phenotype patients from somatoform disease controls. AUC values were significantly higher for serum and CSF NfL compared to other biomarkers. CONCLUSION: NfL and GFAP but not UCHL1 or tTAU serum concentrations are associated with CSF levels of the respective biomarker. NfL exhibits more robust correlations between its serum and CSF concentrations as compared to GFAP independently from BBB integrity, clinical and radiological covariates. Both serum and CSF NfL values differentiate between MS and controls.

Predicting Clinical Outcomes 7-10 Years after Severe Traumatic Brain Injury: Exploring the Prognostic Utility of the IMPACT Lab Model and Cerebrospinal Fluid UCH-L1 and MAP-2.

BACKGROUND: Severe traumatic brain injury (TBI) is a major contributor to disability and mortality in the industrialized world. Outcomes of severe TBI are profoundly heterogeneous, complicating outcome prognostication. Several prognostic models have been validated for acute prediction of 6-month global outcomes following TBI (e.g., morbidity/mortality). In this preliminary observational prognostic study, we assess the utility of the International Mission on Prognosis and Analysis of Clinical Trials in TBI (IMPACT) Lab model in predicting longer term global and cognitive outcomes (7-10 years post injury) and the extent to which cerebrospinal fluid (CSF) biomarkers enhance outcome prediction. METHODS: Very long-term global outcome was assessed in a total of 59 participants (41 of whom did not survive their injuries) using the Glasgow Outcome Scale-Extended and Disability Rating Scale. More detailed outcome information regarding cognitive functioning in daily life was collected from 18 participants surviving to 7-10 years post injury using the Cognitive Subscale of the Functional Independence Measure. A subset (n = 10) of these participants also completed performance-based cognitive testing (Digit Span Test) by telephone. The IMPACT lab model was applied to determine its prognostic value in relation to very long-term outcomes as well as the additive effects of acute CSF ubiquitin C-terminal hydrolase-L1 (UCH-L1) and microtubule associated protein 2 (MAP-2) concentrations. RESULTS: The IMPACT lab model discriminated favorable versus unfavorable 7- to 10-year outcome with an area under the receiver operating characteristic curve of 0.80. Higher IMPACT lab model risk scores predicted greater extent of very long-term morbidity (ß = 0.488 p = 0.000) as well as reduced cognitive independence (ß = - 0.515, p = 0.034). Acute elevations in UCH-L1 levels were also predictive of lesser independence in cognitive activities in daily life at very long-term follow-up (ß = 0.286, p = 0.048). Addition of two CSF biomarkers significantly improved prediction of very long-term neuropsychological performance among survivors, with the overall model (including IMPACT lab score, UCH-L1, and MAP-2) explaining 89.6% of variance in cognitive performance 7-10 years post injury (p = 0.008). Higher acute UCH-L1 concentrations were predictive of poorer cognitive performance (ß = - 0.496, p = 0.029), whereas higher acute MAP-2 concentrations demonstrated a strong cognitive protective effect (ß = 0.679, p = 0.010). CONCLUSIONS: Although preliminary, results suggest that existing prognostic models, including models with incorporation of CSF markers, may be applied to predict outcome of severe TBI years after injury. Continued research is needed examining early predictors of longer-term outcomes following TBI to identify potential targets for clinical trials that could impact long-ranging functional and cognitive outcomes.

Characterization and standardization of multiassay platforms for four commonly studied traumatic brain injury protein biomarkers: a TBI Endpoints Development Study.

Aim: There is a critical need to validate biofluid-based biomarkers as diagnostic and drug development tools for traumatic brain injury (TBI). As part of the TBI Endpoints Development Initiative, we identified four potentially predictive and pharmacodynamic biomarkers for TBI: astroglial markers GFAP and S100B and the neuronal markers UCH-L1 and Tau. Materials & methods: Several commonly used platforms for these four biomarkers were identified and compared on analytic performance and ability to detect gold standard recombinant protein antigens and to pool control versus TBI cerebrospinal fluid (CSF). Results: For each marker, only some assay formats could differentiate TBI CSF from the control CSF. Also, different assays for the same biomarker reported divergent biomarker values for the same biosamples. Conclusion: Due to the variability of TBI marker assay in performance and reported values, standardization strategies are recommended when comparing reported biomarker levels across assay platforms.

Lay abstract Traumatic brain injury (TBI) is a leading cause of mortality and morbidity around the world. There is a critical need to validate biofluid-based biomarker tests as diagnostic and drug development tools. For this study, we focused on four brain-derived proteins called GFAP, S100B, UCH-L1 and Tau. To measure these biomarker proteins in human biofluid, one relies on either commercial or home-brew assays. Here, we attempted to compare the performance of 2­4 assay formats for each biomarker. We compared their assay sensitivity, ability to detect 'gold standard' protein analyte we procured, as well as the ability to differentiated pooled TBI cerebrospinal fluid from healthy control cerebrospinal fluid. We found that there are high variabilities among TBI marker assays in assay performance, reported biomarker values and ability to differentiate TBI versus control biofluid. Thus, a standardization strategy is needed when comparing reported biomarker levels across assay platforms.

Characterization of Cerebrospinal Fluid Ubiquitin C-Terminal Hydrolase L1 as a Biomarker of Human Acute Traumatic Spinal Cord Injury.

A major obstacle for translational research in acute spinal cord injury (SCI) is the lack of biomarkers that can objectively stratify injury severity and predict outcome. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a neuron-specific enzyme that shows promise as a diagnostic biomarker in traumatic brain injury (TBI), but has not been studied in SCI. In this study, cerebrospinal fluid (CSF) and serum samples were collected over the first 72-96 h post-injury from 32 acute SCI patients who were followed prospectively to determine neurological outcomes at 6 months post-injury. UCH-L1 concentration was measured using the Quanterix Simoa platform (Quanterix, Billerica, MA) and correlated to injury severity, time, and neurological recovery. We found that CSF UCH-L1 was significantly elevated by 10- to 100-fold over laminectomy controls in an injury severity- and time-dependent manner. Twenty-four-hour post-injury CSF UCH-L1 concentrations distinguished between American Spinal Injury Association Impairment Scale (AIS) A and AIS B, and AIS A and AIS C patients in the acute setting, and predicted who would remain "motor complete" (AIS A/B) at 6 months with a sensitivity of 100% and a specificity of 86%. AIS A patients who did not improve their AIS grade at 6 months post-injury were characterized by sustained elevations in CSF UCH-L1 up to 96 h. Similarly, the failure to gain >8 points on the total motor score at 6 months post-injury was associated with higher 24-h CSF UCH-L1. Unfortunately, serum UCH-L1 levels were not informative about injury severity or outcome. In conclusion, CSF UCH-L1 in acute SCI shows promise as a biomarker to reflect injury severity and predict outcome.

UCHL1 from serum and CSF is a candidate biomarker for amyotrophic lateral sclerosis.

OBJECTIVE: To identify potential ALS biomarkers in patients and to evaluate their diagnostic performance using cerebrospinal fluid (CSF) and serum. METHOD: We recruited a discovery cohort, comprising 20 ALS patients and 20 controls to screen for potential CSF biomarker, UCHL1, using a Luminex neurodegenerative disease panel. To validate UCHL1's diagnostic performance, we used receiver operating characteristic (ROC) curves to determine the potential for early diagnosis in another cohort comprising 23 CSF and 69 serum ALS samples. Finally, we analyzed its correlation with clinical features. RESULTS: We found significantly elevated levels of CSF-derived UCHL1 in both discovery and validation cohorts (P < 0.05). ROC curves revealed an AUC of 0.8288, with a sensitivity and specificity of 73.91% and 81.25%, respectively, when the cut-off value for UCHL1 was >291.9 pg/mL. A similar result was observed in the serum cohort, with the ALS group exhibiting significantly higher serum UCHL1 levels than the controls (P < 0.05). AUC of the ROC in the serum UCHL1 cohort was 0.7709, with sensitivity and specificity of 61.43% and 79.59%, respectively, when the cut-off value of serum UCHL1 was >15.22 pg/mL. At the early stage CSF and serum UCHL1 were significantly different between ALS patients and controls (P < 0.05). Furthermore, serum UCHL1 levels showed a positive relationship with the burden of UMN and LMN dysfunction, albeit with no statistical significance. INTERPRETATION: Taken together, our findings suggest that ALS patients exhibit significantly elevated CSF- and serum-derived UCHL1. Moreover, our data warrant that UCHL1 displays good diagnostic performance and provide novel options for ALS early diagnosis. However, its prognostic value needs to be further investigated.

Early predictors of perinatal brain damage: the role of neurobiomarkers.

The early detection of perinatal brain damage in preterm and term newborns (i.e. intraventricular hemorrhage, periventricular leukomalacia and perinatal asphyxia) still constitute an unsolved issue. To date, despite technological improvement in standard perinatal monitoring procedures, decreasing the incidence of perinatal mortality, the perinatal morbidity pattern has a flat trend. Against this background, the measurement of brain constituents could be particularly useful in the early detection of cases at risk for short-/long-term brain injury. On this scenario, the main European and US international health-care institutions promoted perinatal clinical and experimental neuroprotection research projects aimed at validating and including a panel of biomarkers in the clinical guidelines. Although this is a promising attempt, there are several limitations that do not allow biomarkers to be included in standard monitoring procedures. The main limitations are: (i) the heterogeneity of neurological complications in the perinatal period, (ii) the small cohort sizes, (iii) the lack of multicenter investigations, (iv) the different techniques for neurobiomarkers assessment, (iv) the lack of consensus for the validation of assays in biological fluids such as urine and saliva, and (v), the lack of reference curves according to measurement technique and biological fluid. In the present review we offer an up-to-date overview of the most promising developments in the use of biomarkers in the perinatal period such as calcium binding proteins (S100B protein), vasoactive agents (adrenomedullin), brain biomarkers (activin A, neuron specific enolase, glial fibrillary acidic protein, ubiquitin carboxyl-terminal hydrolase-L1) and oxidative stress markers.

Targeted Multiple Reaction Monitoring Analysis of CSF Identifies UCHL1 and GPNMB as Candidate Biomarkers for ALS.

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) share some common molecular deficits including disruption of protein homeostasis leading to disease-specific protein aggregation. While insoluble protein aggregates are the defining pathological confirmation of diagnosis, patient stratification based on early molecular etiologies may identify distinct subgroups within a clinical diagnosis that would respond differently in therapeutic development programs. We are developing targeted multiple reaction monitoring (MRM) mass spectrometry methods to rigorously quantify CSF proteins from known disease genes involved in lysosomal, ubiquitin-proteasomal, and autophagy pathways. Analysis of CSF from 21 PD, 21 ALS, and 25 control patients, rigorously matched for gender, age, and age of sample, revealed significant changes in peptide levels between PD, ALS, and control. In patients with PD, levels of two peptides for chromogranin B (CHGB, secretogranin 1) were significantly reduced. In CSF of patients with ALS, levels of two peptides from ubiquitin carboxy-terminal hydrolase like protein 1 (UCHL1) and one peptide each for glycoprotein non-metastatic melanoma protein B (GPNMB) and cathepsin D (CTSD) were all increased. Analysis of patients with ALS separated into two groups based on length of survival after CSF sampling revealed that the increases in GPNMB and UCHL1 were specific for short-lived ALS patients. While analysis of additional cohorts is required to validate these candidate biomarkers, this study suggests methods for stratification of ALS patients for clinical trials and identifies targets for drug efficacy measurements during therapeutic development.

Single-step functionalization of poly-catecholamine nanofilms for ultra-sensitive immunosensing of ubiquitin carboxyl terminal hydrolase-L1 (UCHL-1) in spinal cord injury.

Rapid, selective, and ultra-sensitive detection of brain and spinal cord injury markers in bodily fluids is an unmet clinical need. In this work, Polycatecholamine as a rich source of amine moieties was used for single-step fabrication of ultrasensitive immunosensors for the detection of Ubiquitin carboxyl-terminal hydrolase (UCHL-1) biomarker of brain and spinal cord injuries and address the clinical need. The surface of graphene electrodes was modified by electropolymerizing aqueous solution of dopamine (DA) and norepinephrine (NE) monomers for generating polycatecholamines nanofilms on the surface of graphene screen printed electrodes (GSPE) in a single functionalization step. Amine moieties of the polymer allowed immobilization of UCHL-1 antibody on the electrode. The single-step modification of GSPE offered a simple, ultrasensitive, and stable production of immunosensors for the detection of UCHL-1. The operational range of the UCHL-1 immunosensor developed with Polynorepinephrine pNE-modified is 0.1 pg mL-1 - 105 pg mL-1 (LOD: 1.91 pg mL-1), and 1 pg mL-1 - 105 pg mL-1 (LOD: 0.70 pg mL-1) with Polydopamine (pDA) modification, satisfying the clinical range. Both pNE and pDA modified immunosensors, detected UCHL-1 spiked in phosphate buffer saline, artificial cerebrospinal fluid, and serum. Along with the sensitive detections, selective performances were recorded in the above matrices in the presence of interfering neurotransmitters GABA and Glutamate as well as glial fibrillary acidic protein (GFAP). Upon testing clinical samples of spinal cord injury patients and healthy controls, both pNE and pDA immunosensors, delivered a comparable response for UCHL-1, thereby, making immunosensors useful for clinical settings.

Biofluid biomarkers of traumatic brain injury.

PRIMARY OBJECTIVE: The purpose of this paper is to review the clinical and research utility and applications of blood, cerebrospinal fluid (CSF), and cerebral microdialysis biomarkers in traumatic brain injury (TBI). RESEARCH DESIGN: Not applicable. METHODS AND PROCEDURES: A selective review was performed on these biofluid biomarkers in TBI. MAIN OUTCOME AND RESULTS: Neurofilament heavy chain protein (NF-H), glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCHL1), neuron-specific enolase (NSE), myelin basic protein (MBP), tau, and s100ß blood biomarkers are elevated during the acute phase of severe head trauma but have key limitations in their research and clinical applications to mild TBI (mTBI). CSF biomarkers currently provide the best reflection of the central nervous system (CNS) pathobiological processes in TBI. Both animal and human studies of TBI have demonstrated the importance of serial sampling of biofluids and suggest that CSF biomarkers may be better equipped to characterize both TBI severity and temporal profiles. CONCLUSIONS: The identification of biofluid biomarkers could play a vital role in identifying, diagnosing, and treating the underlying individual pathobiological changes of TBI. CNS-derived exosomes analyzed by ultra-high sensitivity detection methods have the potential to identify blood biomarkers for the range of TBI severity and time course.

Association of ubiquitin carboxy-terminal hydrolase-L1 in cerebrospinal fluid with clinical severity in a cohort of patients with Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is an acute immune-mediated polyneuropathy. Although its pathogenic mechanism has been revealed and various therapeutic trials have been performed, a proportion of patients experience the severe sequelae associated with GBS. In this paper, we investigated whether the amount of the neuron-specific protein, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), in the cerebrospinal fluid of patients with GBS was correlated with the clinical course of the disease. UCH-L1 protein levels were greater in patients with GBS than in controls. The patients with GBS whose UCH-L1 protein levels were higher than those of the controls presented with more severe symptoms at peak. UCH-L1 protein levels tended to become elevated as the total protein levels were increased; however, elevated UCH-L1 without an increase in total protein might be correlated with severe disease course (bedridden or ventilator supported). These results suggest that UCH-L1 could be a biomarker associated with the severity of the disease at the acute phase of GBS.

CSF α-synuclein and UCH-L1 levels in Parkinson's disease and atypical parkinsonian disorders.

INTRODUCTION: There is an unmet need for biomarkers for Parkinson's disease (PD) and atypical parkinsonian disorders (APD). α-Synuclein, linked to the pathogenesis of PD, is a promising biomarker candidate in need of further investigation. The ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a pivotal component of the ubiquitin proteasome system which seems to be disturbed in PD, may also be involved in the pathogenesis of this disorder. METHODS: We investigated cerebrospinal fluid (CSF) α-synuclein and UCH-L1 levels from 22 healthy controls, 52 patients with PD, 34 with multiple system atrophy (MSA), 32 with progressive supranuclear palsy, and 12 with corticobasal degeneration. RESULTS: α-Synuclein levels were significantly decreased in PD and in MSA compared with controls, and in synucleinopathies compared with tauopathies. UCH-L1 levels were significantly decreased in PD, MSA as well as PSP compared with controls, and in PD compared with APD (p < 0.001). Both markers discriminated PD well from controls (p < 0.0001; area under the curve [AUC] = 0.82 and 0.89, respectively). Additionally, CSF α-synuclein separated patients with synucleinopathies from those with tauopathies (p = 0.015; AUC = 0.63), whereas CSF UCH-L1 discriminated between PD and APD (p = 0.0003; AUC = 0.69). Interestingly, α-synuclein and UCH-L1 levels were strongly correlated in PD and synucleinopathies, and weakly in tauopathies. No correlation was found in controls. CONCLUSIONS: CSF levels of α-synuclein and UCH-L1 show distinct patterns in parkinsonian syndromes. Their combined determination may be useful in the differential diagnosis of parkinsonian disorders and provide key to understanding their pathoetiology and clinical course. Further large studies are needed to validate our findings.

Different expression of ubiquitin C-terminal hydrolase-L1 and αII-spectrin in ischemic and hemorrhagic stroke: Potential biomarkers in diagnosis.

The two primary categories of stroke, ischemic and hemorrhagic, both have fundamentally different mechanisms and thus different treatment options. These two stroke categories were applied to rat models to identify potential biomarkers that can distinguish between them. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) without reperfusion while hemorrhagic stroke was induced by injecting collagenase IV into the striatum. Brain hemispheres and biofluids were collected at two time points: 3 and 6h after stroke. Known molecules were tested on the rat samples via quantitative immunoblotting (injured brain, CSF) and Banyan's proprietary ELISA assays (CSF, serum). The injured brain quantitative analyses revealed that αII-spectrin breakdown products (SBDP150, SBDP145) were strongly increased after 6h ischemia. In CSF, SBDP145 and ubiquitin C-terminal hydrolase-L1 (UCH-L1) levels were elevated after 6h ischemic stroke detected by Western blot and ELISA. In serum UCH-L1 levels were increased after 3 and 6h of ischemia detected by ELISA. However, levels of those proteins in hemorrhagic stroke remain normal. In summary, in both the brain and the biofluids, SBDPs and UCH-L1 were elevated after ischemic but not hemorrhagic stroke. These molecules behaved differently in the two stroke models and thus may be capable of being differentiated.

Cerebrospinal fluid and urinary biomarkers in multiple sclerosis.

OBJECTIVES: Biomarkers with the potential for longitudinal measurements are needed in multiple sclerosis (MS). Urine is easy to collect, and repeated sampling is possible. METHODS: 39 paired CSF and urine samples were taken. Oligoclonal bands (OCBs) were measured in CSF. Kappa and lambda free light chain (FLC), neopterin and ubiquitin C-terminal hydrolase-L1 (UCHL1) were measured in CSF and urine. RESULTS: 16/39 samples had OCBs unique to the CSF. CSF FLC levels (P < 0.0001) were higher in OCB-positive subjects, with no difference in urinary FLC. CSF and urinary FLC did not correlate. There were a significant correlation between total CSF FLC and CSF neopterin in MS samples (correlation coefficient = 0.588, P = 0.016) and a strong correlation between CSF lambda FLC and CSF neopterin in MS samples (correlation coefficient = 0.875, P < 0.001). There was a strong correlation between urinary neopterin/creatinine levels and urinary total FLC/protein levels (correlation coefficient = 0.452, P = 0.004). Only three CSF samples (8%) had detectable levels of UCHL1. 18/38 (48%) (8/15 MS and 10/23 control) urine samples had detectable levels of UCLH1. CONCLUSIONS: This study confirms the relationship between CSF OCBs and CSF FLCs, highlighting the importance of intrathecal B- and plasma-cell activation in MS. There is a relationship between CSF FLC and CSF neopterin in MS, highlighting the multifaceted immune activation seen in MS. Correlations in the OCB-positive group highlight the multifaceted immune activation seen in MS. Further studies are required to evaluate CSF and urinary biomarkers.

Cerebrospinal fluid ubiquitin C-terminal hydrolase as a novel marker of neuronal damage after epileptic seizure.

BACKGROUND: Ubiquitin carboxy-terminal hydrolase (UCH-L1) has been established as a reliable and potential biomarker of neuronal damage after acute neurologic insults such as ischemic stroke, subarachnoid hemorrhage, and traumatic brain injury. The effects of seizures on UCH-L1 levels in cerebrospinal fluid (CSF) has not been investigated in epileptic patients. The aim of the present study was to evaluate whether CSF UCH-L1 levels are a reliable marker of brain damage from epileptic seizures. METHODS: Thirty-three patients with epilepsy (mean age 45 years) participated. Twenty-five patients had generalized seizures and eight had partial seizures. CSF was sampled by lumbar puncture. The control samples were obtained from 23 adult patients on whom lumbar puncture was performed to exclude neurological disease. CSF UCH-L1 levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: Patients with epilepsy had significantly elevated CSF levels of UCH-L1 after seizures compared with controls (p<0.001). CSF UCH-L1 levels were significantly higher in patients with generalized seizures than in patients with partial seizures and controls (p<0.001). Moreover, patients with repetitive generalized tonic-clonic (GTC) seizures had higher CSF UCH-L1 levels than those with a single GTC seizure (p<0.001). CSF UCH-L1 levels in seizure patients showed strong correlation with severity of seizures (r=0.56) and seizure duration (r=0.77). Conversely, CSF UCH-L1 levels in seizure patients did not correlate with the age of patients, duration of epilepsy, age of first seizure, time from last seizure or number of seizures. CONCLUSIONS: Our results suggest that UCH-L1 may serve as a novel biomarker for neuronal damage after epileptic seizure.

Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is increased in cerebrospinal fluid and plasma of patients after epileptic seizure.

BACKGROUND: Clinical and experimental studies have demonstrated that seizures can cause molecular and cellular responses resulting in neuronal damage. At present, there are no valid tests for assessing organic damage to the brain associated with seizure. The aim of this study was to investigate cerebrospinal fluid (CSF) and plasma concentrations of Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a sensitive indicator of acute injury to brain neurons, in patients with tonic-clonic or partial secondarily generalized seizures due to various etiologies. METHODS: CSF and plasma concentrations of UCH-L1 were assessed in 52 patients within 48 hours after epileptic seizure and in 19 controls using ELISA assays. RESULTS: CSF obtained within 48 hours after seizure or status epilepticus (SE) presented significantly higher levels of UCH-L1 compared to controls (p = 0.008). Plasma UCH-L1 concentrations were negatively correlated with time to sample withdrawal. An analysis conducted using only the first 12 hours post-seizure revealed significant differences between concentrations of UCH-L1 in plasma and controls (p = 0.025). CSF and plasma concentrations were strongly correlated with age in patients with seizure, but not in control patients. Plasma UCH-L1 levels were also significantly higher in patients after recurrent seizures (n = 4) than in those after one or two seizures (p = 0.013 and p = 0.024, respectively). CONCLUSION: Our results suggest that determining levels of neuronal proteins may provide valuable information on the assessment of brain damage following seizure. These data might allow clinicians to make more accurate therapeutic decisions, to identify patients at risk of progression and, ultimately, to provide new opportunities for monitoring therapy and targeted therapeutic interventions.

Cerebrospinal fluid protein biomarker panel for assessment of neurotoxicity induced by kainic acid in rats.

Glutamate excitotoxicity plays a key role in the etiology of a variety of neurological, psychiatric, and neurodegenerative disorders. The goal of this study was to investigate spatiotemporal distribution in the brain and cerebrospinal fluid (CSF) concentrations of ubiquitin C-terminal hydrolase-1 (UCH-L1), glial fibrillary acidic protein (GFAP), αII-spectrin breakdown products (SBDP150, SBDP145, and SBDP120), and their relationship to neuropathology in an animal model of kainic acid (KA) excitotoxicity. Triple fluorescent labeling and Fluoro-Jade C staining revealed a reactive gliosis in brain and specific localization of degenerating neurons in hippocampus and entorhinal cortex of KA-treated rats. Immunohistochemistry showed upregulation of GFAP expression in hippocampus and cortex beginning 24h post KA injection and peaking at 48h. At these time points concurrent with extensive neurodegeneration all SBDPs were observed throughout the brain. At 24h post KA injection, a loss of structural integrity was observed in cellular distribution of UCH-L1 that correlated with an increase in immunopositive material in the extracellular matrix. CSF levels of UCH-L1, GFAP, and SBDPs were significantly increased in KA-treated animals compared with controls. The temporal increase in CSF biomarkers correlated with brain tissue distribution and neurodegeneration. This study provided evidence supporting the use of CSF levels of glial and neuronal protein biomarkers to assess neurotoxic damage in preclinical animal models that could prove potentially translational to the clinic. The molecular nature of these biomarkers can provide critical information on the underlying mechanisms of neurotoxicity that might facilitate the development of novel drugs and allow physicians to monitor drug safety.

Clinical utility of serum levels of ubiquitin C-terminal hydrolase as a biomarker for severe traumatic brain injury.

BACKGROUND: Brain damage markers released in cerebrospinal fluid (CSF) and blood may provide valuable information about diagnosis and outcome prediction after traumatic brain injury (TBI). OBJECTIVE: To examine the concentrations of ubiquitin C-terminal hydrolase-L1 (UCH-L1), a novel brain injury biomarker, in CSF and serum of severe TBI patients and their association with clinical characteristics and outcome. METHODS: This case-control study enrolled 95 severe TBI subjects (Glasgow Coma Scale [GCS] score, 8). Using sensitive UCH-L1 sandwich ELISA, we studied the temporal profile of CSF and serum UCH-L1 levels over 7 days for severe TBI patients. RESULTS: Comparison of serum and CSF levels of UCH-L1 in TBI patients and control subjects shows a robust and significant elevation of UCH-L1 in the acute phase and over the 7-day study period. Serum and CSF UCH-L1 receiver-operating characteristic curves further confirm strong specificity and selectivity for diagnosing severe TBI vs controls, with area under the curve values in serum and CSF statistically significant at all time points up to 24 hours (P < .001). The first 12-hour levels of both serum and CSF UCH-L1 in patients with GCS score of 3 to 5 were also significantly higher than those with GCS score of 6 to 8. Furthermore, UCH-L1 levels in CSF and serum appear to distinguish severe TBI survivors from nonsurvivors within the study, with nonsurvivors having significantly higher and more persistent levels of serum and CSF UCH-L1. Cumulative serum UCH-L1 levels > 5.22 ng/mL predicted death (odds ratio, 4.8). CONCLUSION: Serum levels of UCH-L1 appear to have potential clinical utility in diagnosing TBI, including correlating to injury severity and survival outcome.

Serum levels of neuron-specific ubiquitin carboxyl-terminal esterase-L1 predict brain injury in a canine model of hypothermic circulatory arrest.

OBJECTIVES: Ubiquitin carboxyl-terminal esterase-L1 (UCHL1) is a protein highly selectively expressed in neurons and has been linked to neurodegenerative disease in humans. We hypothesize that UCHL1 would be an effective serum biomarker for brain injury as tested in canine models of hypothermic circulatory arrest (HCA) and cardiopulmonary bypass (CPB). METHODS: Dogs were exposed to CPB (n = 14), 1 hour of HCA (1h-HCA; n = 11), or 2 hours of HCA (2h-HCA; n = 20). Cerebrospinal fluid and serum were collected at baseline, 8 hours, and 24 hours after treatment. UCHL1 levels were measured using a sandwich enzyme-linked immunosorbent assay. Neurologic function and histopathology were scored at 24 hours, and UCHL1 immunoreactivity was examined at 8 hours. RESULTS: Baseline UCHL1 protein levels in cerebrospinal fluid and serum were similar for all groups. In serum, UCHL1 levels were elevated at 8 hours after treatment for 2h-HCA subjects compared with baseline values (P < .01) and also compared with CPB dogs at 8 hours (P < .01). A serum UCHL1 level above 3.9 ng/(mg total protein) at 8 hours had the best discriminatory power for predicting functional disability. In cerebrospinal fluid, UCHL1 was elevated in all groups at 8 hours after treatment compared with baseline (P < .01). However, UCHL1 levels in cerebrospinal fluid remained elevated at 24 hours only in 2h-HCA subjects (P < .01). Functional and histopathologic scores were closely correlated (Pearson coefficient, 0.66; P < .01) and were significantly worse in 2h-HCA animals. CONCLUSIONS: This is the first report associating elevated serum UCHL1 with brain injury. The novel neuronal biomarker UCHL1 is increased in serum 8 hours after severe neurologic insult in 2h-HCA animals compared with CPB animals. These results support the potential for use in cardiac surgery patients and form the basis for clinical correlation in humans.

Biokinetic analysis of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in severe traumatic brain injury patient biofluids.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a neuron-specific enzyme that has been identified as a potential biomarker of traumatic brain injury (TBI). The study objectives were to determine UCH-L1 exposure and kinetic metrics, determine correlations between biofluids, and assess outcome correlations in severe TBI patients. Data were analyzed from a prospective, multicenter study of severe TBI (Glasgow Coma Scale [GCS] score ≤ 8). Cerebrospinal fluid (CSF) and serum data from samples taken every 6 h after injury were analyzed by enzyme-linked immunosorbent assay (ELISA). UCH-L1 CSF and serum data from 59 patients were used to determine biofluid correlations. Serum samples from 86 patients and CSF from 59 patients were used to determine outcome correlations. Exposure and kinetic metrics were evaluated acutely and up to 7 days post-injury and compared to mortality at 3 months. There were significant correlations between UCH-L1 CSF and serum median concentrations (r(s)=0.59, p<0.001), AUC (r(s)=0.3, p=0.027), Tmax (r(s)=0.68, p<0.001), and MRT (r(s)=0.65, p<0.001). Outcome analysis showed significant increases in median serum AUC (2016 versus 265 ng/mL*min, p=0.006), and Cmax (2 versus 0.4 ng/mL, p=0.003), and a shorter Tmax (8 versus 19 h, p=0.04) in those who died versus those who survived, respectively. In the first 24 h after injury, there was a statistically significant acute increase in CSF and serum median Cmax((0-24h)) in those who died. This study shows a significant correlation between UCH-L1 CSF and serum median concentrations and biokinetics in severe TBI patients, and relationships with clinical outcome were detected.

Ubiquitin C-terminal hydrolase-L1 as a biomarker for ischemic and traumatic brain injury in rats.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), also called neuronal-specific protein gene product 9.5, is a highly abundant protein in the neuronal cell body and has been identified as a possible biomarker on the basis of a recent proteomic study. In this study, we examined whether UCH-L1 was significantly elevated in cerebrospinal fluid (CSF) following controlled cortical impact (CCI) and middle cerebral artery occlusion (MCAO; model of ischemic stroke) in rats. Quantitative immunoblots of rat CSF revealed a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced alphaII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum).