Cerebrospinal Fluid Ubiquitin Research Articles

Elevated cerebrospinal fluid ubiquitin C-terminal hydrolase-L1 levels correlate with phenotypic severity and therapeutic response in Niemann-Pick disease, type C1

BackgroundNiemann-Pick disease, type C1 (NPC1) is an ultrarare, recessive disorder due to pathological variants of NPC1. The NPC1 phenotype is characterized by progressive cerebellar ataxia and cognitive impairment. Although classically a childhood/adolescent disease, NPC1 is heterogeneous with respect to the age of onset of neurological signs and symptoms. While miglustat has shown to be clinically effective, there are currently no FDA approved drugs to treat NPC1. Identification and characterization of biomarkers may provide tools to facilitate therapeutic trials. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is a protein which is highly expressed by neurons and is a biomarker of neuronal damage. We thus measured cerebrospinal fluid (CSF) levels of UCHL1 in individuals with NPC1. MethodsCSF levels of UCHL1 were measured using a Quanterix Neuroplex 4 assay in 94 individuals with NPC1 and 35 age-appropriate comparison samples. Cross-sectional and longitudinal CSF UCHL1 levels were then evaluated for correlation with phenotypic measures and treatment status. ResultsCSF UCHL1 levels were markedly elevated (3.3-fold) in individuals with NPC1 relative to comparison samples. The CSF UCHL1 levels showed statistically significant (adj p < 0.0001), moderate, positive correlations with both the 17- and 5-domain NPC Neurological Severity Scores and the Annual Severity Increment Scores. Miglustat treatment significantly decreased (adj p < 0.0001) CSF UCHL1 levels by 30% (95% CI 17–40%). ConclusionsCSF UCHL1 levels are elevated in NPC1, increase with increasing clinical severity and decrease in response to therapy with miglustat. Based on these data, UCHL1 may be a useful biomarker to monitor disease progression and therapeutic response in individuals with NPC1.

Endo-lysosomal protein concentrations in CSF from patients with frontotemporal dementia caused by CHMP2B mutation.

Increasing evidence implicates proteostatic dysfunction as an early event in the development of frontotemporal dementia (FTD). This study aimed to explore potential cerebrospinal fluid (CSF) biomarkers associated with the proteolytic systems in genetic FTD caused by CHMP2B mutation. Combining solid-phase extraction and parallel reaction monitoring mass spectrometry, a panel of 47 peptides derived from 20 proteins was analyzed in CSF from 31 members of the Danish CHMP2B-FTD family. Compared with family controls, mutation carriers had significantly higher levels of complement C9, lysozyme and transcobalamin II, and lower levels of ubiquitin, cathepsin B, and amyloid precursor protein. Lower CSF ubiquitin concentrations in CHMP2B mutation carriers indicate that ubiquitin levels relate to the specific disease pathology, rather than all-cause neurodegeneration. Increased lysozyme and complement proteins may indicate innate immune activation. Altered levels of amyloid precursor protein and cathepsins have previously been associated with impaired lysosomal proteolysis in FTD. CSF markers of proteostasis were explored in CHMP2B-mediated frontotemporal dementia (FTD).31 members of the Danish CHMP2B-FTD family were included.We used solid-phase extraction and parallel reaction monitoring mass spectrometry.Six protein levels were significantly altered in CHMP2B-FTD compared with controls.Lower CSF ubiquitin levels in patients suggest association with disease mechanisms.

Evaluation of cerebrospinal fluid ubiquitin C-terminal hydrolase-L1, glial fibrillary acidic protein, and neurofilament light protein as novel markers for the diagnosis of neurosyphilis among HIV-negative patients

To evaluate the possibility of using cerebrospinal fluid (CSF) ubiquitin C-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), and neurofilament light protein (NF-L) for the diagnosis of neurosyphilis (NS). A cross-sectional study of 576 subjects was conducted at Zhongshan Hospital from January 2021 to August 2022 to evaluate the diagnostic accuracy of CSF UCH-L1, GFAP, and NF-L for NS and analyze their correlations with CSF rapid plasma reagin (RPR), white blood cells (WBCs), and protein. Patients with NS had higher CSF UCH-L1, GFAP, and NF-L levels than patients with syphilis/non-NS and nonsyphilis. Using a cut-off point of 652.25 pg/ml, 548.89 pg/ml, and 48.38 pg/ml, CSF UCH-L1, GFAP, and NF-L had a sensitivity of 85.11%, 76.60%, and 82.98%, with a specificity of 92.22%, 85.56%, and 91.11%, respectively, for NS diagnosis. Moreover, parallel and serial testing algorithms improved their sensitivity and specificity to 93.62% and 98.89%, respectively. Interestingly, levels between patients with NS who are CSF RPR-positive and -negative did not differ and showed a weak or moderate correlation with WBC and CSF protein in patients with syphilis. CSF UCH-L1, GFAP, and NF-L can be used as novel markers for the diagnosis of NS, independent of CSF RPR, WBC, and proteins.

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.

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 (7-10years post injury) and the extent to which cerebrospinal fluid (CSF) biomarkers enhance outcome prediction. 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 7-10years 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. 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 (β = 0.488 p = 0.000) as well as reduced cognitive independence (β = - 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 (β = 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 7-10years post injury (p = 0.008). Higher acute UCH-L1 concentrations were predictive of poorer cognitive performance (β = - 0.496, p = 0.029), whereas higher acute MAP-2 concentrations demonstrated a strong cognitive protective effect (β = 0.679, p = 0.010). 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 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.

The value of cerebrospinal fluid ubiquitin C-terminal hydrolase-L1 protein as a prognostic predictor of neurologic outcome in post-cardiac arrest patients treated with targeted temperature management

AimWe evaluated the prognostic value of serum- and cerebrospinal fluid (CSF)-ubiquitin carboxyl-terminal esterase L1 protein (UCHL1) measurements in post- post-out of hospital cardiac arrest (OHCA) patients treated with target temperature management (TTM), to predict neurologic outcome. MethodsThis was a prospective single-centre observational cohort study, conducted from April 2018 to September 2019. Serum- and CSF-UCHL1 were obtained immediately (UCHL1initial), 24 h (UCHL124), 48 h (UCHL148), and 72 h (UCHL172) after return of spontaneous circulation (ROSC). The area under the receiver operating characteristic curves (AUROC) and Delong method were used to identify cut-off values of serum- and CSF-UCHL1initial, UCHL124, UCHL148, UCHL172 for predicting neurologic outcomes. ResultsOf 38 patients enrolled, 16 comprised the poor outcome group. The AUROCs for serum- and CSF-UCHL1initial were 0.71 and 0.93 in predicting poor neurological outcomes, respectively (p = 0.01). The AUROCs for serum- and CSF-UCHL124 were 0.85 and 0.91 (p = 0.24). The AUROCs for serum- and CSF-UCHL148 were 0.90 and 0.97 (p = 0.07). The AUROCs for serum- and CSF-UCHL172 were 0.94 and 0.98 (p = 0.25). ConclusionFindings of this study demonstrate that CSF-UCHL1 measured immediately, 24, 48, and 72 h after ROSC is a valuable predictor for evaluating neurologic outcomes, whereas serum-UCHL1 measured at 24, 48, and 72 h after ROSC showed a significant performance in the prognostication of poor outcomes in post-OHCA patients treated with TTM.

CSF Ubiquitin Levels Are Higher in Alzheimer's Disease than in Frontotemporal Dementia and Reflect the Molecular Subtype in Prion Disease.

Disturbances in the ubiquitin-proteasome system seem to play a role in neurodegenerative dementias (NDs). Previous studies documented an increase of cerebrospinal fluid (CSF) free monoubiquitin in Alzheimer’s disease (AD) and Creutzfeldt–Jakob disease (CJD). However, to date, no study explored this biomarker across the heterogeneous spectrum of prion disease. Using a liquid chromatography−multiple reaction monitoring mass spectrometry, we investigated CSF free monoubiquitin in controls (n = 28) and in cases with prion disease (n = 84), AD (n = 38), and frontotemporal dementia (FTD) (n = 30). Furthermore, in CJD subtypes, we evaluated by immunohistochemistry (IHC) the relative extent of brain ubiquitin deposits. Prion disease and, to a lesser extent, AD subjects showed increased levels of CSF free monoubiquitin, whereas FTD cases had median protein values similar to controls. The biomarker showed a good to optimal accuracy in the differential diagnosis between NDs and, most interestingly, between AD and FTD. After stratification, according to molecular subtypes, sporadic CJD VV2 demonstrated significantly higher levels of CSF ubiquitin and more numerous brain ubiquitin deposits at IHC in comparison to the typical and most prevalent MM(V)1 subtype. Moreover, CSF ubiquitin correlated with biomarkers of neurodegeneration and astrogliosis in NDs, and was associated with disease stage but not with survival in prion disease. The differential increase of CSF free monoubiquitin in prion disease subtypes and AD may reflect common, though disease and time-specific, phenomena related to neurodegeneration, such as neuritic damage, dysfunctional proteostasis, and neuroinflammation.

Plasma ubiquitin C-terminal hydrolase L1 levels reflect disease stage and motor severity in Parkinson's disease.

Parkinson’s disease (PD) is characterized by Lewy bodies containing α-synuclein and ubiquitin aggregates, their co-occurrence possibly linked to a failure of the ubiquitin proteasome system. Ubiquitin C-terminal hydrolase L1 (UCHL1) plays an important role in maintenance of nervous system integrity, and overexpression of UCHL1 has been shown to increase ubiquitin levels within neurons. While cerebrospinal fluid ubiquitin levels were reported to be lower in PD vs controls, plasma UCHL1 levels and their relationship with clinical measures in PD has not been reported. We measured plasma UCHL1 levels using single molecule array (Simoa) in 291 subjects (242 PD and 49 healthy controls, HC). We found that UCHL1 levels were significantly higher in PD patients at moderate stages (Hoehn and Yahr, H&Y stage >2) vs milder PD (H&Y ≤2, p<0.001) and HC (p=0.001). There was no significant difference in UCHL1 levels between PD patients at H&Y stages ≤2 vs HC. Across all PD patients, UCHL1 correlated significantly with UPDRS Part III motor scores (β=3.87, 95% CI=0.43-7.31, p=0.028), but not with global cognition. Overall, we found that UCHL1 correlates with motor function in PD, with higher levels seen in later disease stages. These findings will be validated in longitudinal studies.

Mass Spectrometric Analysis of Cerebrospinal Fluid Ubiquitin in Alzheimer's Disease and Parkinsonian Disorders.

PurposeDysfunctional proteostasis, with decreased protein degradation and an accumulation of ubiquitin into aggregated protein inclusions, is a feature of neurodegenerative diseases. Identifying new potential biomarkers in cerebrospinal fluid (CSF) reflecting this process could contribute important information on pathophysiology.Experimental designA developed method combining SPE and PRM‐MS is employed to monitor the concentration of ubiquitin in CSF from subjects with Alzheimer's disease (AD), Parkinson's disease (PD), and progressive supranuclear palsy (PSP). Four independent cross‐sectional studies are conducted, studies 1–4, including controls (n = 86) and participants with AD (n = 60), PD (n = 15), and PSP (n = 11).ResultsThe method shows a repeatability and intermediate precision not exceeding 6.1 and 7.9%, respectively. The determined LOD is 0.1 nm and the LOQ range between 0.625 and 80 nm. The CSF ubiquitin concentration is 1.2–1.5‐fold higher in AD patients compared with controls in the three independent AD‐control studies (Study 1, p < 0.001; Study 2, p < 0.001; and Study 3, p = 0.003). In the fourth study, there is no difference in PD or PSP, compared to controls.Conclusion and clinical relevanceCSF ubiquitin may reflect dysfunctional proteostasis in AD. The described method can be used for further exploration of ubiquitin as a potential biomarker in neurodegenerative diseases.

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

Electrophoretically mediated microanalysis was implemented for on-line fluorescence derivatization of ubiquitin (UBI), a potential biomarker of Alzheimer disease. Ubiquitin was labeled on its amino groups by the Fluoprobes® 488 N-hydroxysuccinimide. The pH of the BGE, the applied electric field, and the washing process of the capillary were optimized. The best derivatization yield was obtained at pH 9.5 under an electric field of ∼210 V/cm. The concentration and volume of the fluorescent dye as well as the sample medium and volume of injected UBI were also optimized. In order to improve further the LOD of UBI, electrophoretically mediated microanalysis was performed in a wider (100 μm id) and longer (110 cm) capillary. As expected, these capillary dimensions improved the analytical sensitivity of UBI when compared to the 50 μm id capillary. Under the optimized conditions, this analytical methodology allowed the analysis of free UBI in standard solution with a LOD of ∼15 nM. Finally, the on-line fluorescent derivatization of UBI was applied to the detection and the quantification of UBI in cerebrospinal fluid (CSF) samples. Due to the high salt concentration of biological samples, desalting of CSF was required prior to the CE analysis. Quantification of UBI in CSF either by a direct evaluation of peak areas or using the standard addition method was achieved.

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

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. 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. 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. Our results suggest that UCH-L1 may serve as a novel biomarker for neuronal damage after epileptic seizure.

Identify Biomarkers of Neuropsychiatric Systemic Lupus Erythematosus by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Combined with Weak Cation Magnetic Beads

To identify proteomic biomarkers in cerebrospinal fluid (CSF) and develop a diagnostic proteomic model for neuropsychiatric systemic lupus erythematosus (NPSLE). CSF proteomic spectra were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with weak cation exchange (WCX) magnetic beads. The spectra were taken from 27 patients with NPSLE before and after treatment, and 27 controls including 17 patients with scoliosis and 10 patients with SLE but without neuropsychiatric manifestation. Discriminating peaks were processed by Biomarker Patterns Software to build a decision tree model for NPSLE classification. In addition, CSF samples of 12 patients with NPSLE, 12 patients with lumbar disc herniation, and 9 patients with other neurological conditions were used as a blind test group to verify the accuracy of the model. Twelve discriminating mass-to-charge (m/z) peaks were identified between NPSLE and controls: m/z peaks 7740, 11962, 8065, 7661, 6637, 5978, 11384, 11744, 8595, 10848, 7170, and 5806. The diagnostic decision tree model, built with a panel of m/z peaks 8595, 7170, 7661, 7740, and 5806, recognized NPSLE with both sensitivity and specificity of 92.6%, based on training group samples, and sensitivity and specificity of 91.7% and 85.7%, respectively, based on the blind test group. In addition, the root node m/z peak 8595 protein, which was downregulated in the CSF of patients with NPSLE after treatment, was identified and confirmed as ubiquitin by immunoprecipitation and ELISA. Potential CSF biomarkers for NPSLE are identified by MALDI-TOF-MS combined with WCX magnetic beads. The novel diagnostic proteomic model with m/z peaks 8595, 7170, 7661, 7740, and 5806 is highly sensitive and relatively specific for NPSLE diagnosis. The level of ubiquitin in CSF is a promising biomarker for active NPSLE.

Ubiquitin as potential cerebrospinal fluid marker of Creutzfeldt–Jakob disease

Until today, a definite diagnosis of Creutzfeldt-Jakob disease (CJD) can only be made neuropathologically. At lifetime the early and differential diagnosis is often a problem. With SELDI we analyzed cerebrospinal fluid (CSF) from 32 CJD patients, 32 patients having other dementive diseases and 31 non-demented control subjects for diagnosis-dependent protein pattern differences. In a screening set of patients, peaks that discriminate best between groups were identified. These peaks were subsequently analyzed using an independent validation set of patients. Diagnostic accuracies were compared with established markers like tau protein and 14-3-3-protein. Potential marker proteins were purified and identified by LC-MS/MS. In the validation set only one peak of 8.6 kDa out of ten in the screening set could be confirmed. This protein was identified to be ubiquitin and increased levels in CSF (but not in serum) of CJD patients were confirmed by Western blot. Ubiquitin allows the correct diagnoses of that CJD cases missed by tau protein or 14-3-3-protein. We conclude that ubiquitin is a promising additional CSF biomarker for diagnosis of CJD, especially in differential diagnostically difficult cases. The selective increase of ubiquitin in CSF of CJD patients might point to an involvement of ubiquitin in pathophysiological process.

Ubiquitin reduces fluid shifts after traumatic brain injury

Ubiquitin has well-described intracellular properties. Recent data also suggest pleiotropic effects of extracellular ubiquitin, including induction of apoptosis, regulation of immune functions, and therapeutic potential during fluid resuscitation from severe trauma. However, the actions of exogenous ubiquitin after traumatic brain injury (TBI) are unknown. Series 1: Thirty-five minutes after TBI and hemorrhage, 1.5 mg ubiquitin/kg (n = 5) or albumin (n = 5) intravenous was followed by fluid resuscitation to maintain mean arterial and cerebral perfusion pressure. Series 2: Ubiquitin (n = 5) or vehicle (n = 6) was administered after TBI only. Ubiquitin was measured with enzyme-linked immunosorbent assay in serum, urine (series 1), and cerebrospinal fluid (series 2) for 300 minutes. Series 1: After intravenous bolus, serum ubiquitin peaked at t = 45 minutes with a half-life of 54 minutes. Recovery in urine was 10%. With albumin versus ubiquitin, 85% more resuscitation fluid was required to stabilize systemic and cerebral hemodynamics (P < .05 for t = 150 to 300 minutes), but hematocrit was similar. With albumin there were progressive increases in intracranial pressure, peak inspiratory pressure, and decreases in oxygenation. All were significantly attenuated by ubiquitin (all P < .05 vs albumin). Series 2: Intravenous ubiquitin altered cerebrospinal fluid ubiquitin with an increased time to peak (t = 88 +/- 13 min vs 45 +/- 7 min, P < .05) and area under the concentration-time curve (82 +/- 22 vs 23 +/- 11 microg/min(1)/mL(-1), P < .05). After TBI, intravenous ubiquitin crossed the blood-brain barrier and significantly reduced third spacing of fluid into the brain and lung during resuscitation.

Ubiquitin immunoreactivity in cerebrospinal fluid after traumatic brain injury: Clinical and experimental findings

Recent data indicate that ubiquitin is increased in serum after trauma and might regulate immune functions. Its cellular source is unknown. Because there have been no previous studies after traumatic brain injury (TBI), we determined whether ubiquitin immunoreactivity is increased in cerebrospinal fluid (CSF) after TBI. Prospective observational study of patients, with a subsequent interventional study of animals. The subjects were 14 patients with TBI, five patients with nontraumatic subarachnoid hemorrhage, ten nonneurologic controls, and seven cross-bred swine. Standardized TBI. Ubiquitin immunoreactivity was analyzed by enzyme-linked immunosorbent assay and immunoblotting. Hemolysis was assessed spectrophotometrically. CSF ubiquitin levels (mean +/- sd) were 19 +/- 3 ng/mL in nonneurologic control patients, 81 +/- 48 ng/mL at 7 +/- 2 hrs after TBI (p = .002), and at the end of operation in patients with nontraumatic subarachnoid hemorrhage they were 104 +/- 68 ng/mL (p = .001). CSF and serum ubiquitin were measured for 7 days in six patients with TBI. In survivors (n = 3), CSF ubiquitin levels progressively recovered, whereas in nonsurvivors (n = 3), the levels increased until death. There was no difference in serum ubiquitin levels between survivors/nonsurvivors and there was no correlation between serum and CSF ubiquitin levels. In swine, CSF ubiquitin levels peaked at 8- to 30-fold higher than baseline at 60 min post-TBI and then declined with a half-life of 1.3 hrs. In CSF with hemolysis, peak ubiquitin levels were five-fold higher than without hemolysis (p < .05). Ubiquitin and hemoglobin correlations in CSF and after in vitro lysis of erythrocytes suggested that erythrolysis could account for no more than 23 +/- 16% of the CSF ubiquitin. CSF ubiquitin levels are increased more than four-fold in patients after TBI and nontraumatic subarachnoid hemorrhage. Peak CSF ubiquitin measurements in patients with TBI probably underestimated the actual peak, on the basis of data from the animal model. The progressive rise in CSF ubiquitin in patients with TBI who died suggests that lack of clearance could reflect lethal progression to irreversible brain damage. Erythrolysis is one potential source of CSF ubiquitin.

A Population-based Study of tau Protein and Ubiquitin in Cerebrospinal Fluid in 85-year-olds: Relation to Severity of Dementia and Cerebral Atrophy, but not to the Apolipoprotein E4 Allele

Alzheimer's disease (AD) is the most common form of dementia, and is characterized by a degeneration of neurones and their synapses, and a higher number of senile plaques (SP) and neurofibrillary tangles (NFT) compared with that found in non-demented individuals of the same age. NFT are composed of a hyperphosphorylated and ubiquitinated form of tau protein. Previous studies have found that in the cerebrospinal fluid (CSF) both tau and ubiquitin are increased in AD. We examined CSF-tau and CSF-ubiquitin in a population based sample of 85-year-olds, 26 demented (11 with probable Alzheimer's disease (AD), 13 with probable vascular dementia (VAD) and 2 with mixed (AD/VAD) type of dementia) and 35 non-demented individuals. CSF-tau was significantly higher both in the probable AD group (254±113 pg/mL; P<0.01), and in the probable VAD group (247±75 pg/mL; P<0.005), than in the non- demented group (171±78 pg/mL), but did not significantly differ between the probable AD and probable VAD groups. In contrast, CSF-ubiquitin did not significantly differ between the probable AD (100±24 ng/mL), probable VAD (102±16 ng/mL), and non-demented (97±27 ng/mL) groups. CSF-tau increased with increasing severity of dementia ( P<0.001), though no such relation was found for CSF-ubiquitin. Neither CSF-tau nor CSF-ubiquitin differed between patients with or without the apolipoprotein E E4 isoform. Higher CSF-tau and CSF-ubiquitin levels were also associated with increasing degree of cortical and central brain atrophy as measured by computerized tomography. The relationships between CSF-tau and severity of dementia and to brain atrophy suggest that CSF-tau may be used as a measure of neuronal/ axonal degeneration in patients with dementia. We have previously shown a marked increase in both CSF-tau and CSF-ubiquitin in younger patients with AD and VAD. The less pronounced increase in CSF-tau and the lack of difference in CSF-ubiquitin in older patients suggest that the severity of the degenerative process is less in older than in younger demented patients.

Ubiquitin in cerebrospinal fluid: a rapid competitive enzyme-linked immunoflow assay.

The aims of this study were to develop a rapid immunoassay to determine the levels of ubiquitin in cerebrospinal fluid and to establish the ubiquitin levels in the spinal fluid of normal aged individuals. A competitive enzyme-linked immunoflow assay was developed. In this assay, ubiquitin is bound to nitrocellulose membrane, after which the primary antibody-test sample mixture and the enzyme-labeled secondary antibody under vacuum are applied sequentially. The final reaction product is collected in a microtiter plate by suction. This competitive assay requires only approximately 4 h and is potentially useful for determining in biological fluids the levels of any antigen or its antibodies that might be present. Employing this immunoassay, cerebrospinal fluid ubiquitin levels were found to be 147.5 +/- 5.2 ng ml-1 in non-neurological aged cases.

Alzheimer disease: correlation of cerebro-spinal fluid and brain ubiquitin levels

Neurofibrillary degeneration is one of the histopathological hallmarks of Alzheimer disease (AD). Previous studies have shown an association of ubiquitin with the cytoskeletal protein pathology in AD. In the present study, we report (i) the measurement of ubiquitin levels in cerebrospinal fluid (CSF) from histopathologically confirmed AD and control cases, using a new rapid immunoassay, the competitive enzyme-linked immunoflow assay (CELIFA), (ii) the determination of ubiquitin levels in brain tissue taken from the same cases, using a competitive enzyme-linked immunosorbent assay (ELISA), and (iii) an evaluation of the correlation between levels of ubiquitin in CSF and in brain tissue. Ubiquitin levels in CSF of AD and neurological control groups are significantly higher than those of non-neurological aged controls. Ubiquitin levels in brain homogenates of the AD group are significantly higher than those of both non-neurological aged and neurological control groups. The source of this increase in brain ubiquitin in AD is the particulate fraction, because ubiquitin levels in the brain cytosol fraction are the same among the three groups. In AD and non-neurological aged controls, there is a significant positive correlation between ubiquitin levels in CSF and in homogenate of cerebral white matter. In contrast, the correlation in the non-AD neurological control group has a negative tendency. These studies suggest that in AD, elevated levels of ubiquitin in the CSF reflect the increased amount of the protein in the brain and, therefore, can serve as a biomarker of the neuropathology in this disease.

Marked increase in cerebrospinal fluid ubiquitin in Creutzfeldt-Jakob disease

We have established the radioimmunoassay for ubiquitin in the cerebrospinal fluid (CSF) and measured the ubiquitin concentration in CSF from 4 cases of neuropathologically verified Creutzfeldt-Jakob disease (CJD), 10 cases of multi-infarct dementia (MID), 7 cases of senile dementia of Alzheimer type (SDAT), and 18 controls. The normal values were determined to range from 7.3 to 21.0 ng/ml, 14.3 ± 1.1 ng/ml in the mean ± S.E.M. The CSF ubiquitin levels in the cases of MID and SDAT were 16.6 ± 6.4 ng/ml and 21.3 ± 6.1 ng/ml. respectively. In the cases of CJD, the CSF ubiquitin was markedly increased at the early and middle stages of the disease (230.6 ng/ml in Case 1, 107.6 ng/ml in Case 2, 212.5 ng/ml in Case 3, and 377.0 ng/ml in Case 4) and these gradually decreased as the disease progressed. The measurement of CSF ubiquitin seems useful to make an early diagnosis of CJD.

69. Memory disorders, cerebrovascular insufficiency, thrombocyte aggregation and thrombocyte aggregation inhibitors

We have established the radioimmunoassay for ubiquitin in the cerebrospinal fluid (CSF) and measured the ubiquitin concentration in CSF from 4 cases of neuropathologically verified Creutzfeldt-Jakob disease (CJD), 10 cases of multi-infarct dementia (MID), 7 cases of senile dementia of Alzheimer type (SDAT), and 18 controls. The normal values were determined to range from 7.3 to 21.0 ng/ml, 14.3 ± 1.1 ng/ml in the mean ± S.E.M. The CSF ubiquitin levels in the cases of MID and SDAT were 16.6 ± 6.4 ng/ml and 21.3 ± 6.1 ng/ml. respectively. In the cases of CJD, the CSF ubiquitin was markedly increased at the early and middle stages of the disease (230.6 ng/ml in Case 1, 107.6 ng/ml in Case 2, 212.5 ng/ml in Case 3, and 377.0 ng/ml in Case 4) and these gradually decreased as the disease progressed. The measurement of CSF ubiquitin seems useful to make an early diagnosis of CJD.