Quantification of total apolipoprotein E and its isoforms in cerebrospinal fluid from patients with neurodegenerative diseases.

BACKGROUND: The human APOE gene, which codes for apolipoprotein E (apoE), has three major polymorphic alleles: ε2, ε3, and ε4 that give rise to amino acid substitutions. APOE-ε4 is a strong risk factor of sporadic Alzheimer's disease (AD) but the reason why is still unknown despite intense research for more than 20 years. The aim of the study was to investigate if the concentrations of total apoE and the specific apoE isoforms in cerebrospinal fluid (CSF) differ between various neurodegenerative diseases and control individuals, as well as among the APOE genotypes. METHODS: Quantification of total apoE and specific apoE isoforms (E2, E3, and E4) in CSF was performed using high-resolution parallel reaction monitoring mass spectrometry. In total, 1820 individuals were involved in the study including clinically diagnosed AD patients (n = 228), cognitively unimpaired (CU) patients (n = 896), and patients with other neurodegenerative disorders (n = 696). Follow-up data was available for 100 individuals, assessed at two time points. Subjects were dichotomized based on an Aß42/40 CSF concentration ratio cut-off into Aß positive (Aß+, < 0.091) and Aß negative (Aß-, > 0.091) groups. RESULTS: Even though there was a significant increase of total apoE in the amyloid ß-positive (Aß+) group compared with amyloid ß-negative (Aß-) individuals (p < 0.001), the magnitude of the effect was very small (AUC = 0.55). Moreover, CSF total apoE concentrations did not differ between Aß- CU controls and clinically diagnosed AD patients. There was a difference in concentration between isoforms in heterozygous individuals in an isoform-dependent manner (E2 < E3 < E4) (p < 0.001, AUC = 0.64-0.69), and these associations remained when dichotomizing the samples into Aß+ and Aß- groups (p < 0.01, AUC = 0.63-0.74). In the cohort with follow-up samples, neither total apoE nor isoform-specific apoE concentrations differed between the two time points (p > 0.05). CONCLUSIONS: The results indicate that neither the concentrations of total apoE nor the different apoE isoforms in CSF are associated with APOE-ε4 carrier status, Aß status, or clinical dementia diagnoses.

Cerebrospinal fluid markers of extracellular matrix remodelling, synaptic plasticity and neuroinflammation before and after cranial radiotherapy.

BACKGROUND: Advances in the treatment of brain tumours have increased the number of long-term survivors, but at the cost of side effects following cranial radiotherapy ranging from neurocognitive deficits to outright tissue necrosis. At present, there are no tools reflecting the molecular mechanisms underlying such side effects, and thus no means to evaluate interventional effects after cranial radiotherapy. Therefore, fluid biomarkers are of great clinical interest. OBJECTIVE: Cerebrospinal fluid (CSF) levels of proteins involved in inflammatory signalling, synaptic plasticity and extracellular matrix (ECM) integrity were investigated following radiotherapy to the brain. METHODS: Patients with small-cell lung cancer (SCLC) eligible for prophylactic cranial irradiation (PCI) were asked to participate in the study. PCI was prescribed either as 2 Gy/fraction to a total dose of 30 Gy (limited disease) or 4 Gy/fraction to 20 Gy (extensive disease). CSF was collected by lumbar puncture at baseline, 3 months and 1 year following PCI. Protein concentrations were measured using immunobased assays or mass spectrometry. RESULTS: The inflammatory markers IL-15, IL-16 and MCP-1/CCL2 were elevated in CSF 3 months following PCI compared to baseline. The plasticity marker GAP-43 was elevated 3 months following PCI, and the same trend was seen for SNAP-25, but not for SYT1. The investigated ECM proteins, brevican and neurocan, showed a decline following PCI. There was a strong correlation between the progressive decline of soluble APPα and brevican levels. CONCLUSION: To our knowledge, this is the first time ECM-related proteins have been shown to be affected by cranial radiotherapy in patients with cancer. These findings may help us to get a better understanding of the mechanisms behind side effects following radiotherapy.

Plasma and cerebrospinal fluid tau and neurofilament concentrations in rapidly progressive neurological syndromes: a neuropathology-based cohort.

BACKGROUND AND PURPOSE: Cerebrospinal fluid (CSF) tau and neurofilament light chain (NF-L) proteins have proved to be reliable biomarkers for neuronal damage; however, there is a strong need for blood-based tests. METHODS: The present study included 132 autopsy cases with rapidly progressive neurological syndromes, including Alzheimer disease (AD) (21), sporadic (65) and genetic (21) Creutzfeldt-Jakob disease (CJD), 25 cases with vascular, neoplastic and inflammatory alterations, and additionally 18 healthy control individuals. CSF tau and NF-L concentrations were measured by enzyme-linked immunosorbent assay. Plasma tau and NF-L concentrations were measured using ultra-sensitive single molecule array technology. RESULTS: Plasma and CSF tau (R = 0.59, P < 0.001) and NF-L (R = 0.69, P < 0.001) levels correlated significantly (Spearman test). Plasma tau and NF-L levels were significantly higher in all disease groups compared to healthy controls (P < 0.001). Receiver operating characteristic curves were used and area under the curve values for comparisons with controls were 0.82 (AD), 0.94 (sporadic CJD), 0.92 (genetic CJD) and 0.83 (other neurological disorders) for plasma tau and 0.99, 0.99, 1.00 and 0.96 for plasma NF-L, respectively. Molecular subtyping of sporadic CJD showed a strong effect (linear logistic regression) on plasma tau (P < 0.001) but not NF-L levels (P = 0.19). CONCLUSION: Plasma tau and NF-L concentrations are strongly increased in CJD and show similar diagnostic performance to the corresponding CSF measure. Molecular subtypes of sporadic CJD show different levels of plasma tau. Although not disease-specific, these findings support the use of plasma tau and NF-L as tools to identify, or to rule out, neurodegeneration.

A targeted proteomic multiplex CSF assay identifies increased malate dehydrogenase and other neurodegenerative biomarkers in individuals with Alzheimer's disease pathology.

Alzheimer's disease (AD) is the most common cause of dementia. Biomarkers are required to identify individuals in the preclinical phase, explain phenotypic diversity, measure progression and estimate prognosis. The development of assays to validate candidate biomarkers is costly and time-consuming. Targeted proteomics is an attractive means of quantifying novel proteins in cerebrospinal and other fluids, and has potential to help overcome this bottleneck in biomarker development. We used a previously validated multiplexed 10-min, targeted proteomic assay to assess 54 candidate cerebrospinal fluid (CSF) biomarkers in two independent cohorts comprising individuals with neurodegenerative dementias and healthy controls. Individuals were classified as 'AD' or 'non-AD' on the basis of their CSF T-tau and amyloid Aß1-42 profile measured using enzyme-linked immunosorbent assay; biomarkers of interest were compared using univariate and multivariate analyses. In all, 35/31 individuals in Cohort 1 and 46/36 in Cohort 2 fulfilled criteria for AD/non-AD profile CSF, respectively. After adjustment for multiple comparisons, five proteins were elevated significantly in AD CSF compared with non-AD CSF in both cohorts: malate dehydrogenase; total APOE; chitinase-3-like protein 1 (YKL-40); osteopontin and cystatin C. In an independent multivariate orthogonal projection to latent structures discriminant analysis (OPLS-DA), these proteins were also identified as major contributors to the separation between AD and non-AD in both cohorts. Independent of CSF Aß1-42 and tau, a combination of these biomarkers differentiated AD and non-AD with an area under curve (AUC)=0.88. This targeted proteomic multiple reaction monitoring (MRM)-based assay can simultaneously and rapidly measure multiple candidate CSF biomarkers. Applying this technique to AD we demonstrate differences in proteins involved in glucose metabolism and neuroinflammation that collectively have potential clinical diagnostic utility.

Cerebrospinal fluid-induced retardation of amyloid ß aggregation correlates with Alzheimer's disease and the APOE ε4 allele.

Misfolding and aggregation of amyloid ß (Aß) are key features of Alzheimer's disease (AD) pathogenesis, but the molecular events controlling this process are not known in detail. In vivo, Aß aggregation and plaque formation occur in the interstitial fluid of the brain extracellular matrix. This fluid communicates freely with cerebrospinal fluid (CSF). Here, we examined the effect of human CSF on Aß aggregation kinetics in relation to AD diagnosis and carrier status of the apolipoprotein E (APOE) ε4 allele, the main genetic risk factor for sporadic AD. The aggregation of Aß was inhibited in the presence of CSF and, surprisingly, the effect was more pronounced in APOE ε4 carriers. However, by fractionation of CSF using size exclusion chromatography, it became evident that it was not the ApoE protein itself that conveyed the inhibition, since the retarding species eluted at lower volume, corresponding to a much higher molecular weight, than ApoE monomers. Cholesterol quantification and immunoblotting identified high-density lipoprotein particles in the retarding fractions, indicating that such particles may be responsible for the inhibition. These results add information to the yet unresolved puzzle on how the risk factor of APOE ε4 functions in AD pathogenesis.

Serum and cerebrospinal fluid urate levels in synucleinopathies versus tauopathies.

BACKGROUND: Low levels of serum urate are associated with a higher risk of Parkinson's disease (PD). Higher serum and cerebrospinal fluid (CSF) urate levels are associated with slower rates of clinical decline in PD and in multiple system atrophy (MSA). AIMS: To compare CSF and blood urate levels in healthy controls, patients with synucleinopathies and with tauopathies. METHODS: We investigated urate levels in serum and CSF from 18 healthy controls, 19 patients with synucleinopathies (six patients with PD and 13 with MSA), and 24 patients with tauopathies (18 with progressive supranuclear palsy and six with corticobasal degeneration). None of the patients were treated with dopaminergic medications. RESULTS: No significant differences were seen when comparing serum and CSF urate levels from controls across the parkinsonian diagnostic groups. However, in men, serum urate levels were significantly lower in the synucleinopathy group compared with the tauopathy group (P = 0.046), although with a broad overlap. CONCLUSION: Our study suggests that urate levels might provide new insights into the potential pathophysiological mechanisms underlying Parkinsonism and thereby contribute to the future management of these disorders.

Changes in CSF acetyl- and butyrylcholinesterase activity after long-term treatment with AChE inhibitors in Alzheimer's disease.

OBJECTIVES: To measure cerebrospinal fluid (CSF) activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in patients with Alzheimer's disease (AD) participating in randomized clinical trials from three European centers, before and after long-term treatment with different AChE inhibitors (AChEIs). MATERIALS AND METHODS: Of the 144 patients included in the study, 104 were treated with donepezil, 15 with galantamine, 16 with rivastigmine, and nine with placebo. CSF AChE and BChE activities were measured at baseline and after 1- year treatment. RESULTS: Donepezil and galantamine groups showed a significant increase in CSF AChE activity at follow-up, while no changes for BChE activity were observed; in donepezil group, a positive correlation between plasma concentration and AChE activity was documented. Conversely, in rivastigmine group, a decrease in CSF activity of both enzymes was observed. CSF AChE and BChE activities were not correlated with the clinical outcome in any group considered. CSF biomarkers did not show any change after treatment. CONCLUSIONS: AChEIs differently influence the activity of target enzymes in CSF independent of their pharmacodynamic effects.

Combination of hippocampal volume and cerebrospinal fluid biomarkers improves predictive value in mild cognitive impairment.

BACKGROUND: Mild cognitive impairment (MCI) is a heterogeneous condition, and the prognosis differs within the group. Recent findings suggest that hippocampal volumetry and CSF biomarkers can be used to predict which MCI patients have an underlying neurodegenerative disorder. OBJECTIVE: To examine the combined predictive value of hippocampal volume and CSF levels of total tau (T-tau) and beta-amyloid(42) (Abeta(42)) in stable and converting MCI patients. The participants (n = 68) included patients with MCI at baseline and who converted to dementia by the time of the 2-year follow-up (n = 21), stable MCI patients (n = 21) and healthy controls (n = 26). METHODS: The Göteborg MCI study is a clinically based longitudinal study with biannual clinical assessments. Hippocampal volumetry was performed manually, based on data from the 0.5-tesla MRI investigations at baseline. Baseline CSF levels of T-tau and Abeta(42) were measured using commercially available, enzyme-linked immunosorbent assays. RESULTS: The converting MCI group had significantly smaller left hippocampi, lower CSF Abeta(42) and higher T-tau compared to both the stable MCI group and the healthy controls. Multivariate analysis revealed that a combination of the variables outperformed the prognostic ability of the separate variables. CONCLUSIONS: Hippocampal volumes supplement the prognostic accuracy of CSF Abeta(42) and T-tau in MCI.

Reduced cerebrospinal fluid BACE1 activity in multiple sclerosis.

BACKGROUND: Cell and animal experiments have shown that beta-site APP-cleaving enzyme 1 (BACE1) may be involved in myelination. OBJECTIVE: Here, we assess the association of cerebrospinal fluid (CSF) BACE1 activity with multiple sclerosis (MS). METHODS: BACE1 activity and levels of secreted amyloid precursor protein (APP) and amyloid-beta (Abeta) isoforms were analyzed in CSF from 100 patients with MS and 114 neurologically healthy controls. Patients with systemic lupus erythematosus (SLE), 26 with and 41 without cerebral engagement, were also included to enable comparisons with regards to another autoimmune disease. A subset of patients with MS and controls underwent a second lumbar puncture after 10 years. RESULTS: MS patients had lower CSF BACE1 activity than controls (P = 0.03) and patients with cerebral SLE (P < 0.001). Patients with cerebral SLE had higher BACE1 activity than any other group (P < 0.05 for all comparisons). BACE1 activity correlated with the different amyloid markers in all study groups. BACE1 activity decreased over 10 years in the MS group (P = 0.039) and correlated weakly with clinical disease severity scores in an inverse manner. CONCLUSIONS: These results suggest an involvement of BACE1 in the MS disease process.

Clinical proteomics in neurodegenerative disorders.

Neurodegenerative disorders are characterized by neuronal impairment that eventually leads to neuronal death. In spite of the brain's known capacity for regeneration, lost neurons are difficult to replace. Therefore, drugs aimed at inhibiting neurodegenerative processes are likely to be most effective if the treatment is initiated as early as possible. However, clinical manifestations in early disease stages are often numerous, subtle and difficult to diagnose. This is where biomarkers that specifically reflect onset of pathology, directly or indirectly, may have a profound impact on diagnosis making in the future. A triplet of biomarkers for Alzheimer's disease (AD), total and hyperphosphorylated tau and the 42 amino acid isoform of beta-amyloid, has already been established for early detection of AD before the onset of dementia. However, more biomarkers are needed both for AD and for other neurodegenerative disorders, such as Parkinson's disease, frontotemporal dementia and amyotrophic lateral sclerosis. This review provides an update on recent advances in clinical neuroproteomics, a biomarker discovery field that has expanded immensely during the last decade, and gives an overview of the most commonly used techniques and the major clinically relevant findings these techniques have lead to.