Takahiko Tokuda

Detection of elevated levels of α-synuclein oligomers in CSF from patients with Parkinson disease

Background: To date, there is no accepted clinical diagnostic test for Parkinson disease (PD) that is based on biochemical analysis of blood or CSF. The discovery of mutations in the SNCA gene encoding α-synuclein in familial parkinsonism and the accumulation of α-synuclein in the PD brain suggested a critical role for this protein in PD etiology. Methods: We investigated total and α-synuclein oligomers levels in CSF from patients clinically diagnosed with PD, progressive supranuclear palsy (PSP), or Alzheimer disease (AD), and age-matched controls, using ELISA developed in our laboratory. Results: The levels of α-synuclein oligomers and oligomers/total-α-synuclein ratio in CSF were higher in the PD group (n = 32; p < 0.0001, Mann-Whitney U test) compared to the control group (n = 28). The area under the receiver operating characteristic curve (AUC) indicated a sensitivity of 75.0% and a specificity of 87.5%, with an AUC of 0.859 for increased CSF α-synuclein oligomers in clinically diagnosed PD cases. However, when the CSF oligomers/total-α-synuclein ratio was analyzed, it provided an even greater sensitivity of 89.3% and specificity of 90.6%, with an AUC of 0.948. In another cross-sectional pilot study, we confirmed that the levels of CSF α-synuclein oligomers were higher in patients with PD (n = 25) compared to patients with PSP (n = 18; p < 0.05) or AD (n = 35; p < 0.001) or control subjects (n = 43; p < 0.05). Conclusion: Our results demonstrate that levels of α-synuclein oligomers in CSF and the oligomers/total-α-synuclein ratio can be useful biomarkers for diagnosis and early detection of PD.

Increased TDP-43 protein in cerebrospinal fluid of patients with amyotrophic lateral sclerosis

There is mounting pathological, biochemical and genetic evidence that the metabolism and aggregation of the 43-kDa transactive response (TAR)-DNA-binding protein (TDP-43) play a crucial role in the pathogenesis of sporadic and some forms of familial amyotrophic lateral sclerosis (ALS). Recently, it was reported using an ELISA system that elevated levels of TDP-43 were detected in plasma samples from patients with Alzheimer’s disease and frontotemporal dementia, compared to healthy controls. To determine whether quantification of TDP-43 in cerebrospinal fluid (CSF) is potentially informative in the diagnosis of ALS, we measured the concentration, by a similar ELISA method, of TDP-43 in CSF from 30 patients with ALS (diagnosed according to the revised El Escorial criteria) and 29 age-matched control patients without any neurodegenerative disease. We found that, as a group, the ALS patients had significantly higher levels of TDP-43 in their CSF than the age-matched controls (6.92xa0±xa03.71xa0ng/ml in ALS versus 5.31xa0±xa00.94xa0ng/ml in controls, pxa0<xa00.05), with levels of TDP-43 in CSF elevated beyond 95% upper confidence level for the control group in six (20%) of the patients with sporadic ALS. All the six patients with higher levels of CSF TDP-43 were examined within 10xa0months of the onset of illness. The patients examined within 10xa0months of onset showed significantly higher levels of CSF TDP-43 (8.24xa0±xa04.72xa0ng/ml) than those examined after 11xa0months or more of onset (5.41xa0±xa00.66xa0ng/ml, pxa0<xa00.05). These results suggest that the levels of TDP-43 in CSF may increase in the early stage of ALS. We also confirmed the existence of the TDP-43 protein in CSF from some patients with ALS, and a control subject, by western blotting of proteins immunocaptured from the CSF samples. Raised TDP-43 levels in the CSF may preempt the formation of TDP-43 pathology in the central nervous system, or correlate with early-stage TDP-43 pathology, and accordingly be a biomarker for the early stage of ALS.

Differential levels of α-synuclein, β-amyloid42 and tau in CSF between patients with dementia with Lewy bodies and Alzheimer's disease

Background The clinical diagnosis of dementia with Lewy bodies (DLB) is made on the basis of consensus criteria; however, the sensitivity of the criteria is relatively low. There are no generally accepted biomarkers to distinguish DLB from other dementias. Here the utility of quantification of α-synuclein, β-amyloid42 (Aβ42) and tau in the CSF of patients with DLB, Alzheimers disease (AD) and other dementias was examined. Methods 86 patients were divided into three age and sex matched groups: DLB (n=34), AD (n=31) and other dementias (n=21). Two patients with α-synuclein gene (SNCA) duplication were also examined. Aβ and tau were quantified using an ELISA kit. A modified sandwich ELISA was developed which enables the sensitive quantification of CSF α-synuclein. Results Total and phosphorylated tau levels as well as Aβ40/42 and tau/Aβ42 ratios were significantly higher in AD patients than in patients with DLB (p<0.01) and other dementias (p<0.01). CSF α-synuclein levels in DLB patients were significantly lower than those in patients with AD (p<0.05) and other dementias (p<0.01). CSF α-synuclein level correlated with the Aβ42 level in DLB patients (p=0.01, r=0.43). Two patients with SNCA duplication exhibited relatively low levels of CSF α-synuclein. Conclusions The study suggests that reduced levels of CSF α-synuclein in DLB may reflect the accumulation of α-synuclein with Lewy pathology in the brain and that quantification of CSF α-synuclein helps in the differentiation of DLB from AD and other dementias in combination with Aβ42 and tau analysis.

CSF α-synuclein levels in dementia with Lewy bodies and Alzheimer's disease

Dementia with Lewy bodies (DLB) is characterized by widespread depositions of alpha-synuclein, which are described as Lewy bodies. Recently, it was shown that neuronal cells in culture constitutively release alpha-synuclein into the culture medium and that alpha-synuclein is normally present in human cerebrospinal fluid (CSF). The aim of the present study was to evaluate the diagnostic value of CSF alpha-synuclein levels in discriminating DLB from Alzheimers disease (AD). Alpha-synuclein was measured in CSF from 16 patients with DLB and 21 patients with AD. Iodine-123 metaiodobenzylguanidine cardiac scintigraphy was also performed to assess Lewy body pathology. CSF alpha-synuclein levels did not differ significantly between DLB and AD patients. However, the duration of illness was associated with lower alpha-synuclein levels (p<0.05) in DLB, while no such association was found in AD. The present data show CSF alpha-synuclein levels are not sensitive diagnostic markers to discriminate DLB from AD. However, the lower alpha-synuclein levels in DLB patients with longer duration suggest a reduction in CSF alpha-synuclein in association with increased severity of alpha-synucleinopathy in the brain.

Diagnostic accuracy of 123I-meta-iodobenzylguanidine myocardial scintigraphy in dementia with Lewy bodies: a multicenter study.

Background and Purpose Dementia with Lewy bodies (DLB) needs to be distinguished from Alzheimer’s disease (AD) because of important differences in patient management and outcome. Severe cardiac sympathetic degeneration occurs in DLB, but not in AD, offering a potential system for a biological diagnostic marker. The primary aim of this study was to investigate the diagnostic accuracy, in the ante-mortem differentiation of probable DLB from probable AD, of cardiac imaging with the ligand 123I-meta-iodobenzylguanidine (MIBG) which binds to the noradrenaline reuptake site, in the first multicenter study. Methods We performed a multicenter study in which we used 123I-MIBG scans to assess 133 patients with clinical diagnoses of probable (n = 61) or possible (n = 26) DLB or probable AD (n = 46) established by a consensus panel. Three readers, unaware of the clinical diagnosis, classified the images as either normal or abnormal by visual inspection. The heart-to-mediastinum ratios of 123I-MIBG uptake were also calculated using an automated region-of-interest based system. Results Using the heart-to-mediastinum ratio calculated with the automated system, the sensitivity was 68.9% and the specificity was 89.1% to differentiate probable DLB from probable AD in both early and delayed images. By visual assessment, the sensitivity and specificity were 68.9% and 87.0%, respectively. In a subpopulation of patients with mild dementia (MMSE ≥ 22, n = 47), the sensitivity and specificity were 77.4% and 93.8%, respectively, with the delayed heart-to-mediastinum ratio. Conclusions Our first multicenter study confirmed the high correlation between abnormal cardiac sympathetic activity evaluated with 123I-MIBG myocardial scintigraphy and a clinical diagnosis of probable DLB. The diagnostic accuracy is sufficiently high for this technique to be clinically useful in distinguishing DLB from AD, especially in patients with mild dementia.

Elevated CSF TDP-43 levels in amyotrophic lateral sclerosis: Specificity, sensitivity, and a possible prognostic value

Abstract TAR DNA binding protein of 43 kDa (TDP-43) is likely to be the major pathogenetic protein in amyotrophic lateral sclerosis (ALS). A previous study has shown that levels of TDP-43 in CSF measured by an ELISA are significantly higher for ALS patients than for controls. The aim of this study was to investigate whether elevated CSF TDP-43 levels are specific to ALS, and are associated with clinical profiles in ALS patients. We measured CSF TDP-43 levels by the same ELISA in 27 ALS patients and 50 neurodegenerative or inflammatory disease controls such as Parkinsons disease, multiple sclerosis, and Guillain-Barré syndrome. Results showed that the CSF TDP-43 levels were increased only in ALS patients. Receiver operating characteristic (ROC) analyses showed a sensitivity of 59.3% and a specificity of 96.0%. We also found that lower CSF TDP-43 levels may be associated with shorter survival time. In conclusion, the CSF TDP-43 is a potential biomarker that supports a diagnosis of ALS. Moreover, among ALS patients, lower levels of CSF TDP-43 may reflect the accumulation of TDP-43 in the cortical and spinal motor neurons and thereby shorter survival time, although this should be confirmed in larger prospective studies.

The 28‐amino acid form of an APLP1‐derived Aβ‐like peptide is a surrogate marker for Aβ42 production in the central nervous system

Surrogate markers for the Alzheimer disease (AD)‐associated 42‐amino acid form of amyloid‐β (Aβ42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1‐derived Aβ‐like peptides (APL1β) that are generated by β‐ and γ‐cleavages at a concentration of ∼4.5u2009nM. These novel peptides, APL1β25, APL1β27 and APL1β28, were not deposited in AD brains. Interestingly, most γ‐secretase modulators (GSMs) and familial AD‐associated presenilin1 mutants that up‐regulate the relative production of Aβ42 cause a parallel increase in the production of APL1β28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1β28 levels are higher than in non‐AD controls, while the relative Aβ42 levels are unchanged or lower. Most strikingly, the relative APL1β28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non‐AD controls. Based on these results, we propose the relative level of APL1β28 in the CSF as a candidate surrogate marker for the relative level of Aβ42 production in the brain.

Extracellular neurosin degrades α-synuclein in cultured cells

Neurosin, also called kallikrein 6, is a trypsin-like serine protease predominantly expressed in the central nervous system. Neurosin may degrade alpha-synuclein, a major component of the Lewy bodies commonly observed in dopaminergic neurons of patients with sporadic Parkinsons disease. In the present study, we investigated the localization and proteolytic activity of human neurosin using cultured cells to elucidate the physiological role of this enzyme at the cellular level. Heterologous expression of pre-pro-neurosin was localized to the endoplasmic reticulum and secreted. The proteolytic activity of neurosin was analyzed by zymography and fluorescent substrate, and showed that extracellular neurosin had protease activity but intracellular neurosin did not. We also coexpressed alpha-synuclein with neurosin and demonstrated that alpha-synuclein was not cleaved within cells, but extracellular alpha-synuclein was degraded by secreted neurosin. These findings suggest that neurosin targets the extracellular alpha-synuclein.

Oligomeric and phosphorylated alpha-synuclein as potential CSF biomarkers for Parkinson’s disease

BackgroundDespite decades of intensive research, to date, there is no accepted diagnosis for Parkinson’s disease (PD) based on biochemical analysis of blood or CSF. However, neurodegeneration in the brains of PD patients begins several years before the manifestation of the clinical symptoms, pointing to serious flaw/limitations in this approach.ResultsTo explore the potential use of alpha-synuclein (α-syn) species as candidate biomarkers for PD, we generated specific antibodies directed against wide array of α-syn species, namely total-, oligomeric- and phosphorylated-Ser129-α-syn (t-, o- and p-S129-α-syn). Next we sought to employ our antibodies to develop highly specific ELISA assays to quantify α-syn species in biological samples. Finally we verified the usefulness of our assays in CSF samples from 46 PD patients and 48 age-matched healthy controls. We also assessed the discriminating power of combining multiple CSF α-syn species with classical Alzheimer’s disease biomarkers. The combination of CSF o-/t-α-syn, p-S129-α-syn and p-tau provided the best fitting predictive model for discriminating PD patients from controls. Moreover, CSF o-α-syn levels correlated significantly with the severity of PD motor symptoms (ru2009=u2009-0.37).ConclusionOur new ELISA assays can serve as research tools to address the unmet need for reliable CSF biomarkers for PD and related disorders.

Evaluation of Factors Associated With Elevated Levels of Platelet-Derived Microparticles in the Acute Phase of Cerebral Infarction

Background: Platelet-derived microparticles (PDMPs) have attracted attention as blood coagulation-promoting, endothelial cell-activating factors. The objective of this study was to determine the parameters associated with elevated PDMP levels and examine their relationship with atherosclerotic lesions of main intracranial and extracranial arteries. Participants and Methods: Participants included a control group (C) of 61 patients with no apparent cerebral vascular lesions and 110 patients with acute-phase cerebral infarction, consisting of a small-vessel occlusion group (S) of 34 patients, a large-artery atherosclerosis group (L) of 41 patients, a cardioembolism group (CE) of 20 patients, and a stroke of undetermined etiology group (U) of 15 patients. Platelet-derived microparticle levels were measured using enzyme-linked immunosorbent assay (ELISA) at the time of admission, and the patients were reclassified into group CP (control level PDMPs), consisting of 70 patients with control PDMP levels, and group HP (high PDMPs), consisting of 40 patients with elevated PDMP levels. All patients underwent cranial magnetic resonance (MR) and carotid ultrasound examinations. Results: Platelet-derived microparticle levels were significantly higher in groups S and L than in group C (P < .01). Concomitant intima-media thickness (IMT; odds ratio [OR] = 1.29, P < .05) and concomitant intracranial stenosis (OR = 3.95, P < .01) were significantly correlated with elevated PDMP levels. Fibrinogen and high-sensitivity CRP levels were significantly higher in group HP than in group CP. Conclusion: Alterations in PDMP levels correlated with the presence of atherothrombotic lesions, and PDMP levels are expected to be useful as a clinical indicator, reflecting the presence of intracranial atherosclerotic lesions in the acute phase of cerebral infarction.