Omar El-Agnaf

Cerebrospinal Fluid Lysosomal Enzymes and Alpha-Synuclein in Parkinson's Disease

To assess the discriminating power of multiple cerebrospinal fluid (CSF) biomarkers for Parkinsons disease (PD), we measured several proteins playing an important role in the disease pathogenesis. The activities of β‐glucocerebrosidase and other lysosomal enzymes, together with total and oligomeric α‐synuclein, and total and phosphorylated tau, were thus assessed in CSF of 71 PD patients and compared to 45 neurological controls. Activities of β‐glucocerebrosidase, β‐mannosidase, β‐hexosaminidase, and β‐galactosidase were measured with established enzymatic assays, while α‐synuclein and tau biomarkers were evaluated with immunoassays. A subset of PD patients (nu2009=u200944) was also screened for mutations in the β‐glucocerebrosidase‐encoding gene (GBA1). In the PD group, β‐glucocerebrosidase activity was reduced (Pu2009<u20090.05) and patients at earlier stages showed lower enzymatic activity (Pu2009<u20090.05); conversely, β‐hexosaminidase activity was significantly increased (Pu2009<u20090.05). Eight PD patients (18%) presented GBA1 sequence variations; 3 of them were heterozygous for the N370S mutation. Levels of total α‐synuclein were significantly reduced (Pu2009<u20090.05) in PD, in contrast to increased levels of α‐synuclein oligomers, with a higher oligomeric/total α‐synuclein ratio in PD patients when compared with controls (Pu2009<u20090.001). A combination of β‐glucocerebrosidase activity, oligomeric/total α‐synuclein ratio, and age gave the best performance in discriminating PD from neurological controls (sensitivity 82%; specificity 71%, area under the receiver operating characteristic curveu2009=u20090.87). These results demonstrate the possibility of detecting lysosomal dysfunction in CSF and further support the need to combine different biomarkers for improving the diagnostic accuracy of PD.

Quantification of α-synuclein in cerebrospinal fluid as a biomarker candidate: review of the literature and considerations for future studies

The pursuit of laboratory tests that allow for the reliable and inexpensive identification of subjects with parkinsonism represents a hot topic in translational neuroscience. This unmet need affects the counseling of presymptomatic, at-risk subjects and delays the accurate diagnosis of already symptomatic individuals. The absence of validated markers that are closely linked to the pathological disease process also compromises the objective monitoring of therapeutic interventions in clinical trials. Typical Parkinsons disease represents a heterogenous syndrome (but the majority of patients suffer from neurodegeneration) that is linked to the misprocessing of α-synuclein (α-Syn). The identification of α-Syn as a bona fide constituent of human cerebrospinal fluid and its quantification in early cross-sectional studies represent the beginning of a new chapter in Parkinsons disease research. It will determine what role, if any, cerebrospinal fluid α-Syn plays as a biomarker candidate in Lewy inclusion-positive forms of parkinsonism. This article focuses on the progress that has been made in seven recently published papers and highlights the challenges that lie ahead. We also provide specific information regarding standardized operating procedures for cerebrospinal fluid collection in PD biomarker research efforts.

Levels of cerebrospinal fluid α-synuclein oligomers are increased in Parkinson’s disease with dementia and dementia with Lewy bodies compared to Alzheimer’s disease

IntroductionThe objective was to study whether α-synuclein oligomers are altered in the cerebrospinal fluid (CSF) of patients with dementia, including Parkinson disease with dementia (PDD), dementia with Lewy bodies (DLB), and Alzheimer disease (AD), compared with age-matched controls.MethodsIn total, 247 CSF samples were assessed in this study, including 71 patients with DLB, 30 patients with PDD, 48 patients with AD, and 98 healthy age-matched controls. Both total and oligomeric α-synuclein levels were evaluated by using well-established immunoassays.ResultsThe levels of α-synuclein oligomers in the CSF were increased in patients with PDD compared with the controls (Pu2009<u20090.05), but not in patients with DLB compared with controls. Interestingly, the levels of α-synuclein oligomers in the CSF were also significantly higher in patients with PDD (Pu2009<u20090.01) and DLB (Pu2009<u20090.05) compared with patients with AD. The levels of CSF α-synuclein oligomers and the ratio of oligomeric/total-α-synuclein could distinguish DLB or PDD patients from AD patients, with areas under the curves (AUCs) of 0.64 and 0.75, respectively. In addition, total-α-synuclein alone could distinguish DLB or PDD patients from AD patients, with an AUC of 0.80.ConclusionsThe levels of α-synuclein oligomers were increased in the CSF from α-synucleinopathy patients with dementia compared with AD cases.

Differential role of CSF alpha-synuclein species, tau, and Aβ42 in Parkinson's disease

There is a great interest in developing cerebrospinal fluid (CSF) biomarkers for diagnosis and prognosis of Parkinsons disease (PD). CSF alpha synuclein (α-syn) species, namely total and oligomeric α-syn (t-α-syn and o-α-syn), have shown to be of help for PD diagnosis. Preliminary evidences show that the combination of CSF t-α-syn and classical Alzheimers disease (AD) biomarkers—β-amyloid 1–42 (Aβ42), total tau (t-tau), phosphorylated tau (p-tau)—differentiate PD patients from controls, and that reduced levels of Aβ42 represent a predictive factor for development of cognitive deterioration in PD. In this prospective study carried out in 44 PD patients and 25 neurological controls we wanted to verify whether the combination of CSF α-synuclein species—t-α-syn and o-α-syn—and classical AD biomarkers may help in differentiating PD from neurological controls, and if these biomarkers may predict cognitive decline. The median of follow-up duration was 3 years (range: 2–6 years). Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) were used for monitoring cognitive changes along time, being administered once a year. Oligo/total α-syn ratio (o/t-α-syn ratio) confirmed its diagnostic value, significantly contributing to the discrimination of PD from neurological controls. A greater diagnostic accuracy was reached when combining o/t-α-syn and Aβ42/tau ratios (Sens = 0.70, Spec = 0.84, AUC = 0.82; PPV = 0.89, NPV = 0.62, LR+ = 4.40, DOR = 12.52). Low CSF Aβ42 level was associated with a higher rate of MMSE and MoCA decline, confirming its role as independent predictive factor for cognitive decline in PD. None of the other biomarkers assessed (t-tau, p-tau, t-α-syn and o-α-syn) showed to have prognostic value. We conclude that combination of CSF o/t-α-syn and Aβ42/tau ratios improve the diagnostic accuracy of PD. PD patients showing low CSF Aβ42 levels at baseline are more prone to develop cognitive decline.

Ginsenoside Rb1 inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils

Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinsons disease (PD) and other synucleinopathies. Identification of compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no β-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.

Elevated levels of cerebrospinal fluid α-synuclein oligomers in healthy asymptomatic LRRK2 mutation carriers

Mutations in the leucine-rich repeat kinase 2 gene are the most common cause of autosomal dominant Parkinson’s disease (PD). To assess the cerebrospinal fluid (CSF) levels of α-synuclein oligomers in symptomatic and asymptomatic leucine-rich repeat kinase 2 mutation carriers, we used enzyme-linked immunosorbent assays (ELISA) to investigate total and oligomeric forms of α-synuclein in CSF samples. The CSF samples were collected from 33 Norwegian individuals with leucine-rich repeat kinase 2 mutations: 13 patients were clinically diagnosed with PD and 20 patients were healthy, asymptomatic leucine-rich repeat kinase 2 mutation carriers. We also included 35 patients with sporadic PD (sPD) and 42 age-matched healthy controls. Levels of CSF α-synuclein oligomers were significantly elevated in healthy asymptomatic individuals carrying leucine-rich repeat kinase 2 mutations (n = 20; P < 0.0079) and in sPD group (n = 35; P < 0.003) relative to healthy controls. Increased α-synuclein oligomers in asymptomatic leucine-rich repeat kinase 2 mutation carriers showed a sensitivity of 63.0% and a specificity of 74.0%, with an area under the curve of 0.66, and a sensitivity of 65.0% and a specificity of 83.0%, with an area under the curve of 0.74 for sPD cases. An inverse correlation between CSF levels of α- synuclein oligomers and disease severity and duration was observed. Our study suggests that quantification of α-synuclein oligomers in CSF has potential value as a tool for PD diagnosis and presymptomatic screening of high-risk individuals.

Increased α-synuclein levels in the cerebrospinal fluid of patients with Creutzfeldt–Jakob disease

Recent studies have shown that cerebrospinal fluid (CSF) levels of α-synuclein (α-syn) are highly elevated in patients with Creutzfeldt–Jakob disease (CJD) compared to controls. However, the diagnostic value of CSF α-syn in CJD has not been established. To confirm whether CSF α-syn is increased in CJD and is a useful marker for this disease, two independent enzyme-linked immunoabsorbent assays (ELISAs) specific for α-syn were used: ELISA 211-FL140, which is specific for full-length α-syn, and ELISA N19-FL140, which is specific for the full-length and associated C-terminal truncated forms of α-syn. CSF samples from 24 patients with CJD and 24 controls were assessed in this study. We found that samples from the CJD patients showed significantly higher levels of CSF α-syn compared to controls in both ELISA (211-FL140 or N19-FL140) tests (Pxa0=xa00.0467 and Pxa0=xa00.0010, respectively). However, there was a considerable overlap in the concentration ranges of the two groups of subjects. We also measured the levels of total tau (t-tau) protein in these samples and found that CSF t-tau levels were 5–10-times higher in the CJD group (Pxa0<xa00.0001) compared with the controls. When the CSF t-tau and α-syn levels were combined, the area under the ROC curve (AUC) was slightly increased in clinically diagnosed CJD cases (AUC of 0.964) relative to an AUC of 0.943 for increased CSF t-tau alone. The combined use of CSF α-syn and t-tau levels may be a useful biomarker for the diagnosis of CJD.

α-Synuclein Levels in Blood Plasma from LRRK2 Mutation Carriers

The diagnosis of Parkinson’s disease (PD) remains primarily a clinical issue, based mainly on phenotypic patterns. The identification of biomarkers capable of permitting the preclinical detection of PD is critically needed. α-Synuclein is a key protein in PD, with missense and multiplication mutations in the gene encoding α-synuclein (SNCA) having been reported in familial cases of PD, and accumulation of the protein identified in Lewy bodies (LBs) and Lewy neurites (LNs) in affected brain regions. With the objective of validating the use of α-synuclein as a clinical or progressive biomarker in an accessible tissue, we used an enzyme-linked immunosorbent assay (ELISA) to measure α-synuclein levels in the peripheral blood plasma of idiopathic PD and LRRK2 mutation carrier patients and compared our findings with healthy control subjects. Compared to healthy controls, we found a significant decrease in plasma total α-synuclein levels in idiopathic PD (iPD) patients (nu200a=u200a134, pu200a=u200a0.010). However, the reduction was less significant in patients who were LRRK2 mutation carriers (nu200a=u200a32, pu200a=u200a0.133). This lack of significance could be due to the small number of individuals employed in this group. No predictive value of total α-synuclein in the diagnosis of PD was found in a receiver operating characteristic (ROC) curve analysis. Although this is a pilot study requiring corroboration on a larger cohort of patients, our results highlight the possible use of plasma α-synuclein as a biomarker for PD.

α-Synuclein aggregation and modulating factors.

Aggregated a-synuclein is the major component of inclusions in Parkinsons disease and other synucleinopathy brains indicating that a-syn aggregation is associated with the pathogenesis of neurodegenerative disorders. Although the mechanisms underlying a-syn aggregation and toxicity are not fully elucidated, it is clear that a-syn undergoes post-translational modifications and interacts with numerous proteins and other macromolecules, metals, hormones, neurotransmitters, drugs and poisons that can all modulate its aggregation propensity. The current and most recent findings regarding the factors modulating a-syn aggregation process are discussed in detail.

Ser129 phosphorylation of endogenous α-synuclein induced by overexpression of polo-like kinases 2 and 3 in nigral dopamine neurons is not detrimental to their survival and function

Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-syn] was found to be the most abundant form in intracellular inclusions in brains from Parkinsons disease (PD) patients. This finding suggests that P(S129)-α-syn plays a central role in the pathogenesis of PD. However, it is at present unclear whether P(S129)-α-syn is pathogenic driving the neurodegenerative process. Rodent studies using neither the phosphomimics of human α-syn nor co-expression of human wild-type α-syn and kinases phosphorylating α-syn at Ser129 gave consistent results. One major concern in interpreting these findings is that human α-syn was expressed above physiological levels inducing neurodegeneration in rat nigral neurons. In order to exclude this confounding factor, we took a different approach and increased the phosphorylation level of endogenous α-syn. For this purpose, we took advantage of recombinant adeno-associated viral (rAAV) vectors to deliver polo-like kinase (PLK) 2 or PLK3 in the substantia nigra and investigated whether increased levels of P(S129)-α-syn compromised the function and survival of nigral dopaminergic neurons. Interestingly, we observed that hyperphosphorylated α-syn did not induce nigral dopaminergic cell death, as assessed at 1 and 4months. Furthermore, histological analysis did not show any accumulation of α-syn protein or formation of inclusions. Using in vivo microdialysis, we found that the only measurable functional alteration was the depolarisation-induced release of dopamine, while the in vivo synthesis rate of DOPA and dopamine baseline release remained unaltered. Taken together, our results suggest that phosphorylation of α-syn at Ser129 does not confer a toxic gain of function per se.