Mustafa T. Ardah

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

Background: The oligomeric state of α-syn in vivo remains unknown. Results: α-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native α-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric α-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease. Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107–110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797–17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.

Cerebrospinal fluid Tau/α-synuclein ratio in Parkinson's disease and degenerative dementias.

Although alpha‐synuclein is the main constituent of Lewy bodies, cerebrospinal fluid determination on its own does not seem fundamental for the diagnosis of synucleinopathies. We evaluated whether the combination of classical biomarkers, Aβ1–42, total tau, phosphorylated tau, and α‐synuclein can improve discrimination of Parkinsons disease, dementia with Lewy bodies, Alzheimers disease, and frontotemporal dementia. Aβ1–42, total tau, phosphorylated tau, and α‐synuclein were measured in a series of patients with Parkinsons disease (n = 38), dementia with Lewy bodies (n = 32), Alzheimers disease (n = 48), frontotemporal dementia (n = 31), and age‐matched control patients with other neurological diseases (n = 32). Mean α‐synuclein levels in cerebrospinal fluid were significantly lower in the pathological groups than in cognitively healthy subjects. An inverse correlation of α‐synuclein with total tau (r = −0.196, P < .01) was observed. In the group of patients with Parkinsons disease, Aβ1–42, total tau, and phosphorylated tau values were similar to controls, whereas total tau/α‐synuclein and phosphorylated tau/α‐synuclein ratios showed the lowest values. Cerebrospinal fluid α‐synuclein alone did not provide relevant information for Parkinsons disease diagnosis, showing low specificity (area under the curve, 0.662; sensitivity, 94%; specificity, 25%). Instead, a better performance was obtained with the total tau/α‐syn ratio (area under the curve, 0.765; sensitivity, 89%; specificity, 61%). Combined determination of α‐synuclein and classical biomarkers in cerebrospinal fluid shows differential patterns in neurodegenerative disorders. In particular, total tau/α‐synuclein and phosphorylated tau/α‐synuclein ratios can contribute to the discrimination of Parkinsons disease.

Nigrostriatal overabundance of α-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity

Abstractα-Synuclein (α-syn) is a presynaptic protein present at most nerve terminals, but its function remains largely unknown. The familial forms of Parkinson’s disease associated with multiplications of the α-syn gene locus indicate that overabundance of this protein might have a detrimental effect on dopaminergic transmission. To investigate this hypothesis, we use adeno-associated viral (AAV) vectors to overexpress human α-syn in the rat substantia nigra. Moderate overexpression of either wild-type (WT) or A30P α-syn differs in the motor phenotypes induced, with only the WT form generating hemiparkinsonian impairments. Wild-type α-syn causes a reduction of dopamine release in the striatum that exceeds the loss of dopaminergic neurons, axonal fibers, and the reduction in total dopamine. At the ultrastructural level, the reduced dopamine release corresponds to a decreased density of dopaminergic vesicles and synaptic contacts in striatal terminals. Interestingly, the membrane-binding-deficient A30P mutant does neither notably reduce dopamine release nor it cause ultrastructural changes in dopaminergic axons, showing that α-syn’s membrane-binding properties are critically involved in the presynaptic defects. To further determine if the affinity of the protein for membranes determines the extent of motor defects, we compare three forms of α-syn in conditions leading to pronounced degeneration. While membrane-binding α-syns (wild-type and A53T) induce severe motor impairments, an N-terminal deleted form with attenuated affinity for membranes is inefficient in inducing motor defects. Overall, these results demonstrate that α-syn overabundance is detrimental to dopamine neurotransmission at early stages of the degeneration of nigrostriatal dopaminergic axons.

Baicalein inhibits formation of α-Synuclein oligomers within living cells and prevents Aβ peptide fibrillation and oligomerisation

Abnormal protein aggregation in the brain is linked to the pathogenesis of neurodegenerative diseases, including Alzheimers disease (AD) and Parkinsons disease (PD). Recent studies revealed that the oligomeric form of aggregates is most likely the toxic species, and thus could be a good therapeutic target. To screen for potent inhibitors that can inhibit both oligomerisation and fibrillation of α‐synuclein (α‐syn), we systematically compared the antioligomeric and antifibrillar activities of eight compounds that were extracted from Chinese herbal medicines through three platforms that can monitor the formation of α‐syn fibrils and oligomers in cell‐free or cellular systems. Our results revealed that baicalein, a flavonoid extracted from the Chinese herbal medicine Scutellaria baicalensis Georgi (“huang qin” in Chinese), is a potent inhibitor of α‐syn oligomerisation both in cell‐free and cellular systems, and is also an effective inhibitor of α‐syn fibrillation in cell‐free systems. We further tested the protective effect of baicalein against α‐syn‐oligomer‐induced toxicity in neuronal cells. Our data showed that baicalein inhibited the formation of α‐syn oligomers in SH‐SY5Y and Hela cells, and protected SH‐SY5Y cells from α‐syn‐oligomer‐induced toxicity. We also explored the effect of baicalein on amyloid‐β peptide (Aβ) aggregation and toxicity. We found that baicalein can also inhibit Aβ fibrillation and oligomerisation, disaggregate pre‐formed Aβ amyloid fibrils and prevent Aβ fibril‐induced toxicity in PC12 cells. Our study indicates that baicalein is a good inhibitor of amyloid protein aggregation and toxicity. Given the role of these processes in neurodegenerative diseases such as AD and PD, our results suggest that baicalein has potential as a therapeutic agent for the treatment of these devastating disorders.

The protective role of AMP-activated protein kinase in alpha-synuclein neurotoxicity in vitro.

In the present study, we investigated the role of the main intracellular energy sensor, AMP-activated protein kinase (AMPK), in the in vitro neurotoxicity of α-synuclein (ASYN), one of the key culprits in the pathogenesis of Parkinsons disease. The loss of viability in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells inducibly overexpressing wild-type ASYN was associated with the reduced activation of AMPK and its activator LKB1, as well as AMPK target Raptor. ASYN-overexpressing rat primary neurons also displayed lower activity of LKB1/AMPK/Raptor pathway. Restoration of AMPK activity by metformin or AICAR reduced the in vitro neurotoxicity of ASYN overexpression, acting independently of the prosurvival kinase Akt or the induction of autophagic response. The conditioned medium from ASYN-overexpressing cells, containing secreted ASYN, as well as dopamine-modified or nitrated recombinant ASYN oligomers, all inhibited AMPK activation in differentiated SH-SY5Y cells and reduced their viability, but not in the presence of metformin or AICAR. The RNA interference-mediated knockdown of AMPK increased the sensitivity of SH-SY5Y cells to the harmful effects of secreted ASYN. AMPK-dependent protection from extracellular ASYN was also observed in rat neuron-like pheochromocytoma cell line PC12. These data demonstrate the protective role of AMPK against the toxicity of both intracellular and extracellular ASYN, suggesting that modulation of AMPK activity may be a promising therapeutic strategy in Parkinsons disease.

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 (r = -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.

Generation and characterization of novel conformation-specific monoclonal antibodies for α-synuclein pathology

α-Synuclein (α-syn), a small protein that has the intrinsic propensity to aggregate, is implicated in several neurodegenerative diseases including Parkinsons disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), which are collectively known as synucleinopathies. Genetic, pathological, biochemical, and animal modeling studies provided compelling evidence that α-syn aggregation plays a key role in the pathogenesis of PD and related synucleinopathies. It is therefore of utmost importance to develop reliable tools that can detect the aggregated forms of α-syn. We describe here the generation and characterization of six novel conformation-specific monoclonal antibodies that recognize specifically α-syn aggregates but not the soluble, monomeric form of the protein. The antibodies described herein did not recognize monomers or fibrils generated from other amyloidogenic proteins including β-syn, γ-syn, β-amyloid, tau protein, islet amyloid polypeptide and ABri. Interestingly, the antibodies did not react to overlapping linear peptides spanning the entire sequence of α-syn, confirming further that they only detect α-syn aggregates. In immunohistochemical studies, the new conformation-specific monoclonal antibodies showed underappreciated small micro-aggregates and very thin neurites in PD and DLB cases that were not observed with generic pan antibodies that recognize linear epitope. Furthermore, employing one of our conformation-specific antibodies in a sandwich based ELISA, we observed an increase in levels of α-syn oligomers in brain lysates from DLB compared to Alzheimers disease and control samples. Therefore, the conformation-specific antibodies portrayed herein represent useful tools for research, biomarkers development, diagnosis and even immunotherapy for PD and related pathologies.

Structure activity relationship of phenolic acid inhibitors of α-synuclein fibril formation and toxicity

The aggregation of α-synuclein (α-syn) is considered the key pathogenic event in many neurological disorders such as Parkinsons disease (PD), dementia with Lewy bodies and multiple system atrophy, giving rise to a whole category of neurodegenerative diseases known as synucleinopathies. Although the molecular basis of α-syn toxicity has not been precisely elucidated, a great deal of effort has been put into identifying compounds that could inhibit or even reverse the aggregation process. Previous reports indicated that many phenolic compounds are potent inhibitors of α-syn aggregation. The aim of the present study was to assess the anti-aggregating effect of gallic acid (GA) (3,4,5-trihydroxybenzoic acid), a benzoic acid derivative that belongs to a group of phenolic compounds known as phenolic acids. By employing an array of biophysical and biochemical techniques and a cell-viability assay, GA was shown not only to inhibit α-syn fibrillation and toxicity but also to disaggregate preformed α-syn amyloid fibrils. Interestingly, GA was found to bind to soluble, non-toxic oligomers with no β-sheet content, and to stabilize their structure. The binding of GA to the oligomers may represent a potential mechanism of action. Additionally, by using structure activity relationship data obtained from fourteen structurally similar benzoic acid derivatives, it was determined that the inhibition of α-syn fibrillation by GA is related to the number of hydroxyl moieties and their position on the phenyl ring. GA may represent the starting point for designing new molecules that could be used for the treatment of PD and related disorders.

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.

Design, Synthesis, and Qualitative Structure–Activity Evaluations of Novel β-Secretase Inhibitors as Potential Alzheimer’s Drug Leads

We have identified highly selective imidazopyridines armed with benzimidazol and/or arylimidazole as potent β-secretase inhibitors. The most effective and selective analogues demonstrated low nanomolar potency for the BACE1 enzyme as measured by FRET and cell-based (ELISA) assays and exhibited comparable affinity (KI) and high ligand efficiency (LE). In addition, these motifs were highly selective (>200) against the structurally related aspartyl protease BACE2. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on the previously reported hydroxyethylene transition state inhibitor derived from isophthalic acid I. Of the most potent compounds, 34 displayed an IC50 for BACE1 of 18 nM and exhibited cellular activity with an EC50 of 37 nM in the cell-based ELISA assay, as well as high affinity (KI=17 nM) and ligand efficiency (LE=1.7 kJ/mol). Compound 34 was found to be 204-fold more selective for BACE1 compared to the closely related aspartyl protease BACE2.