TDP-43 potentiates alpha-synuclein toxicity to dopaminergic neurons in transgenic mice

TDP-43 and α-synuclein are two disease proteins involved in a wide range of neurodegenerative diseases. While TDP-43 proteinopathy is considered a pathologic hallmark of sporadic amyotrophic lateral sclerosis and frontotemporal lobe degeneration, α-synuclein is a major component of Lewy body characteristic of Parkinson's disease. Intriguingly, TDP-43 proteinopathy also coexists with Lewy body and with synucleinopathy in certain disease conditions. Here we reported the effects of TDP-43 on α-synuclein neurotoxicity in transgenic mice. Overexpression of mutant TDP-43 (M337V substitution) in mice caused early death in transgenic founders, but overexpression of normal TDP-43 only induced a moderate loss of cortical neurons in the transgenic mice at advanced ages. Interestingly, concomitant overexpression of normal TDP-43 and mutant α-synuclein caused a more severe loss of dopaminergic neurons in the double transgenic mice as compared to single-gene transgenic mice. TDP-43 potentiated α-synuclein toxicity to dopaminergic neurons in living animals. Our finding provides in vivo evidence suggesting that disease proteins such as TDP-43 and α-synuclein may play a synergistic role in disease induction in neurodegenerative diseases.     

Diet‐induced obesity accelerates the onset of terminal phenotypes in α‐synuclein transgenic mice

Parkinson's disease (PD) and diabetes belong to the most common neurodegenerative and metabolic syndromes, respectively. Epidemiological links between these two frequent disorders are controversial. The neuropathological hallmarks of PD are protein aggregates composed of amyloid‐like fibrillar and serine‐129 phosphorylated (pS129) α‐synuclein (AS). To study if diet‐induced obesity could be an environmental risk factor for PD‐related α‐synucleinopathy, transgenic (TG) mice, expressing the human mutant A30P AS in brain neurons, were subjected after weaning to a lifelong high fat diet (HFD). The TG mice became obese and glucose‐intolerant, as did the wild‐type controls. Upon aging, HFD significantly accelerated the onset of the lethal locomotor phenotype. Coinciding with the premature movement phenotype and death, HFD accelerated the age of onset of brainstem α‐synucleinopathy as detected by immunostaining with antibodies against pathology‐associated pS129. Amyloid‐like neuropathology was confirmed by thioflavin S staining. Accelerated onset of neurodegeneration was indicated by Gallyas silver‐positive neuronal dystrophy as well as astrogliosis. Phosphorylation of the activation sites of the pro‐survival signaling intermediate Akt was reduced in younger TG mice after HFD. Thus, diet‐induced obesity may be an environmental risk factor for the development of α‐synucleinopathies. The molecular and cellular mechanisms remain to be further elucidated.

     

DJ‐1 deficiency impairs autophagy and reduces alpha‐synuclein phagocytosis by microglia

Parkinson's disease (PD) is a progressive neurodegenerative disorder, of which 1% of the hereditary cases are linked to mutations in DJ‐1, an oxidative stress sensor. The pathological hallmark of PD is intercellular inclusions termed Lewy Bodies, composed mainly of α‐Synuclein (α‐Syn) protein. Recent findings have shown that α‐Syn can be transmitted from cell to cell, suggesting an important role of microglia, as the main scavenger cells of the brain, in clearing α‐Syn. We previously reported that the knock down (KD) of DJ‐1 in microglia increased cells’ neurotoxicity to dopaminergic neurons. Here, we discovered that α‐Syn significantly induced elevated secretion of the proinflammatory cytokines IL‐6 and IL‐1β and a significant dose‐dependent elevation in the production of nitric oxide in DJ‐1 KD microglia, compared to control microglia. We further investigated the ability of DJ‐1 KD microglia to uptake and degrade soluble α‐Syn, and discovered that DJ‐1 KD reduces cell‐surface lipid raft expression in microglia and impairs their ability to uptake soluble α‐Syn. Autophagy is an important mechanism for degradation of intracellular proteins and organelles. We discovered that DJ‐1 KD microglia exhibit an impaired autophagy‐dependent degradation of p62 and LC3 proteins, and that manipulation of autophagy had less effect on α‐Syn uptake and clearance in DJ‐1 KD microglia, compared to control microglia. Further studies of the link between DJ‐1, α‐Syn uptake and autophagy may provide useful insights into the role of microglia in the etiology of the PD.

     

Glycation modulates alpha-synuclein fibrillization kinetics: A sweet spot for inhibition

Glycation is a nonenzymatic posttranslational modification (PTM) known to be increased in the brains of hyperglycemic patients. Alpha-synuclein (αSN), a central player in the etiology of Parkinson’s disease, can be glycated at lysine residues, thereby reducing αSN fibril formation in vitro and modulating αSN aggregation in cells. However, the molecular basis for these effects is unclear. To elucidate this, we investigated the aggregation of αSN modified by eight glycating agents, namely the dicarbonyl compound methylglyoxal (MGO) and the sugars ribose, fructose, mannose, glucose, galactose, sucrose, and lactose. We found that MGO and ribose modify αSN to the greatest extent, and these glycation products are the most efficient inhibitors of fibril formation. We show glycation primarily inhibits elongation rather than nucleation of αSN and has only a modest effect on the level of oligomerization. Furthermore, glycated αSN is not significantly incorporated into fibrils. For both MGO and ribose, we discovered that a level of ∼5 modifications per αSN is optimal for inhibition of elongation. The remaining sugars showed a weak but optimal inhibition at ∼2 modifications per αSN. We propose that this optimal level balances the affinity for the growing ends of the fibril (which decreases with the extent of modification) with the ability to block incorporation of subsequent αSN subunits (which increases with modification). Our results are not only relevant for other αSN PTMs but also for understanding PTMs affecting other fibrillogenic proteins and may thus open novel avenues for therapeutic intervention in protein aggregation disorders.     

帕金森病患者血浆alpha-synuclein、Abeta及tau蛋白变化特点

目的：探索帕金森病（Parkinson''s disease,PD）患者血浆中alpha- 突触核蛋白、Abeta及tau 蛋白变化情况。方法：募集2014 年4 月至 2015 年4 月来我院就诊的PD 患者62 例,正常对照人群59 例,采集两组人群的基本临床信息,测定血浆中琢- 突触核蛋白、 Abeta40、Abeta42、pT181-tau 蛋白、pT231-tau 蛋白和总tau 蛋白浓度,比较两组之间的差异,同时进行相关性分析。结果：PD患者血浆 alpha- 突触核蛋白和pT181-tau 蛋白浓度显著高于对照组(P 值分别为0.001,0.019),而两组间Abeta40、Abeta42、pT231-tau 蛋白和总tau 蛋白浓度无明显差异（P>0.05）。相关性分析提示PD 患者血浆alpha-突触核蛋白和pT181-tau 蛋白浓度与患者年龄、性别、教育程度、 病程、高血压、糖尿病、Hoehn/ Yahr 分级及Schwab &England 评分无相关性（P>0.05）。结论：虽然PD患者血浆琢- 突触核蛋白和 pT181-tau 蛋白高于正常对照组,但尚不适宜作为PD 的生物标志物。     

Oligopeptide-mediated acceleration of amyloid fibril formation of amyloid beta(Abeta) and alpha-synuclein fragment peptide (NAC).

The effects of oligopeptides on the secondary structures of Abeta and NAC, a fragment of alpha-synuclein protein, were studied by circular dichroism (CD) spectra. The effects of oligopeptides on the amyloid fibril formation were also studied by fluorescence spectra due to thioflavine-T. The oligopeptides were composed of a fragment of Abeta or NAC and were interposed by acidic or basic amino acid residues. The peptide, Ac-ELVFFAKK-NH2, which involved a fragment Leu-Val-Phe-Phe-Ala at Abeta(17-21), had no effect on the secondary structures of Abeta(1-28) in 60% or 90% trifluoroethanol (TFE) solutions at both pH 3.2 and pH 7.2. However, it showed pronounced effects on the secondary structure of Abeta(1-28) at pH 5.4. The Ac-ELVFFAKK-NH2 reduced the alpha-helical content, while it increased the beta-sheet content of Abeta(1-28). In phosphate buffer solutions at pH 7.0, Ac-ELVFFAKK-NH2 had little effect on the secondary structures of Abeta(1-28). However, it accelerated amyloid fibril formation when monitored by fluorescence spectra due to thioflavine-T. On the other hand, LPFFD, a peptide known as a beta-sheet breaker, caused neither an appreciable extent of change in the secondary structure nor amyloid fibril formation in the same buffer solution. The peptide, Ac-ETVK-NH2, which involved a fragment Thr-Val at NAC(21-22), had no effect on the secondary structure of NAC in 90% TFE and in isotonic phosphate buffer. However, Ac-ETVK-NH2 in water with small amounts of NaN3 and hexafluoroisopropanol greatly increased the beta-sheet content of NAC after standing the solution for more than 1 week. Interestingly, in this solution. Ac-ETVK-NH2, accelerated the fibril formation of NAC. It was concluded that an oligopeptide that involves a fragment of amyloidogenic proteins could be a trigger for the formation of amyloid plaques of the proteins even when it had little effect on the secondary structures of the proteins as monitored by CD spectra for a short incubation time.     

Structural changes in alpha-synuclein affect its chaperone-like activity in vitro

Alpha-synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of alpha-synuclein has not yet been established. We here present biochemical evidence that recombinant alpha-synuclein has a chaperone-like function against thermal and chemical stress in vitro. In our experiments, alpha-synuclein protected glutathione S-transferase (GST) and aldolase from heat-induced precipitation, and alpha-lactalbumin and bovine serum albumin from dithiothreitol (DTT)-induced precipitation like other molecular chaperones. Moreover, preheating of alpha-synuclein, which is believed to reorganize the molecular surface of alpha-synuclein, increased the chaperone-like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, alpha-synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone-like function of the protein. In addition, alpha-synuclein was also rapidly and significantly precipitated by heat in the presence of Zn2+ in vitro, whereas it was not affected by the presence of Ca2+ or Mg2+. Circular dichroism spectra confirmed that alpha-synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that alpha-synuclein could act as a molecular chaperone, and that the conformational change of the alpha-synuclein could explain the aggregation kinetics of alpha-synuclein, which may be related to the abolishment of the chaperonic-like activity.     

Monoclonal antibodies selective for α‐synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α‐synuclein transgenic mice with the disease‐causing A30P mutation

Inclusions of intraneuronal alpha‐synuclein (α‐synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α‐synuclein is a central feature of the disease pathogenesis. Among the different α‐synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α‐synuclein oligomers were generated. These antibodies, which do not bind amyloid‐beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α‐synuclein transgenic mice than linear epitope antibodies. An oligomer‐selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α‐synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α‐synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer‐selective α‐synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.     

N-Terminal acetylation is critical for forming α-helical oligomer of α-synuclein

The aggregation of the protein α-synuclein (AS) is critical to the pathogenesis of Parkinson's disease. Although generally described as an unstructured monomer, recent evidence suggests that the native form of AS may be an α-helical tetramer which resists aggregation. Here, we show that N-terminal acetylation in combination with a mild purification protocol results in an oligomeric form of AS with partial α-helical structure. N-terminal acetylation of AS could have important implications for both the native and pathological structures and functions of AS. Through our demonstration of a recombinant expression system, our results represent an important step toward biochemical and biophysical characterization of this potentially important form of AS.     

Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

Rotenone decreases intracellular aldehyde dehydrogenase activity: implications for the pathogenesis of Parkinson's disease

Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson's disease (PD). Mechanisms of relatively selective rotenone‐induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the ‘catecholaldehyde hypothesis,’ buildup of the autotoxic dopamine metabolite 3,4‐dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F‐labeled catechols were measured in PC12 cells incubated with rotenone (0–1000 nM, 180 min), without or with F‐dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose dependently increased DOPAL, F‐DOPAL, and 3,4‐dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD⁺ was supplied, whereas the direct‐acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone‐induced toxicity in dopaminergic neurons.

     

Measurements of auto‐antibodies to α‐synuclein in the serum and cerebral spinal fluids of patients with Parkinson's disease

Biomarkers for α‐synuclein are needed for diagnosis and prognosis in Parkinson's disease (PD ). Endogenous auto‐antibodies to α‐synuclein could serve as biomarkers for underlying synucleinopathy, but previous assessments of auto‐antibodies have shown variability and inconsistent clinical correlations. We hypothesized that auto‐antibodies to α‐synuclein could be diagnostic for PD and explain its clinical heterogeneity. To test this hypothesis, we developed an enzyme‐linked immunosorbent assay for measuring α‐synuclein auto‐antibodies in human samples. We evaluated 69 serum samples (16 healthy controls (HC ) and 53 PD patients) and 145 CSF samples (52 HC and 93 PD patients) from our Institution. Both serum and CSF were available for 24 participants. Males had higher auto‐antibody levels than females in both fluids. CSF auto‐antibody levels were significantly higher in PD patients as compared with HC , whereas serum levels were not significantly different. CSF auto‐antibody levels did not associate with amyloid‐β_1–42, total tau, or phosphorylated tau. CSF auto‐antibody levels correlated with performance on the Montreal Cognitive Assessment, even when controlled for CSF amyloidβ_1–42. CSF hemoglobin levels, as a proxy for contamination of CSF by blood during lumbar puncture, did not influence these observations. Using recombinant α‐synuclein with N‐ and C‐terminal truncations, we found that CSF auto‐antibodies target amino acids 100 through 120 of α‐synuclein. We conclude that endogenous CSF auto‐antibodies are significantly higher in PD patients as compared with HC , suggesting that they could indicate the presence of underlying synucleinopathy. These auto‐antibodies associate with poor cognition, independently of CSF amyloidβ_1–42, and target a select C‐terminal region of α‐synuclein.

Read the Editorial Highlight for this article on page 433 .

     

Tissue transglutaminase modulates alpha-synuclein oligomerization

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated α-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant α-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective α-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/α-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all α-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal α-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal β-sheet-containing oligomers, the tTG/α-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant α-synuclein variants. We propose that tTG cross-linking imposes structural constraints on α-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.     

Pre‐fibrillar α‐synuclein variants with impaired β‐structure increase neurotoxicity in Parkinson's disease models

The relation of α‐synuclein (αS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure‐based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre‐fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β‐structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function.