Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy.

Synucleinopathies such as dementia with Lewy bodies or Parkinson's disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation.

Neutrophil gelatinase-associated lipocalin and end-stage renal disease: it is not all about the kidneys!

INTRODUCTION: Neutrophil gelatinase-associated lipocalin (NGAL) is a powerful biomarker for the early detection of acute kidney injury. However, recent data suggest that NGAL also plays an important role in chronic kidney disease (CKD), reflecting the level of acute kidney damage within the CKD condition. To study whether elevated NGAL levels in CKD are a consequence of damaged tubular cells or rather due to extrarenal production, we investigated NGAL levels in anephric patients on dialysis. METHODS: Plasma NGAL levels were investigated in 14 dialysis patients who underwent bilateral nephrectomy (anephric group), 18 anuric dialysis patients with remaining kidneys (dialysis group) and 12 healthy patients (healthy group). RESULTS: Plasma NGAL levels were significantly lower in the healthy group compared with the anephric group (143 vs. 981 ng/mL; P < 0·001) or the dialysis group (143 vs. 838 ng/mL; P < 0·001), respectively. However, NGAL levels did not differ between the anephric group and the dialysis group (981 vs. 838 ng/mL; P = 0·19). DISCUSSION: Assuming that NGAL is highly expressed in chronically damaged kidneys due to tubular stress, there should be significantly less NGAL in anephric patients compared with anuric dialysis patients with remaining kidneys. In contrast to this hypothesis, we found no difference in NGAL expression between these two groups, proving the entire extrarenal NGAL production in anephric patients and suggesting that the tubular NGAL expression seems to be negligible in anuric dialysis patients.

Converse modulation of toxic alpha-synuclein oligomers in living cells by N'-benzylidene-benzohydrazide derivates and ferric iron.

Intracellular alpha-synuclein (alpha-syn) aggregates are the pathological hallmark in several neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Recent evidence suggests that small oligomeric aggregates rather than large amyloid fibrils represent the main toxic particle species in these diseases. We recently characterized iron-dependent toxic alpha-syn oligomer species by confocal single molecule fluorescence techniques and used this aggregation model to identify several N'-benzylidene-benzohydrazide (NBB) derivatives inhibiting oligomer formation in vitro. In our current work, we used the bioluminescent protein-fragment complementation assay (BPCA) to directly analyze the formation of toxic alpha-syn oligomers in cell culture and to investigate the effect of iron and potential drug-like compounds in living cells. Similar to our previous findings in vitro, we found a converse modulation of toxic alpha-syn oligomers by NBB derivates and ferric iron, which was characterized by an increase in aggregate formation by iron and an inhibitory effect of certain NBB compounds. Inhibition of alpha-syn oligomer formation by the NBB compound 293G02 was paralleled by a reduction in cytotoxicity indicating that toxic alpha-syn oligomers are present in the BPCA cell culture model and that pharmacological inhibition of oligomer formation can reduce toxicity. Thus, this approach provides a suitable model system for the development of new disease-modifying drugs targeting toxic oligomer species. Moreover, NBB compounds such as 293G02 may provide useful tool compounds to dissect the functional role of toxic oligomer species in cell culture models and in vivo.

Modelling Ser129 phosphorylation inhibits membrane binding of pore-forming alpha-synuclein oligomers.

BACKGROUND: In several neurodegenerative diseases, hyperphosphorylation at position Ser129 is found in fibrillar deposits of alpha-synuclein (asyn), implying a pathophysiological role of asyn phosphorylation in neurodegeneration. However, recent animal models applying asyn phosphorylation mimics demonstrated a protective effect of phosphorylation. Since metal-ion induced asyn oligomers were identified as a potential neurotoxic aggregate species with membrane pore-forming abilities, the current study was undertaken to determine effects of asyn phosphorylation on oligomer membrane binding. METHODS: We investigated the influence of S129 phosphorylation on interactions of metal-ion induced asyn oligomers with small unilamellar lipid vesicles (SUV) composed of POPC and DPPC applying the phosphorylation mimic asyn129E. Confocal single-particle fluorescence techniques were used to monitor membrane binding at the single-particle level. RESULTS: Binding of asyn129E monomers to gel-state membranes (DPPC-SUV) is slightly reduced compared to wild-type asyn, while no interactions with membranes in the liquid-crystalline state (POPC-SUV) are seen for both asyn and asyn129E. Conversely, metal-ion induced oligomer formation is markedly increased in asyn129E. Surprisingly, membrane binding to POPC-SUV is nearly absent in Fe(3+) induced asyn129E oligomers and markedly reduced in Al(3+) induced oligomers. CONCLUSION: The protective effect of pseudophosphorylation seen in animal models may be due to impeded oligomer membrane binding. Phosphorylation at Ser129 may thus have a protective effect against neurotoxic asyn oligomers by preventing oligomer membrane binding and disruption of the cellular electrophysiological equilibrium. Importantly, these findings put a new complexion on experimental pharmaceutical interventions against POLO-2 kinase.

Inhibition and disaggregation of α-synuclein oligomers by natural polyphenolic compounds.

Aggregation of alpha-synuclein (αS) into oligomers is critically involved in the pathogenesis of Parkinson's disease (PD). Using confocal single-molecule fluorescence spectroscopy, we have studied the effects of 14 naturally-occurring polyphenolic compounds and black tea extract on αS oligomer formation. We found that a selected group of polyphenols exhibited potent dose-dependent inhibitory activity on αS aggregation. Moreover, they were also capable of robustly disaggregating pre-formed αS oligomers. Based upon structure-activity analysis, we propose that the key molecular scaffold most effective in inhibiting and destabilizing self-assembly by αS requires: (i) aromatic elements for binding to the αS monomer/oligomer and (ii) vicinal hydroxyl groups present on a single phenyl ring. These findings may guide the design of novel therapeutic drugs in PD.

Generation of ferric iron links oxidative stress to α-synuclein oligomer formation.

BACKGROUND: Synucleinopathies such as Parkinson's disease are characterized by the deposition of aggregated α-synuclein in affected brain areas. As genes involved in mitochondrial function, mitochondrial toxins, and age-related mitochondrial impairment have been implicated in Parkinson's disease pathogenesis, an increase in reactive oxygen species resulting from mitochondrial dysfunction has been speculated to induce α-synuclein aggregation. In vitro, pore-forming, SDS-resistant α-synuclein oligomers are formed in presence of ferric iron and may represent an important toxic particle species. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the interplay of reactive oxygen species, antioxidants and iron oxidation state in regard to α-synuclein aggregation using confocal single particle fluorescence spectroscopy, Phenanthroline spectrometry and thiobarbituric acid reactive substances assay. We found that the formation of α-synuclein oligomers in presence of Fe³âº is due to a direct interaction. In contrast, oxidizing agents and hydroxyl radicals generated in the Fenton reaction did not directly affect α-synuclein oligomerization. However, reactive oxygen species could enhance aggregation via oxidation of ferrous to ferric iron when iron ions were present. CONCLUSIONS/SIGNIFICANCE: Our data thus indicate that oxidative stress affects α-synuclein aggregation via oxidation of iron to the ferric state. This provides a new perspective on the role of mitochondrial toxins and mitochondrial dysfunction in the pathogenesis of Parkinson's disease.