Aims

It is the aim to study the mechanisms of a-synuclein aggregation in cells at atomic resolution to obtain detailed mechanistic insights of the kinetic aggregtaion process including the cellular players involved. For this a structure activity relationship using in vitro and in cell NMR of a-synuclein is established.

Methods

in cell NMR; solution and solid state NMR; cryo EM; recombinant proteins; kinetics; cell biology

Results

It is known and further demonstrated that in cells (and in vitro) monomeric a-synuclein is not entirely unfolded attributed to the interaction between its positively charged N-terminus and its negatively charged C-terminus, which protects from aggregation. Furthermore, a-synuclein is interacting transiently with protein chaperones (such as HSP90), which regulate a-synuclein function and further interfere with aggregation. Upon down regulation of HSP90 a-synuclein is dislocated to the mitochondria where it unfolds further by transient interaction with a positively charged protein AK2 losing the intra-synuclein aggregation protection enabling protein aggregation. Once fibrils are present they interact with monomeric a-synuclein transiently through inter-molecular interaction between the negatively charged C-terminus of the fibrils and the N-terminal positive charged monomer, which again yields an unfolded aggregation-prone monomer yielding the secondary nucleation process, which is a very important acceleration component of the aggregation process.

Burmann et al Nature 2020 577:127-132.

Conclusions

The detailed structural studies show one mechanism of a-synuclein aggregation in cells at near atomic resolution elucidating thereby that the unfolding of a-synuclein is key for aggregation both at the initial step through interaction with other cellular players or during the secondary nucleation mechanism of the aggregation kinetics.

Aims

Although the exact mechanism of neuronal aggregate formation and death remains elusive, recent research suggests a-Syn-mediated alterations in autophagy. In this study, we aim to investigate the effect of a-Syn on autophagy turnover in cultured human DA neurons and in human postmortem brain tissue.

Methods

We investigate the effect of a-Syn overexpression on autophagy using a combination of molecular biology and immunochemistry. Immunohistochemistry was used to study protein abundance in SNc DA neurons from human postmortem brain tissue.

Results

We found a-Syn overexpression to reduce autophagy turnover by compromising the fusion of autophagosomes with lysosomes, thus leading to a decrease in the formation of autophagolysosomes. In accord with a compensatory increase in the plasma membrane fusion of autophagosomes, a-Syn enhanced the number of extracellular vesicles (EV) and the abundance of autophagy-associated proteins in these EVs. Mechanistically, a-Syn interacted with and decreased the abundance of the v-SNARE protein SNAP29, a member of the SNARE complex mediating autophagolysosome fusion. In line, SNAP29 knockdown mimicked the effect of a-Syn on autophagy whereas SNAP29 co-expression reversed the a-Syn-induced changes on autophagy turnover and EV release and ameliorated DA neuronal cell death. In addition, we found a stage-dependent reduction of SNAP29 in SNc DA neurons from human postmortem brain tissue of Lewy body pathology (LBP) cases.

Conclusions

In summary, our results demonstrate a previously unknown effect of a-Syn on intracellular autophagy-associated SNARE proteins and, as a consequence, a reduced autophagolysosome fusion, and will therefore support the investigation of autophagy-associated pathological changes in PD.

Aims

A pro-inflammatory micro-environment has been suggested to promote abnormal folding, aggregation and spread of alpha-synuclein (asyn) in Parkinson's disease (PD). We have previously shown that levels of the major histocompatibility complex class two transactivator (Mhc2ta) regulate MHCII expression, microglial activation, dopaminergic neurodegeneration and motor impairment upon overexpression of human asyn in rats. The purpose of this study was to determine Mhc2ta effects on seeded asyn pathology, local- and peripheral immune profiles.

Methods

We used a human asyn pre-formed fibril (PFF)-seeded model in VRA4-congenic rats, where normal genetic variation in the VRA4 locus make DA.VRA4 rats express lower Mhc2ta levels than DA. PFFs were injected unilaterally to the striatum two weeks after ipsilateral administration of recombinant adeno-associated vectors carrying human asyn to the substantia nigra. We evaluated motor impairment at 2, 5 and 8 weeks and histology and brain- and peripheral immune cell profiles at baseline, 4 and 8 weeks.

Results

Mhc2ta significantly regulated seeded asyn pathology as well as central and peripheral immune profiles. DA.VRA4 had a wider extent and anatomical distribution of insoluble asyn inclusions, enhanced motor impairment and more dopaminergic neurodegeneration compared to DA rats. DA.VRA4 also displayed higher levels of proinflammatory cytokines in serum and higher CD11b+CD45low cell counts in cell-sorted brain tissue, but a lower MHCII expression per cell compared to DA.

Conclusions

We conclude that Mhc2ta regulates seeded asyn pathology as well as local and peripheral immune profiles in vivo. This makes Mhc2ta a promising therapeutic target for the progressive neuropathology in PD.

Aims

Parkinson's disease (PD) characterizes by the selective loss of dopaminergic neurons, mitochondrial damage, oxidative stress (OS), and autophagy disruption. Autophagy is essential for neuronal maintenance. Therefore, we evaluated the protective effect of autophagy's inducers trehalose (Tre) and metformin (Met) in the paraquat (PQ)-induced PD model.

Methods

SH-SY5Y dopaminergic cells were pretreated for 1 h with Tre or Met, followed by PQ for 24 or 48 h. Autophagy was evaluated by transmission electron microscopy and western blot. Cytoplasmic and mitochondrial OS was detected using dihydroethidium and MitoTracker Red CM-H₂XROS, respectively. Peroxiredoxins hyperoxidation (PrxSO₃), which reflects a highly oxidizing environment, was analyzed by immunodetection. Finally, we assessed autophagy inducers on mitochondrial function (MTT assay) and cell viability (trypan blue exclusion).

Results

Autophagy induced by Tre and Met increased LC3-II and autophagosomes number in different maturation stages compared to the control. PQ inhibits autophagy flux, which was prevented by Tre and Met. Next, we evaluated the effect of autophagy inducers on PQ-induced OS. We showed that Tre did not have an antioxidant effect on the cytoplasm, while Met increased OS. However, both decreased PQ-mediated mitochondrial OS. Also, Tre decreased PQ-induced PrxSO3. Mitochondria are the main target of PQ. PQ-mediated decreased in mitochondrial activity was prevented by Tre and Met. Interestingly, autophagy inducers showed a protective effect on PQ-induced cell death.

Conclusions

Autophagy stimulated by Tre and Met has a protective effect against PQ toxicity by decreasing mitochondrial OS and cell death. Therefore, both autophagy inducers have promising potential to prevent PD development.

Aims

Parkinson's disease (PD) patients have shown an increase in copper (Cu) levels in the blood and cerebrospinal fluid. Besides, wildtype or mutated α-Synuclein requires an environmental factor, such as exposure to copper, to increase its cytotoxicity. Therefore, we aimed to elucidate the mechanisms by which Cu regulates dopaminergic cell death in vivo.

Methods

Mice were treated with CuSO₄ at 100, 250, and 500 ppm in drinking water ad libitum for ten months. Next, we evaluated whether exposure to Cu affects mice's motor function by assessing mice gait. Then mice were intracardially perfused, and brain sections were obtained to analyze dopaminergic neurons with a specific anti-tyrosine hydroxylase (TH) antibody by immunofluorescence. Protein brain extracts were also obtained, and TH, α-Syn, and the autophagy marker LC3-II were assessed by western blot (WB).

Results

Mice treated with Cu showed an abnormal pattern in the stride's length and width compared to the control. Importantly, mice exposed to Cu showed a reduction in dopaminergic neurons in a dose-dependent manner. It was also corroborated by WB, observing that Cu decreases the dopaminergic neurons marker (TH). Besides, we found that Cu increases the expression of α-Syn, a characteristic neuronal protein of Lewy bodies developed in PD, and also the LC3-II protein, both in a dose-dependent manner.

Conclusions

Our results show that chronic Cu exposure has a neurotoxic effect that alters the motor function and induces dopaminergic neuronal death.

Aims

Alpha Synuclein (αSyn) is the major component of the hallmark Lewy body aggregates found in Parkinson’s Disease patients. αSyn aggregation and Lewy body formation is hypothesized to drive Parkinson’s pathophysiology and neuron death. The relationship between αSyn and phospholipid metabolism is emerging as critical in its normal function and its propensity for aggregation and toxicity. Here we identify lysophosphatidylcholine acyltransferase 1 (LPCAT1) as a novel regulator of αSyn pathology and aim to characterize this relationship.

Methods

We used transcriptional profiling of primary neuronal cultures transduced with the neurotoxic 3K αSyn (harboring familial E46K and two flanking lysine mutations) for target discovery. We next employed siRNA knockdown and Lentiviral overexpression of LPCAT1 in both the pre-formed fibril (PFF) αSyn aggregation model and 3K toxicity model. We also manipulated phospholipids regulated by LPCAT1 by adding them directly to primary neuron cultures. Additionally, we analyzed human Parkinson’s patient brain tissue.

Results

Transcriptional profiling showed enrichment in differentially regulated genes related to lipid biosynthesis, this included LPCAT1. Knockdown of LPCAT1 reduced 3K αSyn inclusion burden. Overexpression of LPCAT1 or addition of phosphatidylcholine containing phospholipids increased PFF pS129 pathology in neurons. We also found LPCAT1 mRNA to be upregulated in Parkinson’s patient amygdala tissue with confirmed synuclein pathology.

Conclusions

LPCAT1 is a novel lipid regulatory gene that promotes αSyn pathology in vitro and is possibly upregulated in Parkinson’s patient’s brain tissue. Further research is needed to determine how LPCAT1 and the phospholipids it regulates effect αSyn pathology.