FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that over-expression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson's disease, we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP-decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 over-expression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7-induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7-induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of α-synuclein and could be beneficial in patients with Parkinson's disease. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Delayed increase of thrombocyte levels after a single sub-anesthetic dose of ketamine - A randomized trial.

Recently, ketamine has been investigated as a potential antidepressant option for treatment resistant depression. Unlike traditional drugs, it yields immediate effects, most likely via increased glutamatergic transmission and synaptic plasticity. However, ketamine administration in humans is systemic and its long-term impact on blood parameters has not yet been described in clinical studies. Here we investigated potential sustained effects of ketamine administration (0.5 mg/kg ketamine racemate) on hematological and biochemical values in plasma and serum in a randomized double-blinded study. 80 healthy young participants were included and whole blood samples were collected 5 days before, and 14 days after the infusion. To assess the group effect, repeated measure analyses of co-variance (rmANCOVA) were conducted for the following blood parameters: levels of sodium, potassium, calcium, hemoglobin and number of erythrocytes, lymphocytes, and thrombocytes. RmANCOVA revealed a significant time by treatment effect on thrombocyte levels (F1, 74 = 13.54, p < 0.001, eta = 0.155), driven by an increase in the ketamine group (paired t-test, t = -3.51, df = 38, p = 0.001). Specificity of thrombocyte effect was confirmed by logistic regression, and in addition, no other coagulation parameters showed significant interaction. Moreover, the relative increase in the ketamine group was stable across sexes and not predicted by age, BMI, smoking, alcohol or drug use, and contraception. Our results describe aftereffects of sub-anesthetic ketamine administration on blood coagulation parameters, which should be considered especially when targeting psychiatric populations with relevant clinical comorbidities.

Rab7 induces clearance of α-synuclein aggregates.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, we hypothesized that over-expressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis, we expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila melanogaster. In HEK293 cells, EGFP-Rab7-decorated vesicles contain α-synuclein. Rab7 over-expression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Time-lapse microscopy confirmed that particles frequently disappeared with Rab7 over-expression. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 over-expression, presumably representing autolysosomes. Rab7 over-expression reduced apoptosis and the percentage of dead cells in trypan blue staining. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. These beneficial effects were not produced by Rab7 missense mutations causing Charcot Marie Tooth neuropathy, or by the related GTPases Rab5, Rab9, or Rab23. Using mass spectrometry, we identified Rab7 in neuromelanin granules purified from human substantia nigra, indicating that Rab7 might be involved in the biogenesis of these possibly protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease. Cells over-expressing aggregation-prone A53T alpha-synuclein develop cytoplasmic aggregates mimicking changes observed in Parkinson's disease. When following cells in time-lapse microscopy, some few cells are able to remove these aggregates (Opazo et al. 2008). We now show that the percentage of cells clearing all aggregates from their cytosol is greatly increased with Rab7 over-expression, indicating that availability of Rab7 is rate-limiting for autophagic clearance of aggregates. The functional significance of this effect in neurons was confirmed in a Drosophila melanogaster model of Parkinson's disease.

Expression of the postsynaptic scaffold PSD-95 and development of synaptic physiology during giant terminal formation in the auditory brainstem of the chicken.

In the avian nucleus magnocellularis (NM) endbulb of Held giant synapses develop from temporary bouton terminals. The molecular regulation of this process is not well understood. Furthermore, it is unknown how the postsynaptic specialization of the endbulb synapses develops. We therefore analysed expression of the postsynaptic scaffold protein PSD-95 during the transition from bouton-to-endbulb synapses. PSD-95 has been implicated in the regulation of the strength of glutamatergic synapses and could accordingly be of functional relevance for giant synapse formation. PSD-95 protein was expressed at synaptic sites in embryonic chicken auditory brainstem and upregulated between embryonic days (E)12 and E16. We applied immunofluorescence staining and confocal microscopy to quantify pre-and postsynaptic protein signals during bouton-to-endbulb transition. Giant terminal formation progressed along the tonotopic axis in NM, but was absent in low-frequency NM. We found a tonotopic gradient of postsynaptic PSD-95 signals in NM. Furthermore, PSD-95 immunosignals showed the greatest increase between E12 and E15, temporally preceding the bouton-to-endbulb transition. We then applied whole-cell electrophysiology to measure synaptic currents elicited by synaptic terminals during bouton-to-endbulb transition. With progressing endbulb formation postsynaptic currents rose more rapidly and synapses were less susceptible to short-term depression, but currents were not different in amplitude or decay-time constant. We conclude that development of presynaptic specializations follows postsynaptic development and speculate that the early PSD-95 increase could play a functional role in endbulb formation.