Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

Background Parkinson’s disease (PD)-associated E3 ubiquitin ligase Parkin is enriched at glutamatergic synapses, where it ubiquitinates multiple substrates, suggesting that its mutation/loss-of-function could contribute to the etiology of PD by disrupting excitatory neurotransmission. Here, we evaluate the impact of four common PD-associated Parkin point mutations (T240M, R275W, R334C, G430D) on glutamatergic synaptic function in hippocampal neurons. Results We find that expression of these point mutants in cultured hippocampal neurons from Parkin-deficient and Parkin-null backgrounds alters NMDA and AMPA receptor-mediated currents and cell-surface levels and prevents the induction of long-term depression. Mechanistically, we demonstrate that Parkin regulates NMDA receptor trafficking through its ubiquitination of GluN1, and that all four mutants are impaired in this ubiquitinating activity. Furthermore, Parkin regulates synaptic AMPA receptor trafficking via its binding and retention of the postsynaptic scaffold Homer1, and all mutants are similarly impaired in this capacity. Conclusion Our findings demonstrate that pathogenic Parkin mutations disrupt glutamatergic synaptic transmission in hippocampal neurons by impeding NMDA and AMPA receptor trafficking. Such effects may contribute to the pathophysiology of PD in PARK2 patients

Endolysosomal degradation of Tau and its role in glucocorticoid-driven hippocampal malfunction

Emerging studies implicate Tau as an essential mediator of neuronal atrophy and cognitive impairment in Alzheimer's disease (AD), yet the factors that precipitate Tau dysfunction in AD are poorly understood. Chronic environmental stress and elevated glucocorticoids (GC), the major stress hormones, are associated with increased risk of AD and have been shown to trigger intracellular Tau accumulation and downstream Tau-dependent neuronal dysfunction. However, the mechanisms through which stress and GC disrupt Tau clearance and degradation in neurons remain unclear. Here, we demonstrate that Tau undergoes degradation via endolysosomal sorting in a pathway requiring the small GTPase Rab35 and the endosomal sorting complex required for transport (ESCRT) machinery. Furthermore, we find that GC impair Tau degradation by decreasing Rab35 levels, and that AAV-mediated expression of Rab35 in the hippocampus rescues GC-induced Tau accumulation and related neurostructural deficits. These studies indicate that the Rab35/ESCRT pathway is essential for Tau clearance and part of the mechanism through which GC precipitate brain pathology.work was supported by NIH grants R01NS080967and R21MH 104803 to C.L.W., Portuguese Foundation for Science & Technology (FCT) PhD fellowships to J. Vaz-Silva and T. Meira (PD/BD/105938/2014; PD/BD/113700/2015, respectively), and the following grants to I.S.: FCT Investigator grant IF/01799/2013, the Portuguese North Regional Operational Program (ON.2) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER), the Project Estratégico co-funded by FCT (PEst-C/SAU/LA 0026/2013) and the European Regional Development Fund COMPETE (FCOMP-01 -0124-FEDER-037298) as well as the project NORTE- 01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)info:eu-repo/semantics/publishedVersio

An Acidic Motif Retains Vesicular Monoamine Transporter 2 on Large Dense Core Vesicles

The release of biogenic amines from large dense core vesicles (LDCVs) depends on localization of the vesicular monoamine transporter VMAT2 to LDCVs. We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs. Deletion of the acidic cluster promotes the removal of VMAT2 from LDCVs during their maturation. The motif thus acts as a signal for retention on LDCVs. In addition, replacement of the serines by glutamate to mimic phosphorylation promotes the removal of VMAT2 from LDCVs, whereas replacement by alanine to prevent phosphorylation decreases removal. Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism

(A and B) FM5-95 destaining curves for individual EGFP-Synapsin1a puncta at boutons with (A) or without (B) Piccolo

Shown in black are examples of FM destaining curves for which there was either dramatic (>40%; triangles) or minimal (<p><b>Copyright information:</b></p><p>Taken from "Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis"</p><p></p><p>The Journal of Cell Biology 2008;181(5):831-846.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396795.</p><p></p

(A) Schematic diagram of the pZOff/VAMP2-HRP vector used to express VAMP2-HRP with or without Pclo28 shRNA

(B) Hippocampal neurons electroporated at the time of plating with pZOff/VAMP2-HRP or pZOff/VAMP2-HRP/Pclo28 and immunostained at 6 DIV with HRP (green) and Piccolo (red) antibodies. Note the lack of Piccolo immunoreactivity in Pclo28-expressing axons. (C) Transmission EM micrograph of a VAMP2-HRP–positive bouton containing both clear-centered (unlabeled SV) and dark-centered SVs (labeled SV). HRP-labeled vesicles are easily distinguished from 80-nm DCVs and other labeled structures. (D) Synaptic boutons from VAMP2-HRP–expressing (labeled bouton) and untransfected (unlabeled bouton) neurons. Arrowheads denote synaptic junctions, identified based on electron-dense PSDs. (E) An excitatory synapse formed between a pZOff/VAMP2-HRP/Pclo28 (Pclo28)-transfected presynaptic bouton and a postsynaptic spine. (F) An excitatory synapse formed between a pZOff/VAMP2-HRP (control) transfected presynaptic bouton and a postsynaptic spine.<p><b>Copyright information:</b></p><p>Taken from "Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis"</p><p></p><p>The Journal of Cell Biology 2008;181(5):831-846.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396795.</p><p></p

(A) Dispersion kinetics of Synapsin1a in wild-type neurons incubated with (KN62) or without (control) 10 μM KN62 during a 90-s, 10-Hz stimulation

The broken gray line indicates maximal amount of dispersion (expressed as a percentage of initial fluorescence intensity before stimulation) seen in these neurons without KN62 ( > 100 puncta/condition). (B) Dispersion kinetics of Synapsin1a in neurons lacking Piccolo and incubated with (KN62) or without (control) KN62 during a 90-s, 10-Hz stim. For comparison, the broken gray line indicating maximal amount of dispersion observed in wild-type neurons is included ( > 100 puncta per condition). (C) Bar graphs summarizing the percent dispersion of EGFP-Synapsin1a puncta at synapses with (EGFPSyn) or without (EGFPSynPclo28) Piccolo in the presence or absence of KN62. Percent dispersion (100 − the percentage initial fluorescence) was measured at 90 s after a 10-Hz, 90-s stimulation ( = 2 experiments, 905 puncta for control without KN62; three experiments, 1,070 puncta for control + KN62; seven experiments, 2,195 puncta for Pclo28 without KN62; eight experiments, 2,341 puncta for Pclo28 + KN62). (D) Destaining kinetics of FM5-95 at boutons lacking Piccolo in the presence (triangles) or absence (squares) of KN62, during a 5-Hz, 180-s (900 pulse) stimulation ( = 5 experiments, 1,134 puncta for Pclo28 without KN62; two experiments, 437 puncta for Pclo28 + KN62). Error bars indicate SEM.<p><b>Copyright information:</b></p><p>Taken from "Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis"</p><p></p><p>The Journal of Cell Biology 2008;181(5):831-846.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396795.</p><p></p

(A) Images of 14 DIV hippocampal neurons infected with LV/EGFP-Synapsin1a (EGFPSyn, green) or LV/EGFP-Synapsin1a/Pclo28 (EGPFSynPclo28, green) and loaded with FM4-64 (FM, red) with 90 mM K

Note the high degree of match between EGFP-Synapsin1a and FM4-64 in both merged images. Bar, 10 μm. (B) Bar graphs quantifying the percent colocalization between EGFP-Synapsin and FM4-64 puncta in control cultures (EGFPSyn) or those lacking Piccolo (EGFPSynPclo28; > 800 puncta per condition, two experiments). (C) Bar graph of FM4-64 fluorescence intensity at EGFP-Synapsin1a puncta comparing the relative sizes of the TRP of SVs at control synapses (EGFPSyn) and those lacking Piccolo (EGFPSynPclo28). FM intensity values at EGFPSyn-expressing boutons were normalized against those from neighboring uninfected cells to enable cross-coverslip comparison. Mean normalized FM intensity from control boutons was set to 100; that from Pclo28 boutons was ratioed against this value ( > 800 puncta, two experiments). (D and E) Destaining kinetics of the TRP at 10 Hz (D) and 5 Hz (E) comparing synapses with (EGFPSyn) and without (EGFPSynPclo28) Piccolo ( = 5 experiments per condition). (F) Bar graph comparing the size of the RRP of SVs in boutons with (EGFPSyn) or without (EGFPSynPclo28) Piccolo, as determined by either the application of 500 mM sucrose (left) or 2-Hz, 30-s stimulation (right). No significant differences were found ( test, P > 0.5). Sucrose experiments were performed twice for each condition and stimulation experiments three times. (G) Destaining kinetics of the RRP during 2-Hz, 30-s stimulation for control (EGFPSyn) boutons and those lacking Piccolo (EGFPSynPclo28; = 5 experiments per condition). Error bars indicate SEM.<p><b>Copyright information:</b></p><p>Taken from "Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis"</p><p></p><p>The Journal of Cell Biology 2008;181(5):831-846.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396795.</p><p></p