Multidisciplinary Approaches for Characterizing Synaptic Vesicle Proteins

Leenders, Miriam; Gerwin, Claudia; Sheng, Zu‐Hang

doi:10.1002/0471142301.ns0207s28

Cited by 6 publications

(7 citation statements)

References 18 publications

(17 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Major progress has been made toward identifying proteins of the release machinery at the transmitter release site and characterizing the effects of these proteins on synaptic transmission (1,2,4,33,(53)(54)(55)(56)(57)(58)(59)(60). Here we identify CRMP-2 as a novel "neuromodulator" of N-type Ca 2ϩ channels.…”

Section: Discussionmentioning

confidence: 95%

An Atypical Role for Collapsin Response Mediator Protein 2 (CRMP-2) in Neurotransmitter Release via Interaction with Presynaptic Voltage-gated Calcium Channels

Brittain

Piekarz

Wang

et al. 2009

Journal of Biological Chemistry

182

286

View full text Add to dashboard Cite

Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate and axonal growth of neurons through protein-protein interactions. Their functions in presynaptic biology remain unknown. Here, we identify the presynaptic N-type Ca 2؉ channel (CaV2.2) as a CRMP-2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus. Both proteins co-localize within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to enhanced green fluorescent protein caused a significant increase in Ca 2؉ channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca 2؉ current density was not due to a change in channel gating. Rather, cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2-overexpressing neurons. These neurons also exhibited a significant increase in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-enhanced green fluorescent protein-overexpressing neurons elicited a significant increase in release of glutamate compared with control neurons. influx coincides with synapse formation, we sought to identify proteins that might modulate Ca 2ϩ channel behavior and transmitter release during synaptogenesis. A proteomic screen of growth cone-associated proteins (see supplemental Fig. S1) and proteins interacting with the ␣1B subunit of CaV2.2 (8) identified collapsin response mediator protein-2 (CRMP-2) as a candidate CaV2.2-interacting protein.CRMPs are intracellular phosphoproteins implicated in neuronal growth cone advance and migration (9 -13). In neurons, CRMPs are expressed in the lamellipodia and filopodia of growth cones, in the shafts of axons, and in cell bodies. They contribute to axon formation by binding to tubulin heterodimers and promoting assembly of microtubules (14). CRMP phosphorylation causes dissociation from microtubules, leading to axonal outgrowth arrest (10,15,16). Overexpression of CRMP-2 in cultured hippocampal neurons induces extra axons (12), whereas CRMP-3/4/5 have been shown to be necessary for neurite extension (10), branching (17), and growth cone formation (18). The breadth of interactions of CRMP proteins with motor proteins, kinases, enzymes, and endocytosis-exocytosis-related proteins suggests that CRMPs may serve as adaptors/scaffold molecules (10). Although neurotransmission was not affected in CRMP-1 knock-out mice (19), long term potentiation, spatial learning, and memory were affected in both CRMP-1 and CRMP-3 knock-out mice (19,20), suggesting a role for CRMPs in neurotransmission as well as synaptogenesis. As potential binding partners, CRMPs could affect neurotransmission by a physical interaction with either the synaptic machinery or CaV2.2 itself.

show abstract

Section: Discussionmentioning

confidence: 95%

An Atypical Role for Collapsin Response Mediator Protein 2 (CRMP-2) in Neurotransmitter Release via Interaction with Presynaptic Voltage-gated Calcium Channels

Brittain

Piekarz

Wang

et al. 2009

Journal of Biological Chemistry

182

286

View full text Add to dashboard Cite

show abstract

“…Synaptosome preparations from WT and mutant hAPP Tg mouse brains or AD patient brains and control subjects were collected using Percoll gradient centrifugation as described in the protocol (Leenders et al, 2004). Cortex tissues were homogenized in ice cold Sucrose Buffer [5 mM HEPES, 1 mM EDTA, 0.32 M sucrose and protease inhibitors (Roche, Indianapolis, IN), pH 7.4].…”

Section: Methodsmentioning

confidence: 99%

“…15 µg of protein from synaptosome and post nuclear supernatant (PNS) homogenates were resolved by 4–12% SDS-PAGE for sequential Western blots on the same membranes after stripping between each application of antibody. For multiple detection with different antibodies, blots were first stripped in a solution of 62.5 mM Tris-HCl, pH 7.5, 20 mM dithiothreitol and 1% SDS for 15 min at 50°C with agitation and then washed with TBS/0.1% Tween-20 for 2 × 15 min (Leenders et al, 2004; Cai et al, 2010). …”

Section: Methodsmentioning

confidence: 99%

Impaired retrograde transport of axonal autophagosomes contributes to autophagic stress in Alzheimer’s disease neurons

Tammineni

Feng

et al. 2017

eLife

124

102

View full text Add to dashboard Cite

Neurons face unique challenges of transporting nascent autophagic vacuoles (AVs) from distal axons toward the soma, where mature lysosomes are mainly located. Autophagy defects have been linked to Alzheimer’s disease (AD). However, the mechanisms underlying altered autophagy remain unknown. Here, we demonstrate that defective retrograde transport contributes to autophagic stress in AD axons. Amphisomes predominantly accumulate at axonal terminals of mutant hAPP mice and AD patient brains. Amyloid-β (Aβ) oligomers associate with AVs in AD axons and interact with dynein motors. This interaction impairs dynein recruitment to amphisomes through competitive interruption of dynein-Snapin motor-adaptor coupling, thus immobilizing them in distal axons. Consistently, deletion of Snapin in mice causes AD-like axonal autophagic stress, whereas overexpressing Snapin in hAPP neurons reduces autophagic accumulation at presynaptic terminals by enhancing AV retrograde transport. Altogether, our study provides new mechanistic insight into AD-associated autophagic stress, thus establishing a foundation for ameliorating axonal pathology in AD.DOI: http://dx.doi.org/10.7554/eLife.21776.001

show abstract

“…Isolation of synaptosomes from the forebrain of a 6-week-old male Wistar rat was adapted from Leenders et al (39). After harvesting the F4 fraction from the 15%/23% interface of a four step (23%, 15%; 10%, 3%) Percoll gradient (Sigma-Aldrich), the synaptosomes (typical diameter Ϸ0.5 m) were centrifuged for 15 min at 15,000 ϫ g and 4°C and resuspended in 1 ml of RPMI 1640 w/Glut 25 mM Hepes medium (Invitrogen) containing 0.1% BSA (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry

Maurer

Sodeik

Grünewald

2008

Proc. Natl. Acad. Sci. U.S.A.

160

148

View full text Add to dashboard Cite

The concerted action of four viral glycoproteins and at least one cellular receptor is required to catalyze herpes simplex virus 1 entry into host cells either by fusion at the plasma membrane or intracellularly after internalization by endocytosis. Here, we applied cryo electron tomography to capture 3D intermediates from Herpes simplex virus 1 fusion at the plasma membrane in their native environment by using two model systems: adherent cells and synaptosomes. The fusion process was delineated as a series of structurally different steps. The incoming capsid separated from the tegument and was closely surrounded by the cortical cytoskeleton. After entry, the viral membrane curvature changed concomitantly with a reorganization of the envelope glycoprotein spikes. Individual glycoprotein complexes in transitional conformations during pore formation and dilation revealed the complex viral fusion mechanism in action. Snapshots of the fusion intermediates provide unprecedented details concerning the overall structural changes occurring during herpesvirus entry. Moreover, our data suggest that there are two functional ''poles'' of the asymmetric herpesvirion: one related to cell entry, and the other formed during virus assembly.cryo electron tomography ͉ herpesvirus ͉ synaptosomes ͉ glycoproteins M embrane fusion is essential for cell-cell fusion during development, intracellular membrane trafficking, exoand endocytosis as well as virus entry into and egress from cells. Herpes simplex virus type 1 (HSV-1) enters host cells via membrane fusion. HSV-1 is the prototype of the family Herpesviridae, which includes a number of other human pathogens like varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus. It has a complex structure consisting of an icosahedral, DNA-containing capsid, which is asymmetrically located within the virion and surrounded by an amorphous protein layer called the tegument, and a membrane envelope heterogeneously studded with morphologically distinct spikes formed by 12 different glycoprotein species (1). Five of these glycoproteins (gB, gC, gD, and the complex of gH and gL) mediate the entry of HSV-1 into host cells (2). The specific mode of entry is cell-type dependent. Neurons and Vero cells (an African monkey kidney cell line) are infected by direct fusion at the plasma membrane and subsequent release of the capsid into the cytosol, whereas in keratinocytes and HeLa cells, the primary step in entry involves endocytosis (3). After initial attachment by a nonessential interaction of the viral glycoprotein gC with heparan sulfate proteoglycans on the plasma membrane, entry proceeds by binding of gD to one of its three known cellular receptors: herpesvirus entry mediator (HVEM), nectin-1, or modified heparan sulfate (2). The heterodimer gH/gL and the homotrimer gB are required for membrane fusion (2). So far, it is unclear how the interactions between the four essential entry-associated viral glycoproteins are coordinated to mediate membrane fusio...

show abstract

Multidisciplinary Approaches for Characterizing Synaptic Vesicle Proteins

Cited by 6 publications

References 18 publications

An Atypical Role for Collapsin Response Mediator Protein 2 (CRMP-2) in Neurotransmitter Release via Interaction with Presynaptic Voltage-gated Calcium Channels

An Atypical Role for Collapsin Response Mediator Protein 2 (CRMP-2) in Neurotransmitter Release via Interaction with Presynaptic Voltage-gated Calcium Channels

Impaired retrograde transport of axonal autophagosomes contributes to autophagic stress in Alzheimer’s disease neurons

Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry

Contact Info

Product

Resources

About