Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses

Jackson, Rachel E.; Burrone, Juan

doi:10.3389/fnsyn.2016.00021

Cited by 13 publications

(27 citation statements)

References 44 publications

(83 reference statements)

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“…SPLICS s and SypHy-RGECO reporter plasmids were as described [6, 20]; pEGFPC1 was from Clontech. Verified non-targeting control and rat VAPB and PTPIP51 siRNAs were purchased from GE Healthcare Dharmacon (Accell range).…”

Section: Methodsmentioning

confidence: 99%

“…For analyses of SPLICS s , field stimulations were delivered at frequency of 30 Hz for 10 s. For analyses of FM 4–64, 3 field stimulations of 60 s at 20 Hz were applied at 60 s intervals as described [19]. For analyses of SypHy-RGECO signals, 3 field stimulations of 10 s at 30 Hz were applied at 60 s intervals essentially as described [20]. VAPB-PTPIP51 PLA field stimulations were conducted in the culture plates using the insert adaptor delivering stimulations of 30 Hz for 10 s. Neurons were then fixed and processed for PLAs and immunostaining.…”

Section: Methodsmentioning

confidence: 99%

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The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity

Gómez‐Suaga

Perez‐Nievas

Glennon

et al. 2019

acta neuropathol commun

109

117

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Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by “tethering proteins” that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson’s disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson’s disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson’s disease and FTD/ALS. Electronic supplementary material The online version of this article (10.1186/s40478-019-0688-4) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity

Gómez‐Suaga

Perez‐Nievas

Glennon

et al. 2019

acta neuropathol commun

109

117

View full text Add to dashboard Cite

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“…These cultures recapitulate several features of in vivo neuronal networks including outgrowth of dendrites and axons, formation of synapses between pre- and postsynaptic partners, and presence of dendritic spines. Furthermore, they display synchronous NMDA-R-mediated activity, as measured by calcium imaging at the level of the neuronal network ( Verstraelen et al, 2014 ) and of the individual synapse ( Jackson and Burrone, 2016 ). Hence, such a model can be used to assess the molecular mechanisms that govern synaptic connectivity with high resolution and throughput.…”

Section: Introductionmentioning

confidence: 99%

Image-Based Profiling of Synaptic Connectivity in Primary Neuronal Cell Culture

et al. 2018

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Neurological disorders display a broad spectrum of clinical manifestations. Yet, at the cellular level, virtually all these diseases converge into a common phenotype of dysregulated synaptic connectivity. In dementia, synapse dysfunction precedes neurodegeneration and cognitive impairment by several years, making the synapse a crucial entry point for the development of diagnostic and therapeutic strategies. Whereas high-resolution imaging and biochemical fractionations yield detailed insight into the molecular composition of the synapse, standardized assays are required to quickly gauge synaptic connectivity across large populations of cells under a variety of experimental conditions. Such screening capabilities have now become widely accessible with the advent of high-throughput, high-content microscopy. In this review, we discuss how microscopy-based approaches can be used to extract quantitative information about synaptic connectivity in primary neurons with deep coverage. We elaborate on microscopic readouts that may serve as a proxy for morphofunctional connectivity and we critically analyze their merits and limitations. Finally, we allude to the potential of alternative culture paradigms and integrative approaches to enable comprehensive profiling of synaptic connectivity.

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“…Recently developed tools are beginning to enable optogenetic interrogation of synaptic function. Calcium indicators targeted to presynaptic boutons [83] report Ca 2+ entry at the site of vesicle release. pH indicators targeted to the lumen of synaptic vesicles report the de-acidification that occurs during vesicle fusion.…”

Section: Optogenetics For Phenotypic Screeningmentioning

confidence: 99%

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond

Zhang

Cohen

2017

Trends in Biotechnology

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Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, and in particular tools for all-optical electrophysiology, now provide means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline, from target identification and validation, to target-based and phenotypic screens, to clinical trials.

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Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses

Cited by 13 publications

References 44 publications

The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity

The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity

Image-Based Profiling of Synaptic Connectivity in Primary Neuronal Cell Culture

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond

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