Biology subjects

Rosenmund, C.

Publications and source records attributed to Rosenmund, C..

27 records · Page 2Linked to original sources

bioRxiv2021

Targeted sensors for glutamatergic neurotransmission

Optical report of neurotransmitter release allows visualization of excitatory synaptic transmission. Sensitive genetically-encoded glutamate reporters operating with a range of affinities and emission wavelengths are available. However, without targeting to synapses, the specificity of the fluorescent signal is uncertain, compared to sensors directed at vesicles or other synaptic markers. We fused the state-of-the-art reporter iGluSnFR to glutamate receptor auxiliary proteins in order to target it to postsynaptic sites. Chimeras of Stargazin and gamma-8 that we named SnFR-{gamma}2 and SnFR-{gamma}8, retained function and reported spontaneous glutamate release in hippocampal cells, with apparently diffraction-limited spatial precision. In autaptic neurons on micro island cultures, evoked release could be quantitatively detected at tens of synapses in a field of view whilst evoked currents were recorded simultaneously. These experiments revealed a specific postsynaptic deficit from Stargazin overexpression, resulting in synapses with normal release but without postsynaptic responses. This defect was reverted by delaying overexpression. By working at different calcium concentrations, we determined that SnFR-{gamma}2 is a linear reporter of the global quantal parameters and short term synaptic plasticity, whereas iGluSnFR is not. On average, half of iGluSnFR regions of interest showing evoked fluorescence changes had intense rundown, whereas less than 5% of SnFR-{gamma}2 ROIs did. We provide an open-source analysis suite for extracting quantal parameters including release probability from fluorescence time series of individual and grouped synaptic responses. Taken together, postsynaptic targeting improves several properties of iGluSnFR and further demonstrates the importance of subcellular targeting for optogenetic actuators and reporters.

neuroscience↗

bioRxiv2020

Functional architecture of the synaptic transducers at a central glutamatergic synapse

Neuronal synapses transduce information via the consecutive action of three transducers: voltage-gated Ca2+-channels, fusion-competent synaptic vesicles, and postsynaptic receptors. Their physical distance is thought to influence the speed and efficiency of neurotransmission. However, technical limitations have hampered resolving their nanoscale arrangement. Here, we developed a new method for live-labeling proteins for electron microscopy (EM), revealing that release-competent vesicles preferentially align with Ca2+-channels and postsynaptic AMPA receptors within 20-30 nm and thereby forming a transsynaptic tripartite nanocomplex. Using functional EM, we show that single action potentials cause vesicles within the nanocomplex to fuse with a 50% probability. The loss of the presynaptic scaffold disrupts the formation of the tripartite transducers. Strikingly, the forced transsynaptic alignment of the Ca2+-channel subunit 2{delta}1 and AMPA receptors suffice to restore neurotransmission in a scaffold lacking synapse. Our results demonstrate a synaptic transducer nanocomplex that actively contributes to the organization of central synapses.

neuroscience↗

bioRxiv2020

Bi-allelic variants in TSPOAP1, encoding the active zone protein RIMBP1, cause autosomal recessive dystonia

Dystonia is a debilitating hyperkinetic movement disorder, frequently transmitted as a monogenic trait. Here, we describe homozygous frameshift, nonsense and missense variants in TSPOAP1, encoding the active zone RIM-binding protein 1 (RIMBP1), as a novel genetic cause of autosomal recessive dystonia in seven subjects from three unrelated families. Subjects carrying loss-of-function variants presented with juvenile- onset progressive generalized dystonia, associated with intellectual disability and cerebellar atrophy. Conversely, subjects carrying a pathogenic missense variant (p.Gly1808Ser) presented with isolated adult-onset focal dystonia. In mice, complete loss of RIMBP1, known to reduce neurotransmission, led to motor abnormalities reminiscent of dystonia, decreased Purkinje cell dendritic arborization, and reduced numbers of cerebellar synapses. In vitro analysis of the p.Gly1808Ser variant showed larger spike-evoked calcium transients and enhanced neurotransmission, suggesting that RIMBP1-linked dystonia can be caused by either reduced or enhanced rates of spike-evoked release in relevant neural networks. Our findings establish a direct link between presynaptic RIMBP1 dysfunction and dystonia and highlight the critical role played by well-balanced neurotransmission in motor control and disease pathogenesis.

genetics↗

bioRxiv2020

SynaptoPAC, an Optogenetic Tool for Induction of Presynaptic Plasticity

Optogenetic manipulations have transformed neuroscience in recent years. While sophisticated tools now exist for controlling the firing patterns of neurons, it remains challenging to optogenetically define the plasticity state of individual synapses. A variety of synapses in the mammalian brain express presynaptic long-term potentiation (LTP) upon elevation of presynaptic cyclic adenosine monophosphate (cAMP), but the molecular expression mechanisms as well as the impact of presynaptic LTP on network activity and behavior are not fully understood. In order to establish optogenetic control of presynaptic cAMP levels and thereby presynaptic potentiation, we developed synaptoPAC, a presynaptically targeted version of the photoactivated adenylyl cyclase bPAC. In cultures of hippocampal granule cells, activation of synaptoPAC with blue light increases action potential-evoked transmission, an effect not seen in hippocampal cultures of non-granule cells. In acute brain slices, synaptoPAC activation immediately triggers a strong presynaptic potentiation at mossy fiber terminals in CA3, but not at Schaffer collateral synapse in CA1. Following light-triggered potentiation, mossy fiber transmission decreases within 20 minutes, but remains enhanced still after 30 min. Optogenetic potentiation alters the short-term plasticity dynamics of release, reminiscent of presynaptic LTP. SynaptoPAC is the first optogenetic tool that allows acute light-controlled potentiation of transmitter release at specific synapses of the brain, and will enable to investigate the role of presynaptic potentiation in network function and the animals behavior in an unprecedented manner. Significance StatementSynaptoPAC is a novel optogenetic tool that allows increasing synaptic transmission by light-controlled induction of presynaptic plasticity.

neuroscience↗

bioRxiv2019

Vglut2 expression in dopamine neurons contributes to post-lesional striatal reinnervation

In Parkinsons disease, the most vulnerable neurons are found in the ventral tier of the substantia nigra (SN), while the adjacent dopamine (DA) neurons of the ventral tegmental area (VTA) are mostly spared. Although a significant subset of adult VTA DA neurons expresses Vglut2, a vesicular glutamate transporter, and release glutamate as a second neurotransmitter in the striatum, only very few adult SN DA neurons have this capacity. Previous work has demonstrated that lesions created by neurotoxins such as MPTP and 6-hydroxydopamine (6-OHDA) can upregulate the expression of Vglut2 in surviving DA neurons. Currently, the molecular mechanisms explaining the plasticity of Vglut2 expression in DA neurons are unknown, as are the physiological consequences for DA neuron function and survival. Here we aimed to characterize the developmental expression pattern of Vglut2 in DA neurons and the role of this transporter in post-lesional plasticity in these neurons. Using an intersectional genetic lineage-mapping approach, based on Vglut2-Cre and TH-Flpo drivers, we first found that more than 98% of DA neurons expressed Vglut2 at some point in their embryonic development. Expression of this transporter was detectable in most DA neurons until E11.5 and was found to be localized in developing axons. Moderate enhancement of VGLUT2 expression in primary DA neurons caused an increase in axonal arborization length. Compatible with a developmental role, constitutive deletion of Vglut2 caused a regional defect in TH-innervation of the dorsal striatum in E18.5 embryos. Moreover, using an in vitro neurotoxin model, we demonstrate that Vglut2 expression can be upregulated in post-lesional DA neurons by 2.5-fold, arguing that the developmental expression of Vglut2 in DA neurons can be reactivated at postnatal stages and contribute to post-lesional plasticity of dopaminergic axons. In support of this hypothesis, we find fewer mesostriatial dopaminergic projections in the striatum of conditional Vglut2 KO mice 7 weeks after a neurotoxic lesion, compared to control animals. Thus, we propose here that one of the functions of Vglut2 in adult DA neurons is to promote post-lesional recovery of meso-striatal axons.

neuroscience↗

bioRxiv2019

Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

The neuronal protein complexin contains multiple domains that exert both clamping and facilitatory functions to tune spontaneous and action potential triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that prior to fusion the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitter in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

neuroscience↗

bioRxiv2018

RIM-BP2 primes synaptic vesicles via recruitment of Munc13-1 at hippocampal mossy fiber synapses

All synapses require fusion-competent vesicles and coordinated Ca2+-secretion coupling for neurotransmission, yet functional and anatomical properties show a high diversity across different synapse types. We show here that the presynaptic protein RIM-BP2 has diversified functions in neurotransmitter release at different central mammalian synapses and thus contributes to synaptic diversity. At hippocampal pyramidal CA3-CA1 synapses, RIM-BP2 loss has a mild effect on neurotransmitter release, by only regulating Ca2+-secretion coupling. However, at hippocampal mossy fiber synapses RIM-BP2 has a strong impact on neurotransmitter release by promoting vesicle docking/priming via recruitment of Munc13-1. In wild type mossy fiber synapses, the distance between RIM-BP2 clusters and Munc13-1 clusters is larger than in hippocampal pyramidal CA3-CA1 synapses, suggesting that spatial organization may dictate the role a protein plays in synaptic transmission and that differences in active zone architecture is a major determinant factor in the functional diversity of synapses.

neuroscience↗

bioRxiv2018

Light induced synaptic vesicle autophagy

The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated and whether SVs are cleared en-mass together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real time to support synaptic health. To address these questions, we have developed an imaging based system that simultaneously tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light activated reactive oxygen species (ROS) generator, Supernova, it was possible to temporally control the damage to specific SV proteins and assess their consequence to autophagy mediated clearance mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons, presynaptic autophagy can be induced in as little as 5-10 minutes and eliminates primarily the damaged protein rather than the SV en-mass. Importantly, we also find that autophagy is essential for synaptic function, as light-induced damage to e.g. Synaptophysin only compromises synaptic function when autophagy is simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly regulated clearance system to maintain not only synapse integrity, but also synaptic function.

neuroscience↗

bioRxiv2018

Critical role for Piccolo in Synaptic Vesicle Retrieval

Loss of function of the presynaptic active zone protein Piccolo has recently been linked to a devastating disease causing brain atrophy. Here, we address how Piccolo inactivation adversely affects synaptic function and thus may contributes to neuronal loss. Our analysis shows that Piccolo is critical for the activity dependent recycling and maintenance of synaptic vesicles (SVs). Specifically, we find that boutons lacking Piccolo have deficits in the Rab5/EEA1 dependent formation of early endosomes and thus the recycling of SVs. Mechanistically, impaired Rab5 function was caused by the reduced synaptic recruitment of Pra1, known to interact selectively with the zinc fingers of Piccolo. Importantly, over-expression of GTPase deficient Rab5 or the Znf1 domain of Piccolo restores the size and recycling of SV pools. These data provide a molecular link between the active zone and endosome sorting at synapses providing hints to how Piccolo contributes to both developmental and psychiatric disorders.\n\nImpact StatementThe efficient recycling of synaptic vesicle proteins is critical for the integrity and reliability of synaptic transmission. Increasingly genetic and environmental insults have been shown to affect this recycling pathway, resulting in both cognitive impairment in humans and neurodegenerative diseases, yet the underlying mechanisms are poorly understood. Here we could show that the presynaptic active zone protein Piccolo regulates efficient recycling of synaptic vesicles via Pra1 and Rab5, perhaps explaining why Piccolo loss of function contributes to Pontocerebellar Hypoplasia and major depressive disorders.

neuroscience↗