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Lipstein, N.

Publications and source records attributed to Lipstein, N..

9 recordsLinked to original sources

Peroxisome protein import deficiency causes heart failure in mouse and human

Peroxisomes are ubiquitous cellular organelles with potentially vital roles in lipid and reactive oxygen metabolism. The metabolic demands of the heart are substantial; however, the contribution of peroxisomes to cardiac development, health, and their role in heart failure (HF) remain largely unexplored. We developed and examined a mouse and an engineered human myocardium (EHM) model with a deficiency in cardiac peroxisome biogenesis to investigate the role of peroxisomes in cardiac function and pathology. In the EHM, loss of peroxisome protein import and subsequent peroxisomal metabolic impairment trigger mitochondrial damage and compromise cellular respiration and energy production. Peroxisome dysfunction results in incoherent electrical conduction, defective Ca2+-handling, and ultimately presentation of a HF phenotype with pathological force generation. These phenotypes are mirrored in an orthogonal murine model system with defective cardiac peroxisome biogenesis. Preload-dependent deficits in force generation due to insufficient energy supply are eventually fatal. Thus, peroxisomes play an important role in sustaining normal heart operations. Vice versa, peroxisome maintenance is compromised in pressure overload-induced HF, establishing peroxisomes as potential modulators of pathology and targets of therapy.

cell biology↗

Ca2+-phospholipid-dependent regulation of Munc13-1 is essential for post-tetanic potentiation at mossy fiber synapses and supports working memory

Hippocampal mossy fiber (hMF) to CA3 pyramidal cell synapses are thought to support the formation of working memory through presynaptic short-term facilitation (STF) and post-tetanic potentiation (PTP). However, the molecular mechanisms underlying these transient forms of synaptic enhancement remain poorly understood. We show here that Munc13-1-mediated priming of synaptic vesicles (SVs) at active zones controls hMF STF and PTP in response to Ca2+-phospholipid and Ca2+-calmodulin (CaM) signaling. Knock-in mice expressing Munc13-1 variants that are insensitive to Ca2+-phospholipid and Ca2+-CaM signaling exhibit severely impaired STF and PTP at hMF synapses. Moreover, the PTP-induction threshold is strongly increased upon the loss of Ca2+-phospholipid-Munc13-1 signaling. Since these synaptic defects are accompanied by working memory deficits, especially in mice expressing the Ca2+-phospholipid-insensitive Munc13-1 variant, we conclude that Ca2+-dependent regulation of Munc13-1-mediated SV priming co-determines hMF short-term plasticity and working memory formation.

neuroscience↗

Proximity labelling reveals the compartmental proteome of murine sensory neurons

IntroductionUnderstanding the molecular architecture of peripheral sensory neurons is critical as we pursue novel drug targets against pain and neuropathy. Sensory neurons in the dorsal root ganglion (DRG) show extensive compartmentalization, thus understanding each compartment - from the peripheral to central terminals - is key to this effort. MethodsTo systematically profile this spatial complexity, we generated a TurboIDfl/fl transgenic mouse line (ROSA26em1(TurboID)Bros), enabling targeted proximity labelling and deep proteomic profiling of DRG neuron compartments via Tg(Advillin-Cre)+. ResultsOur data reveal distinct proteomic signatures across neuronal compartments that reflect specialized neuronal functions. We provide proteomic insights into previously inaccessible nerve terminals both in the periphery (innervating the skin) and in the spinal cord. Further, using a DRG explant model of chemotherapy-induced peripheral neuropathy, we uncover novel and discrete proteome changes, highlighting neuronal vulnerability. ConclusionTogether, our findings provide a unique proteome atlas of the sensory neuron proteome across anatomical domains and demonstrate the utility of proximity labelling proteomics for detecting compartment-specific molecular alterations in a disease model.

neuroscience↗

Endogenous tagging of Munc13-1 with a SNAP tag as a tool for monitoring synapse nanoarchitecture

ABSTRACTSynaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine the dynamic changes in synaptic proteins nanoarchitecture typically rely on immunolabeling, or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification, but requires fixation. The latter enables monitoring of proteins in live microscopy, but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed neurons and brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 here as a highly-efficient dye for SNAP-tag labeling of extracellular epitopes, and of intracellular proteins in fixed and permeabilized tissue. Finally, we show that Munc13-1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal- and super resolution microscopy. We conclude that the Unc13aSNAP mouse line is a useful tool for the analysis of presynaptic nanoarchitectural dynamics, with a potential for wide adoption.

neuroscience↗

A minor impact of VGLUT1 expression level on quantal size revealed through the characterization of VGLUT1mEos2 knock-down new mouse model

Synaptic vesicles (SVs) are small organelles secreting neurotransmitters at synapses. By fusing a photoactivated fluorescent protein to VGLUT1, we generated a VGLUT1mEos2 knock-in mouse. VGLUT1mEos2 knock-in mice are viable and healthy, but exhibit a severe reduction in VGLUT1 expression levels. Using VGLUT1mEos2 expressing neurons, we established paradigms to trace individual SV mobility at the single-molecule level or via massive photoconversion. Hippocampal neurons with significantly diminished VGLUT1 expression maintain unaltered miniature glutamate release characteristics in terms of quantal size and frequency. We demonstrate that VGLUT1 expression level are not correlated in a linear fashion with the vesicular glutamate content. In conclusion, the VGLUT1mEos2 mouse line serves as a powerful tool for exploring SV mobility properties and elucidating the contributions of VGLUT1 to excitatory neurotransmission and cognitive processes.

neuroscience↗

A one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application and super-resolution imaging

Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ( dye shuttle) conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling.

pharmacology and toxicology↗

Ketamine potentiates a glutamatergic presynapse

Ketamine produces rapid and sustained antidepressant effects after brief exposure to a single dose. Counterintuitively, while ketamine acts primarily as a blocker of postsynaptic N-methyl-D-aspartate receptors (NMDARs), increased signalling at glutamatergic synapses has been reported. Due to technical limitations, however, it remains unclear whether ketamine directly increases presynaptic glutamate release or acts via postsynaptic or network-level mechanisms. To address this knowledge gap, we used presynaptic capacitance measurements to directly monitor glutamate release in a cerebellar synapse. Ketamine increased glutamate release within minutes and this effect persisted >30 minutes after washout. MK-801, another NMDAR blocker, had no effect on glutamate release. Mechanistically, we show that the ketamine-mediated enhancement of presynaptic release results from an increase in both calcium influx and the number of release-ready vesicles. Our data uncover a rapid effect of ketamine on key presynaptic properties of central glutamatergic synapses, which has important implications for the development of antidepressant drugs.

neuroscience↗

Phorbolester-activated Munc13-1 and ubMunc13-2 exert opposing effects on dense-core vesicle secretion

Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs exist, but their differential roles are not well understood. We studied the interdependence of phorbolesters (DAG mimics) with Munc13-1 and ubMunc13-2 in mouse adrenal chromaffin cells. Although expression of either Munc13-1 or ubMunc13-2 stimulated secretion, phorbolester was only stimulatory for secretion when ubMunc13-2 expression dominated, but inhibitory when Munc13-1 dominated. Accordingly, phorbolester stimulated secretion in wildtype cells, or cells overexpressing ubMunc13-2, but inhibited secretion in Munc13-2/Unc13b knockout (KO) cells or in cells overexpressing Munc13-1. Phorbolester was more stimulatory in the Munc13-1/Unc13a KO than in WT littermates, showing that endogenous Munc13-1 limits the effects of phorbolester. Imaging showed that ubMunc13-2, but not Munc13-1, traffics to the plasma membrane with a time-course matching Ca2+-dependent secretion, and trafficking is independent of synaptotagmin-7 (syt7). However, in the absence of syt7, phorbolester became inhibitory for both Munc13-1 and ubMunc13-2 driven secretion, indicating that stimulatory phorbolester x Munc13-2 interaction depends obligatorily on functional pairing with syt7. Overall, DAG/phorbolester, ubMunc13-2 and syt7 form a stimulatory triad for dense-core vesicle priming.

neuroscience↗

Molecular and functional architecture of striatal dopamine release sites

Dopamine controls striatal circuit function, but its transmission mechanisms are not well understood. We recently showed that dopamine secretion requires RIM, suggesting that it occurs at active zone-like sites similar to conventional synapses. Here, we establish using a systematic conditional gene knockout approach that Munc13 and Liprin-, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Correspondingly, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-, respectively, are needed to restore dopamine release in RIM knockout mice. In contrast, and different from conventional synapses, the active zone scaffolds RIM-BP and ELKS, and the RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-, but other active zone proteins are dispensable. Our work establishes that molecularly simple but efficient release site architecture mediates fast dopamine exocytosis.

neuroscience↗