Biology subjects

Kaeser, P. S.

Publications and source records attributed to Kaeser, P. S..

15 recordsLinked to original sources

bioRxiv2026

Engineered proteins to restore and enhance neurotransmitter release

Synapse dysfunction is a hallmark feature of many neurological diseases. Synaptic transmission is initiated in nerve terminals, where active zone protein complexes control vesicle fusion for neurotransmitter release. Here, we engineer proteins that mediate active zone functions and demonstrate their use for reconstituting and strengthening neurotransmission. A protein composed of CaV{beta}4 and the RIM1 zinc finger, {beta}4-Zn, restored neurotransmitter release after active zone disruption through recruitment of Munc13. We develop {beta}4-MC, derived from the Munc13-1 MUN and C2C domains, which bypasses Munc13 recruitment and directly mediates vesicle docking, priming and fusion after active zone disruption. In {beta}4-MC, MUN domain functions are dispensable for docking, but active MUN and C2C domains are both necessary for neurotransmitter release. In wild-type mouse and human neurons, {beta}4-MC but not {beta}4-Zn strengthens neurotransmitter release. Furthermore, {beta}4-Zn and {beta}4-MC restore and enhance dopamine release in the mouse striatum, respectively. Hence, {beta}4 proteins are engineered secretory proteins with therapeutic potential as enhancers of synaptic function.

neuroscience↗

bioRxiv2025

The synaptic vesicle priming protein Munc13 mediates evoked somatodendritic dopamine release

Midbrain dopamine neurons secrete dopamine from their somata and dendrites in addition to their axonal release. Shared and distinct properties have been proposed for somatodendritic and axonal release, but the mechanisms of somatodendritic release remain unclear. We here used mouse genetics, electrophysiology, and imaging to define roles of the synaptic vesicle priming protein Munc13 in somatodendritic release in comparison to axonal secretion. Munc13 ablation in dopamine neurons decreased evoked but not spontaneous somatodendritic dopamine transmission measured as D2 receptor-mediated currents. Imaging with a fluorescent dopamine sensor confirmed this finding and established comparable importance for Munc13 in somatodendritic and axonal secretion. Pharmacological experiments revealed that release from adrenergic terminals contributes to D2 receptor-mediated currents, and their relative contribution was enhanced after Munc13 knockout. Altogether, these data establish important roles for Munc13 in evoked somatodendritic release. These roles are similar to Munc13 functions in axonal dopamine release and at fast synapses. Spontaneous midbrain dopamine release does not necessitate Munc13 in dopamine neurons and may rely on a release pathway that is independent of the prototypical release machinery employed at synapses.

neuroscience↗

bioRxiv2025

Deployment of endocytic machinery to periactive zones of nerve terminals is independent of active zone assembly and evoked release

In presynaptic nerve terminals, the endocytic apparatus rapidly restores synaptic vesicles after neurotransmitter release. Many endocytic proteins localize to the periactive zone, a loosely defined area adjacent to active zones. A prevailing model posits that recruitment of these endocytic proteins to the periactive zone is activity-dependent. We here show that periactive zone targeting of endocytic proteins is largely independent of active zone machinery and synaptic activity. At mouse hippocampal synapses and Drosophila neuromuscular junctions, pharmacological or genetic silencing resulted in unchanged or increased levels of endocytic proteins including Dynamin, Amphiphysin, Nervous Wreck, Endophilin A, Dap160/Intersectin, PIPK1{gamma} and AP-180. Similarly, disruption of active zone assembly via genetic ablation of active zone scaffolds at each synapse did not impair the localization of endocytic proteins. Overall, our work indicates that endocytic proteins are constitutively deployed to the periactive zone and supports the existence of independent assembly pathways for active zones and periactive zones.

neuroscience↗

bioRxiv2024

Innervation density governs crosstalk of GPCR-based norepinephrine and dopamine sensors

GPCR-based fluorescent sensors are widely used to correlate neuromodulatory signaling with brain function. While experiments in transfected cells often reveal selectivity for individual neurotransmitters, sensor specificity in the brain frequently remains uncertain. Pursuing experiments in brain slices and in vivo, we find that norepinephrine and dopamine cross-activate the respective sensors. Non-specific activation occurred when innervation of the cross-reacting transmitter was high, and silencing of specific innervation was indispensable for interpreting sensor fluorescence.

neuroscience↗

bioRxiv2023

The intracellular C-terminus confers compartment-specific targeting of voltage-gated Ca2+ channels

To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify the mechanisms that target voltage-gated Ca2+ channels (CaVs) to distinct subcellular compartments. In hippocampal neurons, CaV2s trigger neurotransmitter release at the presynaptic active zone, and CaV1s localize somatodendritically. After knockout of all three CaV2s, expression of CaV2.1, but not of CaV1.3, restores neurotransmitter release. Chimeric CaV1.3 channels with CaV2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release fully sensitive to blockade of CaV1 channels. This dominant targeting function of the CaV2.1 C-terminus requires an EF hand in its proximal segment, and replacement of the CaV2.1 C-terminus with that of CaV1.3 abolishes CaV2.1 active zone localization. We conclude that the intracellular C-termini mediate compartment-specific CaV targeting.

neuroscience↗

bioRxiv2023

Molecular definition of distinct active zone protein machineries for Ca2+ channel clustering and synaptic vesicle priming

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions. In heterologous cells, Liprin- and RIM from co- assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-1-4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTP{sigma} recruits Liprin-, RIM and Munc13 into priming complexes without co- clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin- and PTP{sigma} specifically support priming site assembly.

neuroscience↗

bioRxiv2022

Protein composition of axonal dopamine release sites in the striatum

Mechanisms of neuromodulatory transmission in the brain remain ambiguous. Dopamine is a prototypical neuromodulator, and it was recently found that its secretion relies on active zone-like release site assemblies. Here, we use in vivo biotin-identification (iBioID) proximity proteomics in mouse striatum to isolate dopamine release site proteins enriched over the general dopamine axonal protein content. Using three bait proteins, we identified 527 proteins that fall into several synaptic protein classes, including active zone, Ca2+ regulatory and synaptic vesicle proteins. We also detected many proteins not previously associated with synaptic exocytosis. Knockout of the presynaptic organizer protein RIM profoundly disrupted dopamine release site composition assessed by iBioID, while Synaptotagmin-1 knockout did not. -Synuclein, a protein linked to Parkinsons disease, was enriched at release sites, and this enrichment was lost in both tested mutants. We conclude that RIM organizes scaffolded dopamine release sites and we define the protein composition of these sites.

neuroscience↗

bioRxiv2022

Munc13 supports vesicle fusogenicity after disrupting active zone scaffolds and synaptic vesicle docking

Active zones consist of protein scaffolds that are tightly attached to the presynaptic plasma membrane. They dock and prime synaptic vesicles, couple them to Ca2+ entry, and target neurotransmitter release to postsynaptic receptor domains. Simultaneous RIM+ELKS ablation disrupts these scaffolds, abolishes vesicle docking and removes active zone-targeted Munc13, but some vesicles remain releasable. This enduring vesicular fusogenicity may be Munc13-independent or be mediated by non-active zone-anchored Munc13. We tested its Munc13-dependence by ablating Munc13-1 and Munc13-2 on top of RIM+ELKS in cultured hippocampal neurons. The hextuple knockout synapses lacked docked vesicles, but other ultrastructural features were near-normal despite the strong genetic manipulation. Removing Munc13 in addition to RIM+ELKS further impaired action potential-evoked release by decreasing the remaining pool of releasable vesicles. We conclude that Munc13 can support some fusogenicity without RIM and ELKS, and that presynaptic recruitment of Munc13, even without active zone-anchoring, suffices to generate some fusion-competent vesicles.

neuroscience↗

bioRxiv2021

Reconstructing essential active zone functions within a synapse

Active zones are molecular machines that control neurotransmitter release through synaptic vesicle docking and priming, and through coupling of these vesicles to Ca2+ entry. The complexity of active zone machinery has made it challenging to determine which mechanisms drive these roles in release. Here, we induce RIM+ELKS knockout to eliminate active zone scaffolding networks, and then reconstruct each active zone function. Re-expression of RIM1-Zn fingers positioned Munc13 on undocked vesicles and rendered them release-competent. Reconstitution of release-triggering required docking of these vesicles to Ca2+ channels. Fusing RIM1-Zn to CaV{beta}4-subunits sufficed to restore docking, priming and release-triggering without reinstating active zone scaffolds. Hence, exocytotic activities of the 80 kDa CaV{beta}4-Zn fusion protein bypassed the need for megadalton-sized secretory machines. These data define key mechanisms of active zone function, establish that fusion competence and docking are mechanistically separable, and reveal that active zone scaffolding networks are not required for release.

neuroscience↗

bioRxiv2021

Short-term plasticity persists in the absence of PKC phosphorylation of Munc18-1

Post tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knockin mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1 SA mice). Expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3 to CA1 synapse, and the granule cell parallel fiber to Purkinje cell (PF to PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1 SA mice. In Munc18-1 SA mice, 70% of PTP remained at CA3 to CA1 synapses, and the amplitude of PTP was not reduced at PF to PC synapses. These findings indicate that at both CA3 to CA1 and PF to PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1. Significance StatementA leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C phosphorylation of Munc18-1. This study tests this mechanism by creating a knock in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3 to CA1 synapses, or at cerebellar granule cell to Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.

neuroscience↗

bioRxiv2021

Intact synapse structure and function after combined knockout of PTPδ, PTPσ and LAR

It has long been proposed that Leukocyte common Antigen-Related Receptor Protein Tyrosine Phosphatases (LAR-RPTPs) are cell-adhesion proteins for the control of synapse assembly. Their synaptic nanoscale localization, however, has not been established, and the fine structure of synapses after knockout of the three vertebrate genes for LAR-RPTPs (PTP{delta}, PTP{sigma} and LAR) has not been tested. Here, we find that PTP{delta} is precisely apposed to postsynaptic scaffolds at excitatory and inhibitory synapses using superresolution microscopy. We generated triple-conditional knockout mice for PTP{delta}, PTP{sigma} and LAR to test whether they are essential for synapse structure. While mild effects on synaptic vesicle clustering and active zone architecture were detected, synapse numbers and their overall structure were unaffected, membrane anchoring of the active zone persisted, and vesicle docking and release were normal. We conclude that LAR-RPTPs, despite their localization at synaptic appositions, are dispensable for the organization and function of presynaptic nerve terminals.

neuroscience↗

bioRxiv2020

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↗

bioRxiv2020

Phosphorylation triggers presynaptic phase separation of Liprin-α3 to control active zone structure

Liquid-liquid phase separation enables the assembly of membrane-less subcellular compartments, but testing its biological functions has been difficult. The presynaptic active zone, protein machinery in nerve terminals that defines sites for neurotransmitter release, may be organized through phase separation. Here, we discover that the active zone protein Liprin-3 rapidly and reversibly undergoes phase separation upon phosphorylation by PKC at a single site. RIM and Munc13 are co-recruited to membrane-attached condensates, and phospho-specific antibodies establish Liprin-3 phosphorylation in vivo. At synapses of newly generated Liprin-2/3 double knockout mice, RIM, Munc13 and the pool of releasable vesicles were reduced. Re-expression of Liprin-3 restored these defects, but mutating the Liprin-3 phosphorylation site to abolish phase condensation prevented rescue. Finally, PKC activation acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-3. We conclude that Liprin-3 phosphorylation rapidly triggers presynaptic phase separation to modulate active zone structure and function.

neuroscience↗

bioRxiv2018

Highly multiplexed in situ protein imaging with signal amplification by Immuno-SABER

Probing the molecular organization of tissues requires in situ analysis by microscopy. However current limitations in multiplexing, sensitivity, and throughput collectively constitute a major barrier for comprehensive single-cell profiling of proteins. Here, we report Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), a rapid, highly multiplexed signal amplification method that simultaneously tackles these key challenges. Immuno-SABER utilizes DNA-barcoded antibodies and provides a method for highly multiplexed signal amplification via modular orthogonal DNA concatemers generated by Primer Exchange Reaction. This approach offers the capability to preprogram and control the amplification level independently for multiple targets without in situ enzymatic reactions, and the intrinsic scalability to rapidly amplify and image a large number of protein targets. We validated our approach in diverse sample types including cultured cells, cryosections, FFPE sections, and whole mount tissues. We demonstrated independently tunable 5-180-fold amplification for multiple targets, covering the full signal range conventionally achieved by secondary antibodies to tyramide signal amplification, as well as simultaneous signal amplification for 10 different proteins using standard equipment and workflow. We further combined Immuno-SABER with Expansion Microscopy to enable rapid and highly multiplexed super-resolution tissue imaging. Overall, Immuno-SABER presents an effective and accessible platform for rapid, multiplexed imaging of proteins across scales with high sensitivity.

cell biology↗

bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗