The two paralogs of the calcium-dependent activator protein for secretion (CAPS) are priming factors for synaptic vesicles (SVs) and neuropeptide containing large dense-core vesicles (LDCVs). Yet, it is unclear whether CAPS1 and CAPS2 regulate exocytosis of these two vesicle types differentially in dorsal root ganglion (DRG) neurons, wherein synaptic transmission and neuropeptide release are of equal importance. These sensory neurons transfer information from the periphery to the spinal cord (SC), releasing glutamate as the primary neurotransmitter, with co-transmission via neuropeptides in a subset of so called peptidergic neurons. Neuropeptides are key components of the information-processing machinery of pain perception and neuropathic pain generation. Here, we compared the ability of CAPS1 and CAPS2 to support priming of both vesicle types in single and double knock-out mouse (DRG) neurons using a variety of high-resolution live cell imaging methods. While CAPS1 was localized to synapses of all DRG neurons and promoted synaptic transmission, CAPS2 was found exclusively in peptidergic neurons and mediated LDCV exocytosis. Intriguingly, ectopic expression of CAPS2 empowered non-peptidergic neurons to drive LDCV fusion, thereby identifying CAPS2 as an essential molecular determinant for peptidergic signaling. Our results reveal that these distinct functions of both CAPS paralogs are based on their differential subcellular localization in DRG neurons. Our data suggest a major role for CAPS2 in neuropathic pain via control of neuropeptide release.

SNAP-25 is an essential component of SNARE complexes driving fast Ca2+-dependent exocytosis. Yet, the functional implications of the tandem-like structure of SNAP-25 are unclear. Here, we have investigated the mechanistic role of the acylated "linker" domain that concatenates the two SNARE motifs within SNAP-25. Refuting older concepts of an inert connector, our detailed structure-function analysis in murine chromaffin cells demonstrates that linker motifs play a crucial role in vesicle priming, triggering, and fusion pore expansion. Mechanistically, we identify two synergistic functions of the SNAP-25 linker: First, linker motifs support t-SNARE interactions and accelerate ternary complex assembly. Second, the acylated N-terminal linker segment engages in local lipid interactions that facilitate fusion triggering and pore evolution, putatively establishing a favorable membrane configuration by shielding phospholipid headgroups and affecting curvature. Hence, the linker is a functional part of the fusion complex that promotes secretion by SNARE interactions as well as concerted lipid interplay.

Myelin, the electrically insulating sheath on axons, undergoes dynamic changes over time. However, it is composed of proteins with long lifetimes. This raises the question how such a stable structure is renewed. Here, we study the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survive recombination, continue to express myelin genes, but they fail to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualize myelin-like membranes failing to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicates degradation of existing myelin at the abaxonal side and the inner tongue of the sheath. Thinning of compact myelin and shortening of internodes result in the loss of about 50% of myelin and axonal pathology within 20 weeks post recombination. In summary, our data suggest that functional axon-myelin units require the continuous incorporation of new myelin membranes.

The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons.

Peptidergic dorsal root ganglion (DRG) neurons transmit sensory and nociceptive information from the periphery to the central nervous system. Their synaptic activity is profoundly affected by neuromodulatory peptides stored and released from large dense-core vesicles (LDCVs). However, the mechanism of peptide secretion from DRG neurons is poorly understood. Using total internal reflection fluorescence microscopy (TIRFM), we visualized individual LDCVs loaded with fluorescent neuropeptide Y (NPY) and analyzed their stimulation-dependent release. We tested several protocols and found an overall low stimulation-secretion coupling that increased after raising intracellular Ca2+ concentration by applying a weak pre-stimulus. Interestingly, the stimulation protocol also influenced the mechanism of LDCV fusion. Depolarization of DRG neurons with a solution containing 60mM KCl triggered full fusion, kiss-and-run, and kiss-and-stay exocytosis with equal frequency. In contrast, field electrode stimulation primarily induced full fusion exocytosis. Finally, our results indicate that NPY can promote LDCV secretion. These results shed new light on the mechanism of NPY action during modulation of DRG neuron activity, an important pathway in the treatment of chronic pain.

Human myelin disorders are commonly studied in mouse models. Since both clades evolutionarily diverged approximately 85 million years ago, it is critical to know to what extent the myelin protein composition has remained similar. Here, we use quantitative proteomics to analyze myelin purified from human white matter and find that the relative abundance of the structural myelin proteins PLP, MBP, CNP, and SEPTIN8 correlates well with that in C57Bl/6N mice. Conversely, multiple other proteins were identified exclusively or predominantly in human or mouse myelin. This is exemplified by peripheral myelin protein 2 (PMP2), which was specific to human central nervous system myelin, while tetraspanin-2 (TSPAN2) and connexin-29 (CX29/GJC3) were confined to mouse myelin. Assessing published scRNA-seq-datasets, human and mouse oligodendrocytes display well-correlating transcriptome profiles but divergent expression of distinct genes, including Pmp2, Tspan2, and Gjc3. A searchable web interface is accessible via www.mpinat.mpg.de/myelin. Species-dependent diversity of oligodendroglial mRNA expression and myelin protein composition can be informative when translating from mouse models to humans.

Neuronal transmission relies on the regulated secretion of neurotransmitters, which are packed in synaptic vesicles (SVs). Hundreds of SVs accumulate at synaptic boutons. Despite being held together, SVs are highly mobile, so that they can be recruited to the plasma membrane for their rapid release during neuronal activity. However, how such confinement of SVs corroborates with their motility remains unclear. To bridge this gap, we employ ultrafast single-molecule tracking (SMT) in the reconstituted system of native SVs and in living neurons. SVs and synapsin 1, the most highly abundant synaptic protein, form condensates with liquid-like properties. In these condensates, synapsin 1 movement is slowed in both at short (i.e., 60-nm) and long (i.e., several hundred-nm) ranges, suggesting that the SV-synapsin 1 interaction raises the overall packing of the condensate. Furthermore, two-color SMT and super-resolution imaging in living axons demonstrate that synapsin 1 drives the accumulation of SVs in boutons. Even the short intrinsically-disordered fragment of synapsin 1 was sufficient to restore the native SV motility pattern in synapsin triple knock-out animals. Thus, synapsin 1 condensation is sufficient to guarantee reliable confinement and motility of SVs, allowing for the formation of mesoscale domains of SVs at synapses in vivo.

To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type-specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.

A proper equilibrium of post-translational protein modifications is essential for normal cell physiology, and alteration in these processes is key in neurodegenerative disorders such as Alzheimer's disease. Recently, for instance, alteration in protein SUMOylation has been linked to amyloid pathology. In this work, we aimed to elucidate the role of protein SUMOylation during aging and increased amyloid burden in vivo using a His6 -HA-SUMO1 knock-in mouse in the 5XFAD model of Alzheimer's disease. Interestingly, we did not observe any alteration in the levels of SUMO1-conjugation related to Alzheimer's disease. SUMO1 conjugates remained localized to neuronal nuclei upon increased amyloid burden and during aging and were not detected in amyloid plaques. Surprisingly however, we observed age-related alterations in global levels of SUMO1 conjugation and at the level of individual substrates using quantitative proteomic analysis. The identified SUMO1 candidate substrates are dominantly nuclear proteins, mainly involved in RNA processing. Our findings open novel directions of research for studying a functional link between SUMOylation and its role in guarding nuclear functions during aging.

Advances in genome sequencing technologies have favored the identification of rare de novo mutations linked to neurological disorders in humans. Recently, a de novo autosomal dominant mutation in NACC1 was identified (NM_052876.3: c.892C > T, NP_443108.1; p.Arg298Trp), associated with severe neurological symptoms including intellectual disability, microcephaly, and epilepsy. As NACC1 had never before been associated with neurological diseases, we investigated how this mutation might lead to altered brain function. We examined neurotransmission in autaptic glutamatergic mouse neurons expressing the murine homolog of the human mutant NACC1, i.e., Nacc1-R284W. We observed that expression of Nacc1-R284W impaired glutamatergic neurotransmission in a cell-autonomous manner, likely through a dominant negative mechanism. Furthermore, by screening for Nacc1 interaction targets in the brain, we identified SynGAP1, GluK2A, and several SUMO E3 ligases as novel Nacc1 interaction partners. At a biochemical level, Nacc1-R284W exhibited reduced binding to SynGAP1 and GluK2A, and also showed greatly increased SUMOylation. Ablating the SUMOylation of Nacc1-R284W partially restored its interaction with SynGAP1 but did not restore binding to GluK2A. Overall, these data indicate a role for Nacc1 in regulating glutamatergic neurotransmission, which is substantially impaired by the expression of a disease-associated Nacc1 mutant. This study provides the first functional insights into potential deficits in neuronal function in patients expressing the de novo mutant NACC1 protein.

Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the expansion of the samples up to 10-fold, in a single expansion step, through high-temperature homogenization (X10ht). The resulting gels exhibit a higher fluorescence intensity than gels homogenized using enzymatic digestion (based on proteinase K). This enables the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6-8 nm in neuronal cell cultures or isolated vesicles. X10ht also enables the expansion of 100-200 µm thick brain samples, up to 6-fold. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.

iPSC-derived human neurons are expected to revolutionize studies on brain diseases, but their functional heterogeneity still poses a problem. Key sources of heterogeneity are the different cell culture systems used. We show that an optimized autaptic culture system, with single neurons on astrocyte feeder islands, is well suited to culture, and we analyze human iPSC-derived neurons in a standardized, systematic, and reproducible manner. Using classically differentiated and transcription factor-induced human glutamatergic and GABAergic neurons, we demonstrate that key features of neuronal morphology and function, including dendrite structure, synapse number, membrane properties, synaptic transmission, and short-term plasticity, can be assessed with substantial throughput and reproducibility. We propose our optimized autaptic culture system as a tool to study functional features of human neurons, particularly in the context of disease phenotypes and experimental therapy.