Proteases are common components of extracellular vesicles (EVs), yet the extent and functional relevance of ongoing proteolytic activity on EV surfaces remain largely unexplored. Such activity could significantly influence EV function and identity, with likely implications for EV-mediated signalling, recipient cell targeting, cargo delivery, and even translational applications ranging from biomarker discovery to therapeutic approaches. Here, we investigated the impact of sustained proteolysis on the composition of brain cell-derived EVs, focusing on A Disintegrin And Metalloprotease 10 (ADAM10), a key sheddase for signalling and adhesion proteins involved in neuronal and synaptic processes. Using primary rat cortical cultures, we found that numerous known ADAM10 substrates are part of small EVs (sEVs), and that their associated functions overlap with major sEV-mediated roles such as nervous system development, cell adhesion, and neurite outgrowth. Applying N-terminal proteomics to monitor sEV-derived cleavage fragments over time, we identified novel substrate candidates and demonstrated that sEV-associated ADAM10 activity remodels surface proteins involved in EV-cell interactions while generating soluble factors implicated in neuronal development. These findings suggest a previously unrecognised role for ADAM10 as a modulator of sEV composition and potentially cell-targeting specificity in the brain and position EVs as dynamic platforms for proteolytic processing 'on the move'.

Abstract The brain stores information by changing the strength of its synapses, a process that has at least two phases: Late long-term potentiation (L-LTP) is thought to result from the consolidation of early LTP (E-LTP), just as long-term memory requires the prior establishment of short-term memory. Recently, inhibitory avoidance experiments under CaMKII inhibition have challenged this notion, demonstrating long-term fear memory without measurable short-term memory. Here we use optogenetic activation and inhibition of CaMKII during induction of spike-timing-dependent potentiation (tLTP) to dissect the signaling pathways. While CaMKII activation in CA1 neurons was sufficient to induce E-LTP, growth of the postsynaptic density and spine neck expansion, we found that CaMKII-induced LTP does not give rise to L-LTP. Conversely, inhibition of CaMKII during tLTP induction prevented E-LTP, but FOS and L-LTP were still expressed, driven by CaMKK and PKMζ. Thus, both long-term memory and L-LTP form in the absence of CaMKII activation.

The lateral septum is a key subcortical structure and has been implicated in social memory. One aspect of social memory, the ability to recognize relatives, is conserved across vertebrate species and reflected in stable, lifelong memories. Synapses are considered to be the smallest unit of memory storage. Excitatory synapses are typically found on dendritic spines, whereas inhibitory synapses are mostly located on the dendritic shaft. Here, we investigate the synaptic architecture of unusual somatic spines, an apparent synaptic specialization of septal GABAergic neurons. We uncover the formation and molecular organization of septal somatic spines found in the lateral septum in in vivo and in vitro model systems using various microscopy approaches. We use classical label-free methods such as transmission electron microscopy and Golgi stainings and established new culturing methods for dissociated and organotypic septal slices that were kept in culture over multiple weeks. We describe the presence, morphology, ultrastructure, and molecular composition of excitatory somatic spines across multiple developmental stages in various model systems. We propose that the ability to develop such spines is an intrinsic feature of somatospiny neurons that does not depend on extra-septal connectivity. While smaller than dendritic spines, somatic spines exhibited distinct features, frequently containing secretory organelles such as autophagosomes, multivesicular bodies, and endosomes, but often lacking a spine apparatus and ribosomes. Our findings offer insights into the molecular architecture of septal somatic spines and establish a basis for further investigations into the somatic spines of the lateral septum.

Despite substantial clinical benefit from immune checkpoint inhibitors (ICI), advanced melanoma remains challenging due to frequent treatment resistance. Resistance may be intrinsic (primary) or emerge over time (secondary). Biomarkers predicting distinct resistance phenotypes before therapy are lacking. As key mediators of cellular communication, extracellular vesicles (EVs) represent promising biomarkers. This study aimed to identify baseline EV proteome-derived pathways and biomarkers associated with overall, primary, and secondary resistance to ICI in advanced melanoma and to derive biomarker signatures predictive of progression-free survival (PFS). EVs were isolated from pretreatment plasma samples of 46 patients with advanced melanoma using size exclusion chromatography and ultracentrifugation. Proteomic profiling was performed by liquid chromatography-mass spectrometry using DIA-NN. Pathway enrichment and network analyses were conducted using Reactome, Metascape, Cytoscape, and DAVID. Resistance-associated proteins were integrated into composite biomarker signatures and evaluated for association with PFS. Overall resistance was characterized by enrichment of platelet- and complement-associated pathways. Primary resistance was associated with enhanced Fc gamma receptor (FCGR) signaling and downregulation of KSRP-associated post-transcriptional regulatory processes. In contrast, secondary resistance was preceded by distinct baseline EV proteomic patterns involving complement activation and reduced hemostasis- and platelet-related pathways. EV-derived biomarker signatures for overall, primary, and secondary resistance independently discriminated patients according to PFS. Baseline plasma EV proteomics reveals distinct systemic biological programs associated with different resistance phenotypes to ICI in advanced melanoma. EV-derived biomarker signatures enable stratification by PFS and warrant validation in larger, multicentric cohorts.

Extracellular vesicles (EVs) are membranous structures that cells release into the extracellular space. EVs carry various molecules such as proteins, lipids, and nucleic acids, and serve as specialized transporters to influence other cells. In the central nervous system, EVs have been linked to many important processes, including intercellular communication, but molecular details of their physiological functions are not fully understood. Our study aimed to investigate how EVs are released by neuronal cells, and how they affect the neuronal activity of other recipient neurons. We show that mature primary cortical neurons release EVs from both their soma and dendrites. EVs released from neurons closely resemble non-neuronal EVs regarding size and marker proteins, and proteomic analyses showed that neuronally released EVs contain proteins typically acting in pre- and post-synaptic compartments. Interestingly, our analysis revealed that EVs alter spontaneous activity in target neurons by increasing the amplitude of postsynaptic potentials. In summary, our findings elaborate on the role of EVs in synaptic activity modulation in neurons mediated by glutamate receptors.

Repeated sequential activation of connected neurons causes lasting changes in synaptic strength, a process known as spike-timing-dependent plasticity (STDP). Recently, sequential spike patterns have been induced without electrodes, using two spectrally separated channelrhodopsins. However, due to the difficulty of labeling and localizing the few connecting synapses between the stimulated preand postsynaptic neurons (similar to 1-5 per neuron pair), ultrastructural analysis after STDP has not been reported. Here, we optogenetically induce STDP at CA3-CA1 hippocampal synapses and identify stimulated boutons and spines in CA1 using transmission electron microscopy (TEM). Presynaptic CA3 neurons express vesicle-targeted horseradish peroxidase, cre recombinase, and cre-dependent ChrimsonR, a red light-activatable channelrhodopsin. Postsynaptic neurons express violet light-activatable CheRiff and dAPEX2, an enhanced ascorbate peroxidase. In TEM, presynaptic boutons and postsynaptic spines are readily identifiable with well-preserved ultra-structural features. Our labeling strategy allows ultrastructural analysis of optogenetically manipulated neurons and their synapses.

Mutations in the lysosomal membrane protein CLN3 cause Juvenile Neuronal Ceroid Lipofuscinosis (JNCL). Activation of the lysosomal ion channel TRPML1 has previously been shown to be beneficial in several neurodegenerative disease models. Here, we tested whether TRPML1 activation rescues disease-associated phenotypes in CLN3-deficient retinal pigment epithelial (ARPE-19 CLN3-KO) cells. ARPE-19 CLN3-KO cells accumulate LAMP1 positive organelles and show lysosomal storage of mitochondrial ATPase subunit C (SubC), globotriaosylceramide (Gb3), and glycerophosphodiesters (GPDs), whereas lysosomal bis(monoacylglycero)phosphate (BMP/LBPA) lipid levels were significantly decreased. Activation of TRPML1 reduced lysosomal storage of Gb3 and SubC but failed to restore BMP levels in CLN3-KO cells. TRPML1-mediated decrease of storage was TFEB-independent, and we identified TRPML1-mediated enhanced lysosomal exocytosis as a likely mechanism for clearing storage including GPDs. Therefore, ARPE-19 CLN3-KO cells represent a human cell model for CLN3 disease showing many of the described core lysosomal deficits, some of which can be improved using TRPML1 agonists.