Pyramidal neuron dendrites integrate synaptic input from multiple partners. Different inputs converging on the same dendrite have distinct structural and functional features, but the molecular mechanisms organizing input-specific properties are poorly understood. We identify the orphan receptor GPR158 as a binding partner for the heparan sulfate proteoglycan (HSPG) glypican 4 (GPC4). GPC4 is enriched on hippocampal granule cell axons (mossy fibers), whereas postsynaptic GPR158 is restricted to the proximal segment of CA3 apical dendrites receiving mossy fiber input. GPR158-induced presynaptic differentiation in contacting axons requires cell-surface GPC4 and the co-receptor LAR. Loss of GPR158 increases mossy fiber synapse density but disrupts bouton morphology, impairs ultrastructural organization of active zone and postsynaptic density, and reduces synaptic strength of this connection, while adjacent inputs on the same dendrite are unaffected. Our work identifies an input-specific HSPG-GPR158 interaction that selectively organizes synaptic architecture and function of developing mossy fiber-CA3 synapses in the hippocampus.

Synapses are often far from the soma and independently cope with proteopathic stress induced by intense neuronal activity. However, how presynaptic compartments turn over proteins is poorly understood. We show that the synapse-enriched protein EndophilinA, thus far studied for its role in endocytosis, induces macroautophagy at presynaptic terminals. We find that EndophilinA executes this unexpected function at least partly independent of its role in synaptic vesicle endocytosis. EndophilinA-induced macroautophagy is activated when the kinase LRRK2 phosphorylates the EndophilinA-BAR domain and is blocked in animals where EndophilinA cannot be phosphorylated. EndophilinA-phosphorylation promotes the formation of highly curved membranes, and reconstitution experiments show these curved membranes serve as docking stations for autophagic factors, including Atg3. Functionally, deregulation of the EndophilinA phosphorylation state accelerates activity-induced neurodegeneration. Given that EndophilinA is connected to at least three Parkinson's disease genes (LRRK2, Parkin and Synaptojanin), dysfunction of EndophilinA-dependent synaptic macroautophagy may be common in this disorder.

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P2, but this function appears normal in SynaptojaninRQ knock-in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P2, two lipids found in autophagosomal membranes. Using advanced imaging, we show that SynaptojaninRQ mutants accumulate the PI(3)P/PI(3,5)P2-binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell-derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in SynaptojaninRQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic-specific autophagy defects to Parkinson's disease.

Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Searching and evaluating the Human Protein Atlas for transmembrane proteins enabled us to identify an integral membrane protein, TMEM115, that is enriched in the Golgi complex. Biochemical and cell biological analysis suggested that TMEM115 has four candidate transmembrane domains located in the N-terminal region. Both the N- and C-terminal domains are oriented towards the cytoplasm. Immunofluorescence analysis supports that TMEM115 is enriched in the Golgi cisternae. Functionally, TMEM115 knockdown or overexpression delays Brefeldin-A-induced Golgi-to-ER retrograde transport, phenocopying cells with mutations or silencing of the conserved oligomeric Golgi (COG) complex. Co-immunoprecipitation and in vitro binding experiments reveals that TMEM115 interacts with the COG complex, and might self-interact to form dimers or oligomers. A short region (residues 206-229) immediately to the C-terminal side of the fourth transmembrane domain is both necessary and sufficient for Golgi targeting. Knockdown of TMEM115 also reduces the binding of the lectins peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA), suggesting an altered O-linked glycosylation profile. These results establish that TMEM115 is an integral membrane protein of the Golgi stack regulating Golgi-to-ER retrograde transport and is likely to be part of the machinery of the COG complex.

Although both insulin and glucagon are intimately involved in the regulation of glucose homeostasis, the intrinsic control of glucagon secretion, including the biogenesis and exocytosis of glucagon-containing granules, is far less understood compared with that of insulin. As Brefeldin A-inhibited guanine nucleotide exchange protein 3 (BIG3) is a negative regulator of insulin-granule biogenesis and insulin secretion, we investigated whether BIG3 plays any role in alpha-cells and glucagon secretion.

Corticotropin-releasing factor (CRF) and urocortin (UCN) are closely related multifunctional regulators, governing, among other processes, Purkinje cell development. Here, we investigate the effects of CRF and UCN on Purkinje cells in organotypic slices. We show that both peptides upregulate delta2 ionotropic glutamate receptor gene expression, and increase the abundance of the receptor in the postsynaptic density. However, only UCN treatment results in increased delta2 protein level per Purkinje cell, implying the existence of posttranscriptional regulation of GluRdelta2 mRNA. CRF, in contrast, reduces the number of delta2-positive dendritic shafts per cell, implying that the increase of GluRdelta2 in remaining synapses may be mainly due to its retargeting. We further observed different patterns of GluRdelta2 distribution in the zone of postsynaptic density upon CRF and UCN treatment. CRF treatment results in a clustered distribution of GluRdelta2 along the postsynaptic density, whereas UCN treatment provides a linear distribution.

The neuropeptide corticotropin-releasing factor (CRF) exerts a pivotal role in modulating neuronal activity in the mammalian brain. The effects of CRF exhibit notable variations, depending on factors such as duration of exposure, concentration, and anatomical location. In the CA1 region of the hippocampus, the impact of CRF is dichotomous: chronic exposure to CRF impairs synapse formation and dendritic integrity, whereas brief exposure enhances synapse formation and plasticity. In the current study, we demonstrate long-term effects of acute CRF on the density and stability of mature mushroom spines ex vivo. We establish that both CRF receptors are present in this hippocampal region, and we pinpoint their precise subcellular localization within synapses by electron microscopy. Furthermore, both in vivo and ex vivo data collectively demonstrate that a transient surge of CRF in the CA1 activates the cyclin-dependent kinase 5 (Cdk5)-pathway. This activation leads to a notable augmentation in CRF-dependent spine formation. Overall, these data suggest that upon acute release of CRF in the CA1-SR synapse, both CRF-Rs can be activated and promote synaptic plasticity via activating different downstream signaling pathways, such as the Cdk5-pathway.

Endocytosis directs molecular cargo along three main routes: recycling to the cell surface, transport to the Golgi apparatus or degradation in endolysosomes. Pseudomonas exotoxin A (PE) is a bacterial protein that typically traffics to the Golgi and then the endoplasmic reticulum before translocating to the cytosol. Here we show that a substantial fraction of internalized PE is also located in nuclear envelope-associated endosomes (NAE), which display limited mobility, exhibit a propensity to undergo fusion and readily discharge their contents into the nuclear envelope. Electron microscopy and protein trapping in the nucleus indicate that NAE mediate PE transfer into the nucleoplasm. RNAi screening further revealed that NAE-mediated transfer depends on the nuclear envelope proteins SUN1 and SUN2, as well as the Sec61 translocon complex. These data reveal a novel endosomal route from the cell surface to the nucleoplasm that facilitates the accumulation of extracellular and cell surface proteins in the nucleus.

The brain cytoplasmic (BC1) RNA is a non-coding RNA (ncRNA) involved in neuronal translational control. Absence of BC1 is associated with altered glutamatergic transmission and maladaptive behavior. Here, we show that pyramidal neurons in the barrel cortex of BC1 knock out (KO) mice display larger excitatory postsynaptic currents and increased spontaneous activity in vivo. Furthermore, BC1 KO mice have enlarged spine heads and postsynaptic densities and increased synaptic levels of glutamate receptors and PSD-95. Of note, BC1 KO mice show aberrant structural plasticity in response to whisker deprivation, impaired texture novel object recognition and altered social behavior. Thus, our study highlights a role for BC1 RNA in experience-dependent plasticity and learning in the mammalian adult neocortex, and provides insight into the function of brain ncRNAs regulating synaptic transmission, plasticity and behavior, with potential relevance in the context of intellectual disabilities and psychiatric disorders.Brain cytoplasmic (BC1) RNA is a non-coding RNA that has been implicated in translational regulation, seizure, and anxiety. Here, the authors show that in the cortex, BC1 RNA is required for sensory deprivation-induced structural plasticity of dendritic spines, as well as for correct sensory learning and social behaviors.

Biological responses to stress are complex and highly conserved. Corticotropin-releasing factor (CRF) plays a central role in regulating these lifesaving physiological responses to stress. We show that, in mice, CRF rapidly changes Schaffer Collateral (SC) input into hippocampal CA1 pyramidal cells (PC) by modulating both functional and structural aspects of these synapses. Host exposure to acute stress, in vivo CRF injection, and ex vivo CRF application all result in fast de novo formation and remodeling of existing dendritic spines. Functionally, CRF leads to a rapid increase in synaptic strength of SC input into CA1 neurons, e.g., increase in spontaneous neurotransmitter release, paired-pulse facilitation, and repetitive excitability and improves synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD). In line with the changes in synaptic function, CRF increases the number of presynaptic vesicles, induces redistribution of vesicles towards the active zone, increases active zone size, and improves the alignment of the pre- and postsynaptic compartments. Therefore, CRF rapidly enhances synaptic communication in the hippocampus, potentially playing a crucial role in the enhanced memory consolidation in acute stress.

Matrix metalloproteinases (MMP) play critical roles in a variety of immune reactions by facilitating cell migration, and affect cell communication by processing both cytokines and cell surface receptors. Based on published data indicating that MMP-9 is upregulated upon T cell activation and also in the thymus upon the induction of negative selection, we investigated the contribution of MMP-9 into mouse T cell development and differentiation in the thymus. Our data suggest that MMP-9 deficiency does not result in major abnormalities in the development of any conventionally selected or agonist selected subsets and does not interfere with thymocyte apoptosis and clearance, and that MMP-9 expression is not induced in immature T cells at any stage of their thymic development.

Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now find that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and membrane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deforming function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release.

The Zonula Occludens proteins ZO-1 and ZO-2 are cell-cell junction-associated adaptor proteins that are essential for the structural and regulatory functions of tight junctions in epithelial cells and their absence leads to early embryonic lethality in mouse models. Here, we use the embryoid body, an in vitro peri-implantation mouse embryogenesis model, to elucidate and dissect the roles ZO-1 and ZO-2 play in epithelial morphogenesis and de novo tight junction assembly. Through the generation of individual or combined ZO-1 and ZO-2 null embryoid bodies, we show that their dual deletion prevents tight junction formation, resulting in the disorganization and compromised barrier function of embryoid body epithelial layers. The disorganization is associated with poor microvilli development, fragmented basement membrane deposition and impaired cavity formation, all of which are key epithelial tissue morphogenetic processes. Expression of Podocalyxin, which positively regulates the formation of microvilli and the apical membrane, is repressed in embryoid bodies lacking both ZO-1 and ZO-2 and this correlates with an aberrant submembranous localization of Ezrin. The null embryoid bodies thus give an insight into how the two ZO proteins influence early mouse embryogenesis and possible mechanisms underlying the embryonic lethal phenotype.

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.

Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPLL isoform localizes to mitochondria, whereas PREPLS remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPLL is involved in mitochondrial homeostasis.

Neuronal activity causes use-dependent decline in protein function. However, it is unclear how this is coupled to local quality control mechanisms. We show in Drosophila that the endocytic protein Endophilin-A (EndoA) connects activity-induced calcium influx to synaptic autophagy and neuronal survival in a Parkinson disease-relevant fashion. Mutations in the disordered loop, including a Parkinson disease-risk mutation, render EndoA insensitive to neuronal stimulation and affect protein dynamics: when EndoA is more flexible, its mobility in membrane nanodomains increases, making it available for autophagosome formation. Conversely, when EndoA is more rigid, its mobility reduces, blocking stimulation-induced autophagy. Balanced stimulation-induced autophagy is required for dopagminergic neuron survival, and a variant in the human ENDOA1 disordered loop conferring risk to Parkinson disease also blocks nanodomain protein mobility and autophagy both in vivo and in human-induced dopaminergic neurons. Thus, we reveal a mechanism that neurons use to connect neuronal activity to local autophagy and that is critical for neuronal survival.

Pyramidal neurons express rich repertoires of leucine-rich repeat (LRR)-containing adhesion molecules with similar synaptogenic activity in culture. The in vivo relevance of this molecular diversity is unclear. We show that hippocampal CA1 pyramidal neurons express multiple synaptogenic LRR proteins that differentially distribute to the major excitatory inputs on their apical dendrites. At Schaffer collateral (SC) inputs, FLRT2, LRRTM1, and Slitrk1 are postsynaptically localized and differentially regulate synaptic structure and function. FLRT2 controls spine density, whereas LRRTM1 and Slitrk1 exert opposing effects on synaptic vesicle distribution at the active zone. All LRR proteins differentially affect synaptic transmission, and their combinatorial loss results in a cumulative phenotype. At temporoammonic (TA) inputs, LRRTM1 is absent; FLRT2 similarly controls functional synapse number, whereas Slitrk1 function diverges to regulate postsynaptic AMPA receptor density. Thus, LRR proteins differentially control synaptic architecture and function and act in input-specific combinations and a context-dependent manner to specify synaptic properties.

Analysis of neuronal arborization and connections is a powerful tool in fundamental and clinical neuroscience. Changes in neuronal morphology are central to brain development and plasticity and are associated with numerous diseases. Golgi staining is a classical technique based on a deposition of metal precipitate in a random set of neurons. Despite their versatility, Golgi methods have limitations that largely precluded their use in advanced microscopy. We combined Golgi staining with fluorescent labeling and tissue clearing techniques in an Alzheimer's disease model. We further applied 3D electron microscopy to visualize entire Golgi-stained neurons, while preserving ultrastructural details of stained cells, optimized Golgi staining for use with block-face scanning electron microscopy, and developed an algorithm for semi-automated neuronal tracing of cells displaying complex staining patterns. Our method will find use in fundamental neuroscience and the study of neuronal morphology in disease.