Cdk5-dependent rapid formation and stabilization of dendritic spines by corticotropin-releasing factor.

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.

EndophilinA-dependent coupling between activity-induced calcium influx and synaptic autophagy is disrupted by a Parkinson-risk mutation.

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.

Mitochondria metabolism sets the species-specific tempo of neuronal development.

Neuronal development in the human cerebral cortex is considerably prolonged compared with that of other mammals. We explored whether mitochondria influence the species-specific timing of cortical neuron maturation. By comparing human and mouse cortical neuronal maturation at high temporal and cell resolution, we found a slower mitochondria development in human cortical neurons compared with that in the mouse, together with lower mitochondria metabolic activity, particularly that of oxidative phosphorylation. Stimulation of mitochondria metabolism in human neurons resulted in accelerated development in vitro and in vivo, leading to maturation of cells weeks ahead of time, whereas its inhibition in mouse neurons led to decreased rates of maturation. Mitochondria are thus important regulators of the pace of neuronal development underlying human-specific brain neoteny.

Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function.

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.

Corticotropin-releasing factor induces functional and structural synaptic remodelling in acute stress.

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.

Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and lipid-independent mechanisms.

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.

A workflow for streamlined acquisition and correlation of serial regions of interest in array tomography.

BACKGROUND: Array tomography (AT) is a high-resolution imaging method to resolve fine details at the organelle level and has the advantage that it can provide 3D volumes to show the tissue context. AT can be carried out in a correlative way, combing light and electron microscopy (LM, EM) techniques. However, the correlation between modalities can be a challenge and delineating specific regions of interest in consecutive sections can be time-consuming. Integrated light and electron microscopes (iLEMs) offer the possibility to provide well-correlated images and may pose an ideal solution for correlative AT. Here, we report a workflow to automate navigation between regions of interest. RESULTS: We use a targeted approach that allows imaging specific tissue features, like organelles, cell processes, and nuclei at different scales to enable fast, directly correlated in situ AT using an integrated light and electron microscope (iLEM-AT). Our workflow is based on the detection of section boundaries on an initial transmitted light acquisition that serves as a reference space to compensate for changes in shape between sections, and we apply a stepwise refinement of localizations as the magnification increases from LM to EM. With minimal user interaction, this enables autonomous and speedy acquisition of regions containing cells and cellular organelles of interest correlated across different magnifications for LM and EM modalities, providing a more efficient way to obtain 3D images. We provide a proof of concept of our approach and the developed software tools using both Golgi neuronal impregnation staining and fluorescently labeled protein condensates in cells. CONCLUSIONS: Our method facilitates tracing and reconstructing cellular structures over multiple sections, is targeted at high resolution ILEMs, and can be integrated into existing devices, both commercial and custom-built systems.

An accelerated procedure for approaching and imaging of optically branded region of interest in tissue.

Many areas of biology have benefited from advances in light microscopy (LM). However, one limitation of the LM approach is that numerous critically important aspects of subcellular machineries are well beyond the resolution of conventional LM. For studying these, electron microscopy (EM) remains the technique of choice to visualize and identify macromolecules at the ultrastructural level. The most powerful approach is combining both techniques, LM and EM (i.e., to apply correlative light/electron microscopy, CLEM) to image exactly the same region of interest. This combination allows, for example, to immuno-localize proteins by LM and then to visualize the ultrastructural context of the same region of the sample. However, the identification and correlation of the regions of interest (ROIs) at the levels of LM and EM remains a major challenge, mostly due to the difficulties with correlation along the Z-axis for both modalities. In this chapter, we address this difficulty and describe an approach for performing CLEM in tissue samples using marks from near-infrared branding as indicators of a ROI, and then using serial block face-scanning electron microscopy (SBF-SEM) to identify and approach this ROI. Once a ROI has been approached, serial sections are collected on grids for high-resolution imaging by transmission EM, and subsequent correlation with LM images showing labeled proteins.

Loss of Furin in ß-Cells Induces an mTORC1-ATF4 Anabolic Pathway That Leads to ß-Cell Dysfunction.

FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, but its specific role in pancreatic ß-cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, whereas PCs that potentially could provide redundancy are expressed at considerably lower levels. ß-cell-specific Furin knockout (ßFurKO) mice are glucose intolerant as a result of smaller islets with lower insulin content and abnormal dense-core secretory granule morphology. mRNA expression analysis and differential proteomics on ßFurKO islets revealed activation of activating transcription factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). ßFurKO cells show impaired cleavage or shedding of vacuolar-type ATPase (V-ATPase) subunits Ac45 and prorenin receptor, respectively, and impaired lysosomal acidification. Blocking V-ATPase pharmacologically in ß-cells increased mTORC1 activity, suggesting involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in ß-cells lacking Furin, causing ß-cell dysfunction.

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy.

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer.

Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.

Corticotropin releasing factor-binding protein (CRF-BP) as a potential new therapeutic target in Alzheimer's disease and stress disorders.

Alzheimer's disease is the most common cause of dementia and one of the most complex human neurodegenerative diseases. Numerous studies have demonstrated a critical role of the environment in the pathogenesis and pathophysiology of the disease, where daily life stress plays an important role. A lot of epigenetic studies have led to the conclusion that chronic stress and stress-related disorders play an important part in the onset of neurodegenerative disorders, and an enormous amount of research yielded valuable discoveries but has so far not led to the development of effective treatment strategies for Alzheimer's disease. Corticotropin-releasing factor (CRF) is one of the major hormones and at the same time a neuropeptide acting in stress response. Deregulation of protein levels of CRF is involved in the pathogenesis of Alzheimer's disease, but little is known about the precise roles of CRF and its binding protein, CRF-BP, in neurodegenerative diseases. In this review, we summarize the key evidence for and against the involvement of stress-associated modulation of the CRF system in the pathogenesis of Alzheimer's disease and discuss how recent findings could lead to new potential treatment possibilities in Alzheimer's disease by using CRF-BP as a therapeutic target.

Differentiation but not ALS mutations in FUS rewires motor neuron metabolism.

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.

Modernization of Golgi staining techniques for high-resolution, 3-dimensional imaging of individual neurons.

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.

Gold-substituted Silver-intensified Peroxidase Immunolabeling for FIB-SEM Imaging.

Modern electron microscopy offers a wide variety of tools to investigate the ultrastructural organization of cells and tissues and to accurately pinpoint intracellular localizations of macromolecules of interest. New volumetric electron microscopy techniques and new instrumentation provide unique opportunities for high-throughput analysis of comparatively large volumes of tissue and their complete reconstitution in three-dimensional (3D) electron microscopy. However, due to a variety of technical issues such as the limited penetration of label into the tissue, low antigen preservation, substantial electron density of secondary detection reagents, and many others, the adaptation of immuno-detection techniques for use with such 3D imaging methods as focused ion beam-scanning electron microscopy (FIB-SEM) has been challenging. Here, we describe a sample preparation method for 3D FIB-SEM, which results in an optimal preservation and staining of ultrastructural details at a resolution necessary for tracing immunolabeled neuronal structures and detailed reconstruction of synapses. This technique is applicable to neuronal and non-neuronal cells, tissues, and a wide variety of antigens.

PARL deficiency in mouse causes Complex III defects, coenzyme Q depletion, and Leigh-like syndrome.

The mitochondrial intramembrane rhomboid protease PARL has been implicated in diverse functions in vitro, but its physiological role in vivo remains unclear. Here we show that Parl ablation in mouse causes a necrotizing encephalomyelopathy similar to Leigh syndrome, a mitochondrial disease characterized by disrupted energy production. Mice with conditional PARL deficiency in the nervous system, but not in muscle, develop a similar phenotype as germline Parl KOs, demonstrating the vital role of PARL in neurological homeostasis. Genetic modification of two major PARL substrates, PINK1 and PGAM5, do not modify this severe neurological phenotype. Parl-/- brain mitochondria are affected by progressive ultrastructural changes and by defects in Complex III (CIII) activity, coenzyme Q (CoQ) biosynthesis, and mitochondrial calcium metabolism. PARL is necessary for the stable expression of TTC19, which is required for CIII activity, and of COQ4, which is essential in CoQ biosynthesis. Thus, PARL plays a previously overlooked constitutive role in the maintenance of the respiratory chain in the nervous system, and its deficiency causes progressive mitochondrial dysfunction and structural abnormalities leading to neuronal necrosis and Leigh-like syndrome.

An Input-Specific Orphan Receptor GPR158-HSPG Interaction Organizes Hippocampal Mossy Fiber-CA3 Synapses.

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.

Hsp90 Mediates Membrane Deformation and Exosome Release.

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.

A Modular Organization of LRR Protein-Mediated Synaptic Adhesion Defines Synapse Identity.

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.

The non-coding RNA BC1 regulates experience-dependent structural plasticity and learning.

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.