Synapses are often far from their cell bodies and must largely independently cope with dysfunctional proteins resulting from synaptic activity and stress. To identify membrane-associated machines that can engulf synaptic targets destined for degradation, we performed a large-scale in vitro liposome-based screen followed by functional studies. We identified a presynaptically enriched chaperone Hsc70-4 that bends membranes based on its ability to oligomerize. This activity promotes endosomal microautophagy and the turnover of specific synaptic proteins. Loss of microautophagy slows down neurotransmission while gain of microautophagy increases neurotransmission. Interestingly, Sgt, a cochaperone of Hsc70-4, is able to switch the activity of Hsc70-4 from synaptic endosomal microautophagy toward chaperone activity. Hence, Hsc70-4 controls rejuvenation of the synaptic protein pool in a dual way: either by refolding proteins together with Sgt, or by targeting them for degradation by facilitating endosomal microautophagy based on its membrane deforming activity.

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided.

The mechanisms behind Aβ-peptide accumulation in non-familial Alzheimer's disease (AD) remain elusive. Proteins of the tetraspanin family modulate Aβ production by interacting to γ-secretase.

While axons within the central nervous system (CNS) do not regenerate following injury, those in the peripheral nervous system (PNS) do, although not in a clinically satisfactory manner as only a small proportion of axons exhibit long-distance regeneration. Moreover, functional recovery is hampered by excessive axonal sprouting and aberrant reinnervation of target tissue. In order to investigate the mechanisms governing the regrowth of axons following injury, previous studies have used lesion paradigms of peripheral nerves in rat or mouse models, and reagents or cells have been administered to the lesion site through nerve conduits, aiming to improve early-stage regeneration. Morphological analysis of such in vivo experiments has however been limited by the incompatibility of synthetic nerve conduits with existing tissue-clearing and imaging techniques. We present herein a novel experimental approach that allows high-resolution imaging of individual axons within nerve conduits, together with quantitative assessment of fiber growth. We used a GFP-expressing mouse strain in a lesion model of the sciatic nerve to describe a strategy that combines nerve clearing, chemical treatment of chitosan nerve conduits, and long working distance confocal microscopy with image processing and analysis. This novel experimental setup provides a means of documenting axon growth within the actual conduit during the critical initial stage of regeneration. This will greatly facilitate the development and evaluation of treatment regimens to improve axonal regeneration following nerve damage.

Although complex inflammatory-like alterations are observed around the amyloid plaques of Alzheimer's disease (AD), little is known about the molecular changes and cellular interactions that characterize this response. We investigate here, in an AD mouse model, the transcriptional changes occurring in tissue domains in a 100-μm diameter around amyloid plaques using spatial transcriptomics. We demonstrate early alterations in a gene co-expression network enriched for myelin and oligodendrocyte genes (OLIGs), whereas a multicellular gene co-expression network of plaque-induced genes (PIGs) involving the complement system, oxidative stress, lysosomes, and inflammation is prominent in the later phase of the disease. We confirm the majority of the observed alterations at the cellular level using in situ sequencing on mouse and human brain sections. Genome-wide spatial transcriptomics analysis provides an unprecedented approach to untangle the dysregulated cellular network in the vicinity of pathogenic hallmarks of AD and other brain diseases.

Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease.

Recent evidence links dysfunctional lipid metabolism to the pathogenesis of Parkinson's disease, but the mechanisms are not resolved. Here, we generated a new Drosophila knock-in model of DNAJC6/Auxilin and find that the pathogenic mutation causes synaptic dysfunction, neurological defects and neurodegeneration, as well as specific lipid metabolism alterations. In these mutants, membrane lipids containing long-chain polyunsaturated fatty acids, including phosphatidylinositol lipid species that are key for synaptic vesicle recycling and organelle function, are reduced. Overexpression of another protein mutated in Parkinson's disease, Synaptojanin-1, known to bind and metabolize specific phosphoinositides, rescues the DNAJC6/Auxilin lipid alterations, the neuronal function defects and neurodegeneration. Our work reveals a functional relation between two proteins mutated in Parkinsonism and implicates deregulated phosphoinositide metabolism in the maintenance of neuronal integrity and neuronal survival.

Understanding the lower urinary tract (LUT) and development of highly needed novel therapies to treat LUT disorders depends on accurate techniques to monitor LUT (dys)function in preclinical models. We recently developed videocystometry in rodents, which combines intravesical pressure measurements with X-ray-based fluoroscopy of the LUT, allowing the in vivo analysis of the process of urine storage and voiding with unprecedented detail. Videocystometry relies on the precise contrast-based determination of the bladder volume at high temporal resolution, which can readily be achieved in anesthetized or otherwise motion-restricted mice but not in awake and freely moving animals. To overcome this limitation, we developed a machine-learning method, in which we trained a neural network to automatically detect the bladder in fluoroscopic images, allowing the automatic analysis of bladder filling and voiding cycles based on large sets of time-lapse fluoroscopic images (>3 hr at 30 images/s) from behaving mice and in a noninvasive manner. With this approach, we found that urethane, an injectable anesthetic that is commonly used in preclinical urological research, has a profound, dose-dependent effect on urethral relaxation and voiding duration. Moreover, both in awake and in anesthetized mice, the bladder capacity was decreased ~fourfold when cystometry was performed acutely after surgical implantation of a suprapubic catheter. Our findings provide a paradigm for the noninvasive, in vivo monitoring of a hollow organ in behaving animals and pinpoint important limitations of the current gold standard techniques to study the LUT in mice.

γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.

Hematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies.

Oligodendrocyte dysfunction has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motor neuron loss. The failure of trophic support provided by oligodendrocytes is associated with a concomitant reduction in oligodendroglial monocarboxylate transporter 1 (MCT1) expression and is detrimental for the long-term survival of motor neuron axons. Therefore, we established an adeno-associated virus 9 (AAV9)-based platform by which MCT1 was targeted mostly to white matter oligodendrocytes to investigate whether this approach could provide a therapeutic benefit in the SOD1G93A mouse model of ALS. Despite good oligodendrocyte transduction and AAV-mediated MCT1 transgene expression, the disease outcome of SOD1G93A mice was not altered. Our study further increases our current understanding about the complex nature of oligodendrocyte pathology in ALS and provides valuable insights into the future development of therapeutic strategies to efficiently modulate these cells.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

Increasing evidence for a direct contribution of astrocytes to neuroinflammatory and neurodegenerative processes causing Alzheimer's disease comes from molecular and functional studies in rodent models. However, these models may not fully recapitulate human disease as human and rodent astrocytes differ considerably in morphology, functionality, and gene expression.

Current mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays. This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples-for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples-cannot be assessed. We have developed "ALMOST," a novel optical method for 3D surface imaging of reflective opaque objects utilizing an optical projection tomography device in combination with oblique illumination and optical filters.

Genetic ablation or pharmacological inhibition of the heat-activated cation channel TRPM3 alleviates inflammatory heat hyperalgesia, but the underlying mechanisms are unknown. We induced unilateral inflammation of the hind paw in mice, and directly compared expression and function of TRPM3 and two other heat-activated TRP channels (TRPV1 and TRPA1) in sensory neurons innervating the ipsilateral and contralateral paw. We detected increased Trpm3 mRNA levels in dorsal root ganglion neurons innervating the inflamed paw, and augmented TRP channel-mediated calcium responses, both in the cell bodies and the intact peripheral endings of nociceptors. In particular, inflammation provoked a pronounced increase in nociceptors with functional co-expression of TRPM3, TRPV1 and TRPA1. Finally, pharmacological inhibition of TRPM3 dampened TRPV1- and TRPA1-mediated responses in nociceptors innervating the inflamed paw, but not in those innervating healthy tissue. These insights into the mechanisms underlying inflammatory heat hypersensitivity provide a rationale for developing TRPM3 antagonists to treat pathological pain.

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.

Dendritic filopodia are dynamic structures thought to be the precursors of spines during synapse development. Morphological maturation to spines is associated with the stabilization and strengthening of synapses, and can be altered in various neurological disorders. Telencephalin (TLN/intercellular adhesion molecule-5 (ICAM5)) localizes to dendritic filopodia, where it facilitates their formation/maintenance, thereby slowing spine morphogenesis. As spines are largely devoid of TLN, its exclusion from the filopodia surface appears to be required in this maturation process. Using HeLa cells and primary hippocampal neurons, we demonstrate that surface removal of TLN involves internalization events mediated by the small GTPase ADP-ribosylation factor 6 (ARF6), and its activator EFA6A. This endocytosis of TLN affects filopodia-to-spine transition, and requires Rac1-mediated dephosphorylation/release of actin-binding ERM proteins from TLN. At the somato-dendritic surface, TLN and EFA6A are confined to distinct, flotillin-positive membrane subdomains. The co-distribution of TLN with this lipid raft marker also persists during its endosomal targeting to CD63-positive late endosomes. This suggests a specific microenvironment facilitating ARF6-mediated mobilization of TLN that contributes to promotion of dendritic spine development.

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.

Phospholipase D3 (PLD3) polymorphisms are linked to late-onset Alzheimer's disease (LOAD). Being a lysosomal 5'-3' exonuclease, its neuronal substrates remained unknown as well as how a defective lysosomal nucleotide catabolism connects to AD-proteinopathy. We identified mitochondrial DNA (mtDNA) as a major physiological substrate and show its manifest build-up in lysosomes of PLD3-defective cells. mtDNA accretion creates a degradative (proteolytic) bottleneck that presents at the ultrastructural level as a marked abundance of multilamellar bodies, often containing mitochondrial remnants, which correlates with increased PINK1-dependent mitophagy. Lysosomal leakage of mtDNA to the cytosol activates cGAS-STING signaling that upregulates autophagy and induces amyloid precursor C-terminal fragment (APP-CTF) and cholesterol accumulation. STING inhibition largely normalizes APP-CTF levels, whereas an APP knockout in PLD3-deficient backgrounds lowers STING activation and normalizes cholesterol biosynthesis. Collectively, we demonstrate molecular cross-talks through feedforward loops between lysosomal nucleotide turnover, cGAS-STING and APP metabolism that, when dysregulated, result in neuronal endolysosomal demise as observed in LOAD.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.