GDF15 controls primary cilia morphology and function thereby affecting progenitor proliferation.

We recently reported that growth/differentiation factor 15 (GDF15) and its receptor GDNF family receptor alpha-like (GFRAL) are expressed in the periventricular germinal epithelium thereby regulating apical progenitor proliferation. However, the mechanisms are unknown. We now found GFRAL in primary cilia and altered cilia morphology upon GDF15 ablation. Mutant progenitors also displayed increased histone deacetylase 6 (Hdac6) and ciliary adenylate cyclase 3 (Adcy3) transcript levels. Consistently, microtubule acetylation, endogenous sonic hedgehog (SHH) activation and ciliary ADCY3 were all affected in this group. Application of exogenous GDF15 or pharmacological antagonists of either HDAC6 or ADCY3 similarly normalized ciliary morphology, proliferation and SHH signalling. Notably, Gdf15 ablation affected Hdac6 expression and cilia length only in the mutant periventricular niche, in concomitance with ciliary localization of GFRAL. In contrast, in the hippocampus, where GFRAL was not expressed in the cilium, progenitors displayed altered Adcy3 expression and SHH signalling, but Hdac6 expression, cilia morphology and ciliary ADCY3 levels remained unchanged. Thus, ciliary signalling underlies the effect of GDF15 on primary cilia elongation and proliferation in apical progenitors.

Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule interaction.

Tauopathies such as Alzheimer's disease are characterized by aggregation and increased phosphorylation of the microtubule-associated protein tau. Tau's pathological changes are closely linked to neurodegeneration, making tau a prime candidate for intervention. We developed an approach to monitor pathological changes of aggregation-prone human tau in living neurons. We identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that interact with tau and modulate tau kinases. We found that PHOX15 inhibits tau aggregation, restores tau's physiological microtubule interaction, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and suggest that PHOX15 binding reduces the protofilament's ability to adopt a PHF-like conformation by modifying a key glycine triad. Our data demonstrate that live-cell imaging of a tauopathy model enables screening of compounds that modulate tau-microtubule interaction and allows identification of a promising polypharmacological drug candidate that simultaneously inhibits tau aggregation and reduces tau phosphorylation.

TwinF interface inhibitor FP802 stops loss of motor neurons and mitigates disease progression in a mouse model of ALS.

Toxic signaling by extrasynaptic NMDA receptors (eNMDARs) is considered an important promoter of amyotrophic lateral sclerosis (ALS) disease progression. To exploit this therapeutically, we take advantage of TwinF interface (TI) inhibition, a pharmacological principle that, contrary to classical NMDAR pharmacology, allows selective elimination of eNMDAR-mediated toxicity via disruption of the NMDAR/TRPM4 death signaling complex while sparing the vital physiological functions of synaptic NMDARs. Post-disease onset treatment of the SOD1G93A ALS mouse model with FP802, a modified TI inhibitor with a safe pharmacology profile, stops the progressive loss of motor neurons in the spinal cord, resulting in a reduction in the serum biomarker neurofilament light chain, improved motor performance, and an extension of life expectancy. FP802 also effectively blocks NMDA-induced death of neurons in ALS patient-derived forebrain organoids. These results establish eNMDAR toxicity as a key player in ALS pathogenesis. TI inhibitors may provide an effective treatment option for ALS patients.

NECAB2 is an endosomal protein important for striatal function.

Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.

Distinct domains in Ndc1 mediate its interaction with the Nup84 complex and the nuclear membrane.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1's transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1's C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1's amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.

N-methyl-d-aspartate Receptor-mediated Preconditioning Mitigates Excitotoxicity in Human Induced Pluripotent Stem Cell-derived Brain Organoids.

Studies in rodent models of acute and chronic neurodegenerative disorders have uncovered that glutamate-induced excitotoxic cell death is mediated primarily by extrasynaptic N-methyl-d-aspartate receptors (NMDARs). Rodent neurons can also build up in an activity-dependent manner a protective shield against excitotoxicity. This form of acquired neuroprotection is induced by preconditioning with low doses of NMDA or by activation of synaptic NMDARs triggered by bursts of action potentials. Whether NMDARs in human neurons have similar dichotomous actions in cell death and survival is unknown. To investigate this, we established an induced pluripotent stem cell (iPSC)-derived forebrain organoid model for excitotoxic cell death and explored conditions of NMDAR activation that promote neuronal survival when applied prior to a toxic insult. We found that glutamate-induced excitotoxicity in human iPSC-derived neurons is mediated by NMDARs. Treatment of organoids with high concentrations of glutamate or NMDA caused the typical excitotoxicity pathology, comprising structural disintegration, neurite blebbing, shut-off of the transcription factor CRE binding protein (CREB), and cell death. In contrast, bath-applied low doses of NMDA elicited synaptic activity, a robust and sustained increase in CREB phosphorylation as well as function, and upregulation of immediate-early genes, including neuroprotective genes. Moreover, we found that conditions of enhanced synaptic activity increased survival of human iPSC-derived neurons if applied as pre-treatment before toxic NMDA application. These results revealed that both toxic and protective actions of NMDARs are preserved in human neurons. The experimental platform described in this study may prove useful for the validation of neuroprotective gene products and drugs in human neurons.

Structural characterization of an Arf dimer interface: molecular mechanism of Arf-dependent membrane scission.

Dimerization of the small GTPase Arf is prerequisite for the scission of COPI-coated transport vesicles. Here, we quantify the monomer/dimer equilibrium of Arf within the membrane and show that after membrane scission, Arf dimers are restricted to donor membranes. By hydrogen exchange mass spectrometry, we define the interface of activated dimeric Arf within its switch II region. Single amino acid exchanges in this region reduce the propensity of Arf to dimerize. We suggest a mechanism of membrane scission by which the dimeric form of Arf is segregated to the donor membrane. Our data are consistent with the previously reported absence of dimerized Arf in COPI vesicles and could explain the presence of one single scar-like noncoated region in each COPI vesicle.

Epsin but not AP-2 supports reconstitution of endocytic clathrin-coated vesicles.

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based in vitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.

Shaping the heart: Structural and functional maturation of iPSC-cardiomyocytes in 3D-micro-scaffolds.

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) represent the best cell source for cardiac regenerative purposes but retain an immature phenotype after differentiation with significant limitations compared to adult cardiomyocytes. Apart from an incomplete cardiomyocyte-specific structure and microarchitecture, cells show at the level of Ca2+ signaling only slow Ca2+ release and reuptake properties. Here, we investigated the effect of restructuring single iPSC-CMs in specially designed 3D-micro-scaffolds on cell morphology and Ca2+ handling. Using direct laser writing, rectangular-shaped scaffolds were produced and single iPSC-CMs were seeded into these forms. Structural analyses revealed strong sarcolemmal remodeling processes and myofilament reorientation in 3D-shaped cells leading to enhanced clustered expression of L-type Ca2+ channels and ryanodine receptors and consequently, to faster Ca2+ transient kinetics. Spontaneous beating activity was enhanced and Ca2+ handling was more robust compared to non-patterned cells. Overall, our data demonstrate for the first time significant improvement of Ca2+ signaling properties in reshaped iPSC-CMs indicative of functional maturation by structural remodeling.

Proteomic Profiling of Mammalian COPII and COPI Vesicles.

Intracellular transport and homeostasis of the endomembrane system in eukaryotic cells depend on the formation and fusion of vesicular carriers. Coat protein complex (COP) II vesicles export newly synthesized secretory proteins from the endoplasmic reticulum (ER), whereas COPI vesicles facilitate traffic from the Golgi to the ER and intra-Golgi transport. Mammalian cells express various isoforms of COPII and COPI coat proteins. To investigate the roles of coat protein paralogs, we have combined in vitro vesicle reconstitution from semi-intact cells with SILAC-based mass spectrometric analysis. Here, we describe the core proteomes of mammalian COPII and COPI vesicles. Whereas the compositions of COPII vesicles reconstituted with various isoforms of the cargo-binding subunit Sec24 differ depending on the paralog used, all of the isoforms of the COPI coat produce COPI-coated vesicles with strikingly similar protein compositions.

Cutis laxa, exocrine pancreatic insufficiency and altered cellular metabolomics as additional symptoms in a new patient with ATP6AP1-CDG.

Congenital disorders of glycosylation (CDG) are genetic defects in the glycoconjugate biosynthesis. >100 types of CDG are known, most of them cause multi-organ diseases. Here we describe a boy whose leading symptoms comprise cutis laxa, pancreatic insufficiency and hepatosplenomegaly. Whole exome sequencing identified the novel hemizygous mutation c.542T>G (p.L181R) in the X-linked ATP6AP1, an accessory protein of the mammalian vacuolar H+-ATPase, which led to a general N-glycosylation deficiency. Studies of serum N-glycans revealed reduction of complex sialylated and appearance of truncated diantennary structures. Proliferation of the patient's fibroblasts was significantly reduced and doubling time prolonged. Additionally, there were alterations in the fibroblasts' amino acid levels and the acylcarnitine composition. Especially, short-chain species were reduced, whereas several medium- to long-chain acylcarnitines (C14-OH to C18) were elevated. Investigation of the main lipid classes revealed that total cholesterol was significantly enriched in the patient's fibroblasts at the expense of phophatidylcholine and phosphatidylethanolamine. Within the minor lipid species, hexosylceramide was reduced, while its immediate precursor ceramide was increased. Since catalase activity and ACOX3 expression in peroxisomes were reduced, we assume an ATP6AP1-dependent impact on the ß-oxidation of fatty acids. These results help to understand the complex clinical characteristics of this new patient.

Synaptic Activity Protects Neurons Against Calcium-Mediated Oxidation and Contraction of Mitochondria During Excitotoxicity.

AIMS: Excitotoxicity triggered by extrasynaptic N-methyl-d-aspartate-type glutamate receptors has been implicated in many neurodegenerative conditions, including Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and stroke. Mitochondrial calcium overload leading to mitochondrial dysfunction represents an early event in excitotoxicity. Neurons are rendered resistant to excitotoxicity by previous periods of synaptic activity that activates a nuclear calcium-driven neuroprotective gene program. This process, termed acquired neuroprotection, involves transcriptional repression of the mitochondrial calcium uniporter leading to a reduction in excitotoxcity-associated mitochondrial calcium load. As mitochondrial calcium and the production of reactive oxygen species may be linked, we monitored excitotoxicity-associated changes in the mitochondrial redox status using the ratiometric glutathione redox potential indicator, glutaredoxin 1 (GRX1)-redox-sensitive green fluorescent protein (roGFP)2, targeted to the mitochondrial matrix. Aim of this study was to investigate if suppression of oxidative stress underlies mitoprotection afforded by synaptic activity. RESULTS: We found that synaptic activity protects primary rat hippocampal neurons against acute excitotoxicity-induced mitochondrial oxidative stress and mitochondrial contraction associated with it. Downregulation of the mitochondrial uniporter by genetic means mimics the protective effect of synaptic activity on mitochondrial redox status. These findings indicate that oxidative stress acts downstream of mitochondrial calcium overload in excitotoxicity. Innovation and Conclusion: We established mito-GRX1-roGFP2 as a reliable and sensitive tool to monitor rapid redox changes in mitochondria during excitotoxicity. Our results highlight the importance of developing means of blocking mitochondrial calcium overload for therapeutic targeting of oxidative stress and mitochondrial dysfunction in neurodegenerative diseases. Antioxid. Redox. Signal. 29, 1109-1124.

A Flow Cytometry-Based Approach for the Isolation and Characterization of Neural Stem Cell Primary Cilia.

In the adult mammalian brain, the apical surface of the subependymal zone (SEZ) is covered by many motile ependymal cilia and a few primary cilia originating from rare intermingled neural stem cells (NSCs). In NSCs the primary cilia are key for the transduction of essential extracellular signals such as Sonic hedgehog (SHH) and platelet-derived growth factor (PDGF). Despite their importance, the analysis of NSC primary cilia is greatly hampered by the fact that they are overwhelmingly outnumbered by the motile cilia. We here take advantage of flow cytometry to purify the two cilia types and allow their molecular characterization. Primary cilia were identified based on immunoreactivity to the marker adenylate cyclase type III (AC3) and differential levels of prominin-1 whereas motile cilia displayed immunoreactivity only to the latter. Consistent with the morphological differences between the two classes of cilia, enrichment of motile cilia positively correlated with size. Moreover, we observed age-dependent variations in the abundance of the two groups of ciliary organelles reflecting the changes associated with their development. The two cilia groups also differed with respect to the expression of signaling molecules, since PDGF receptor (PDGFR)α, smoothened (Smo) and CXC chemokine receptor (CXCR)4 were only detected in isolated primary but not motile cilia. Thus, our novel method of cilia isolation and characterization by flow cytometry has the potential to be extended to the study of cilia from different tissues and organs, providing a powerful tool for the investigation of primary cilia in physiological and pathological conditions.

Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol.

The high pathogenicity of the Ebola virus reflects multiple concurrent processes on infection. Among other important determinants, Ebola fusogenic glycoprotein (GP) has been associated with the detachment of infected cells and eventually leads to vascular leakage and haemorrhagic fever. Here we report that the membrane-anchored GP is sufficient to induce the detachment of adherent cells. The results show that the detachment induced through either full-length GP1,2 or the subunit GP2 depends on cholesterol and the structure of the transmembrane domain. These data reveal a novel molecular mechanism in which GP regulates Ebola virus assembly and suggest that cholesterol-reducing agents could be useful as therapeutics to counteract GP-mediated cell detachment.

Golgi phosphoprotein 3 triggers signal-mediated incorporation of glycosyltransferases into coatomer-coated (COPI) vesicles.

Newly synthesized membrane and secreted proteins undergo a series of posttranslational modifications in the Golgi apparatus, including attachment of carbohydrate moieties. The final structure of so-formed glycans is determined by the order of execution of the different glycosylation steps, which seems intimately related to the spatial distribution of glycosyltransferases and glycosyl hydrolases within the Golgi apparatus. How cells achieve an accurate localization of these enzymes is not completely understood but might involve dynamic processes such as coatomer-coated (COPI) vesicle-mediated trafficking. In yeast, this transport is likely to be regulated by vacuolar protein sorting 74 (Vps74p), a peripheral Golgi protein able to interact with COPI coat as well as with a binding motif present in the cytosolic tails of some mannosyltransferases. Recently, Golgi phosphoprotein 3 (GOLPH3), the mammalian homolog of Vps74, has been shown to control the Golgi localization of core 2 N-acetylglucosamine-transferase 1. Here, we highlight a role of GOLPH3 in the spatial localization of α-2,6-sialyltransferase 1. We show, for the first time, that GOLPH3 supports incorporation of both core 2 N-acetylglucosamine-transferase 1 and α-2,6-sialyltransferase 1 into COPI vesicles. Depletion of GOLPH3 altered the subcellular localization of these enzymes. In contrast, galactosyltransferase, an enzyme that does not interact with GOLPH3, was neither incorporated into COPI vesicles nor was dependent on GOLPH3 for proper localization.

The nuclear calcium signaling target, activating transcription factor 3 (ATF3), protects against dendrotoxicity and facilitates the recovery of synaptic transmission after an excitotoxic insult.

The focal swellings of dendrites ("dendritic beading") are an early morphological hallmark of neuronal injury and dendrotoxicity. They are associated with a variety of pathological conditions, including brain ischemia, and cause an acute disruption of synaptic transmission and neuronal network function, which contribute to subsequent neuronal death. Here, we show that increased synaptic activity prior to excitotoxic injury protects, in a transcription-dependent manner, against dendritic beading. Expression of activating transcription factor 3 (ATF3), a nuclear calcium-regulated gene and member of the core gene program for acquired neuroprotection, can protect against dendritic beading. Conversely, knockdown of ATF3 exacerbates dendritic beading. Assessment of neuronal network functions using microelectrode array recordings revealed that hippocampal neurons expressing ATF3 were able to regain their ability for functional synaptic transmission and to participate in coherent neuronal network activity within 48 h after exposure to toxic concentrations of NMDA. Thus, in addition to attenuating cell death, synaptic activity and expression of ATF3 render hippocampal neurons more resistant to acute dendrotoxicity and loss of synapses. Dendroprotection can enhance recovery of neuronal network functions after excitotoxic insults.

Proliferation and cilia dynamics in neural stem cells prospectively isolated from the SEZ.

Neural stem cells (NSCs) generate new neurons in vivo and in vitro throughout adulthood and therefore are physiologically and clinically relevant. Unveiling the mechanisms regulating the lineage progression from NSCs to newborn neurons is critical for the transition from basic research to clinical application. However, the direct analysis of NSCs and their progeny is still elusive due to the problematic identification of the cells. We here describe the isolation of highly purified genetically unaltered NSCs and transit-amplifying precursors (TAPs) from the adult subependymal zone (SEZ). Using this approach we show that a primary cilium and high levels of epidermal growth factor receptor (EGFR) at the cell membrane characterize quiescent and cycling NSCs, respectively. However, we also observed non-ciliated quiescent NSCs and NSCs progressing into the cell cycle without up-regulating EGFR expression. Thus, the existence of NSCs displaying distinct molecular and structural conformations provides more flexibility to the regulation of quiescence and cell cycle progression.

Scission of COPI and COPII vesicles is independent of GTP hydrolysis.

Intracellular transport and maintenance of the endomembrane system in eukaryotes depends on formation and fusion of vesicular carriers. A seeming discrepancy exists in the literature about the basic mechanism in the scission of transport vesicles that depend on GTP-binding proteins. Some reports describe that the scission of COP-coated vesicles is dependent on GTP hydrolysis, whereas others found that GTP hydrolysis is not required. In order to investigate this pivotal mechanism in vesicle formation, we analyzed formation of COPI- and COPII-coated vesicles utilizing semi-intact cells. The small GTPases Sar1 and Arf1 together with their corresponding coat proteins, the Sec23/24 and Sec13/31 complexes for COPII and coatomer for COPI vesicles were required and sufficient to drive vesicle formation. Both types of vesicles were efficiently generated when GTP hydrolysis was blocked either by utilizing the poorly hydrolyzable GTP analogs GTPγS and GMP-PNP, or with constitutively active mutants of the small GTPases. Thus, GTP hydrolysis is not required for the formation and release of COP vesicles.

Virus-Like Particles Harboring CCL19, IL-2 and HPV16 E7 Elicit Protective T Cell Responses in HLA-A2 Transgenic Mice.

Infection by high-risk genotypes of human papillomaviruses (HR-HPVs) is the cause of cancer of the uterine cervix. Although prophylactic vaccines directed against the two most prevalent HR-HPV types (HPV16 and 18) have been commercialized recently, there is a need for effective therapeutic vaccines against HR-HPVs. We have tested in mice a chimeric protein composed of the hepatitis B small surface antigen (HBsAg(S)) flanked at its N-terminus by chemokine CC ligand 19/macrophage inflammatory protein-3ß (CCL19/MIP-3ß), and at the C-terminus by interleukin 2 (IL-2) and an artificial HPV16 E7 polytope. This protein is assembled into nanoparticles and both CCL19 and IL-2 conserve their functionality. HLA-A2 (AAD) transgenic mice immunized with a plasmid encoding this protein mounted specific T cell responses against E7 without the need of an adjuvant. Furthermore, vaccination prevented the development of tumors after implantation of the E6/E7-expressing TC-1/A2 tumor cell line. Our results suggest that vaccines based on HBsAg(S) nanoparticles carrying short E7 epitopes and immune-stimulatory domains might be of therapeutic value in the treatment of patients suffering from cervical pre-cancer or cancer lesions caused by HR-HPVs.

Nuclear factor erythroid-derived 2 (Nfe2) regulates JunD DNA-binding activity via acetylation: a novel mechanism regulating trophoblast differentiation.

We recently demonstrated that the bZip transcription factor nuclear factor erythroid-derived 2 (Nfe2) represses protein acetylation and expression of the transcription factor glial cell missing 1 (Gcm1) in trophoblast cells, preventing excess syncytiotrophoblast formation and permitting normal placental vascularization and embryonic growth. However, the Gcm1 promoter lacks a Nfe2-binding site and hence the mechanisms linking Nfe2 and Gcm1 expression remained unknown. Here we show that Nfe2 represses JunD DNA-binding activity to the Gcm1 promoter during syncytiotrophoblast differentiation. Interventional studies using knockdown and knockin approaches show that enhanced JunD DNA-binding activity is required for increased expression of Gcm1 and syncytiotrophoblast formation as well as impaired placental vascularization and reduced growth of Nfe2(-/-) embryos. Induction of Gcm1 expression requires binding of JunD to the -1441 site within the Gcm1 promoter, which is distinct from the -1314 site previously shown to induce Gcm1 expression by other bZip transcription factors. Nfe2 modulates JunD binding to the Gcm1 promoter via acetylation, as reducing JunD acetylation using the histone acetyltransferase inhibitor curcumin reverses the increased JunD DNA-binding activity observed in the absence of Nfe2. This identifies a novel mechanism through which bZip transcription factors interact. Within the placenta this interaction regulates Gcm1 expression, syncytiotrophoblast formation, placental vascularization, and embryonic growth.