Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

The interplay between anatomical connectivity and dynamics in neural networks plays a key role in the functional properties of the brain and in the associated connectivity changes induced by neural diseases. However, a detailed experimental investigation of this interplay at both cellular and population scales in the living brain is limited by accessibility. Alternatively, to investigate the basic operational principles with morphological, electrophysiological and computational methods, the activity emerging from large in vitro networks of primary neurons organized with imposed topologies can be studied. Here, we validated the use of a new bio-printing approach, which effectively maintains the topology of hippocampal cultures in vitro and investigated, by patch-clamp and MEA electrophysiology, the emerging functional properties of these grid-confined networks. In spite of differences in the organization of physical connectivity, our bio-patterned grid networks retained the key properties of synaptic transmission, short-term plasticity and overall network activity with respect to random networks. Interestingly, the imposed grid topology resulted in a reinforcement of functional connections along orthogonal directions, shorter connectivity links and a greatly increased spiking probability in response to focal stimulation. These results clearly demonstrate that reliable functional studies can nowadays be performed on large neuronal networks in the presence of sustained changes in the physical network connectivity.

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.

The pathogenesis and clinical heterogeneity of Parkinson's disease (PD) have been evaluated from molecular, pathophysiological, and clinical perspectives. High-throughput proteomic analysis of cerebrospinal fluid (CSF) opened new opportunities for scrutinizing this heterogeneity. To date, this is the most comprehensive CSF-based proteomics profiling study in PD with 569 patients (350 idiopathic patients, 65 GBA + mutation carriers and 154 LRRK2 + mutation carriers), 534 controls, and 4135 proteins analyzed. Combining CSF aptamer-based proteomics with genetics we determined protein quantitative trait loci (pQTLs). Analyses of pQTLs together with summary statistics from the largest PD genome wide association study (GWAS) identified 68 potential causal proteins by Mendelian randomization. The top causal protein, GPNMB, was previously reported to be upregulated in the substantia nigra of PD patients. We also compared the CSF proteomes of patients and controls. Proteome differences between GBA + patients and unaffected GBA + controls suggest degeneration of dopaminergic neurons, altered dopamine metabolism and increased brain inflammation. In the LRRK2 + subcohort we found dysregulated lysosomal degradation, altered alpha-synuclein processing, and neurotransmission. Proteome differences between idiopathic patients and controls suggest increased neuroinflammation, mitochondrial dysfunction/oxidative stress, altered iron metabolism and potential neuroprotection mediated by vasoactive substances. Finally, we used proteomic data to stratify idiopathic patients into "endotypes". The identified endotypes show differences in cognitive and motor disease progression based on previously reported protein-based risk scores.Our findings not only contribute to the identification of new therapeutic targets but also to shape personalized medicine in CNS neurodegeneration.

Genetic defects in myelin formation and maintenance cause leukodystrophies, a group of white matter diseases whose mechanistic underpinnings are poorly understood. Hypomyelination and congenital cataract (HCC), one of these disorders, is caused by mutations in FAM126A, a gene of unknown function. We show that FAM126A, also known as hyccin, regulates the synthesis of phosphatidylinositol 4-phosphate (PtdIns(4)P), a determinant of plasma membrane identity. HCC patient fibroblasts exhibit reduced PtdIns(4)P levels. FAM126A is an intrinsic component of the plasma membrane phosphatidylinositol 4-kinase complex that comprises PI4KIIIα and its adaptors TTC7 and EFR3 (refs 5,7). A FAM126A-TTC7 co-crystal structure reveals an all-α-helical heterodimer with a large protein-protein interface and a conserved surface that may mediate binding to PI4KIIIα. Absence of FAM126A, the predominant FAM126 isoform in oligodendrocytes, destabilizes the PI4KIIIα complex in mouse brain and patient fibroblasts. We propose that HCC pathogenesis involves defects in PtdIns(4)P production in oligodendrocytes, whose specialized function requires massive plasma membrane expansion and thus generation of PtdIns(4)P and downstream phosphoinositides. Our results point to a role for FAM126A in supporting myelination, an important process in development and also following acute exacerbations in multiple sclerosis.

Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-gamma(35)S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50 = 0.15 microM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90alpha inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.

Modulation of synaptic vesicle retrieval is considered to be potentially important in steady-state synaptic performance. Here we show that at physiological temperature endocytosis kinetics at hippocampal and cortical nerve terminals show a bi-phasic dependence on electrical activity. Endocytosis accelerates for the first 15-25 APs during bursts of action potential firing, after which it slows with increasing burst length creating an optimum stimulus for this kinetic parameter. We show that activity-dependent acceleration is only prominent at physiological temperature and that the mechanism of this modulation is based on the dephosphorylation of dynamin 1. Nerve terminals in which dynamin 1 and 3 have been replaced with dynamin 1 harboring dephospho- or phospho-mimetic mutations in the proline-rich domain eliminate the acceleration phase by either setting endocytosis at an accelerated state or a decelerated state, respectively. DOI:http://dx.doi.org/10.7554/eLife.00845.001.

The exocytosis of synaptic vesicles (SVs) elicited by potent stimulation is rapidly compensated by bulk endocytosis of SV membranes leading to large endocytic vacuoles ('bulk' endosomes). Subsequently, these vacuoles disappear in parallel with the reappearance of new SVs. We have used synapses of dynamin 1 and 3 double knock-out neurons, where clathrin-mediated endocytosis (CME) is dramatically impaired, to gain insight into the poorly understood mechanisms underlying this process. Massive formation of bulk endosomes was not defective, but rather enhanced, in the absence of dynamin 1 and 3. The subsequent conversion of bulk endosomes into SVs was not accompanied by the accumulation of clathrin coated buds on their surface and this process proceeded even after further clathrin knock-down, suggesting its independence of clathrin. These findings support the existence of a pathway for SV reformation that bypasses the requirement for clathrin and dynamin 1/3 and that operates during intense synaptic activity.

The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P2 phosphatases conserved from yeast to humans and the only PI(4,5)P2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL-Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin.