Intersectin and Endophilin condensates prime synaptic vesicles for release site replenishment.

Neurotransmitter is released from dedicated sites of synaptic vesicle fusion within a synapse. Following fusion, the vacated sites are replenished immediately by new vesicles for subsequent neurotransmission. These replacement vesicles are assumed to be located near release sites and used by chance.

ERR2 and ERR3 promote the development of gamma motor neuron functional properties required for proprioceptive movement control.

The ability of terrestrial vertebrates to effectively move on land is integrally linked to the diversification of motor neurons into types that generate muscle force (alpha motor neurons) and types that modulate muscle proprioception, a task that in mammals is chiefly mediated by gamma motor neurons. The diversification of motor neurons into alpha and gamma types and their respective contributions to movement control have been firmly established in the past 7 decades, while recent studies identified gene expression signatures linked to both motor neuron types. However, the mechanisms that promote the specification of gamma motor neurons and/or their unique properties remained unaddressed.

Spatial and Temporal Aspects of Exocytosis Studied on the Isolated Plasma Membranes.

Exocytosis of large-dense core vesicles in neuroendocrine cells is a highly regulated, calcium-dependent process, mediated by networks of interrelated proteins and lipids. Here, I describe experimental procedures for studies of selective spatial and temporal aspects of exocytosis at the plasma membrane, or in its proximity, using adrenal chromaffin cells. The assay utilizes primary cells subjected to a brief ultrasonic pulse, resulting in the formation of thin, flat inside-out plasma membranes with attached secretory vesicles and elements of cell cytoskeleton.

ATP hydrolysis by yeast Hsp104 determines protein aggregate dissolution and size in vivo.

Signs of proteostasis failure often entwine with those of metabolic stress at the cellular level. Here, we study protein sequestration during glucose deprivation-induced ATP decline in Saccharomyces cerevisiae. Using live-cell imaging, we find that sequestration of misfolded proteins and nascent polypeptides into two distinct compartments, stress granules, and Q-bodies, is triggered by the exhaustion of ATP.

Vps34 derived phosphatidylinositol 3-monophosphate modulates megakaryocyte maturation and proplatelet production through late endosomes/lysosomes.

Background: Development of platelet precursor cells, megakaryocytes (MKs), implies an increase in their size; formation of the elaborate demarcation membrane system (DMS); and extension of branched cytoplasmic structures, proplatelets, that will release platelets. The membrane source(s) for MK expansion and proplatelet formation have remained elusive.

Objective: We hypothesized that traffic of membranes regulated by phosphatidylinositol 3-monophosphate (PI3P) contributes to MK maturation and proplatelet formation.

Regulation of synaptic vesicle acidification at the neuronal synapse.

The vacuolar H -adenosine triphosphatases (vATPases) acidify multiple intracellular organelles, including synaptic vesicles (SVs) and secretory granules. Acidification of SVs represents a critical point during the SV cycle: without acidification, neurotransmitters cannot be loaded into SVs. Despite the obvious importance of the vesicle acidification process for neurotrasmission and the life of complex organisms, little is known about the regulation of vATPase at the neuronal synapse.

Cells Control BIN1-Mediated Membrane Tubulation by Altering the Membrane Charge.

The Bridging integrator 1 (BIN1)/Amphiphysin/Rvs (BAR) protein family is an essential part of the cell's machinery to bend membranes. BIN1 is a muscle-enriched BAR protein with an established role in muscle development and skeletal myopathies. Here, we demonstrate that BIN1, on its own, is able to form complex interconnected tubular systems in vitro, reminiscent of t-tubule system in muscle cells.

Endophilin-A regulates presynaptic Ca influx and synaptic vesicle recycling in auditory hair cells.

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea.

Spatial and Temporal Aspects of Phosphoinositides in Endocytosis Studied in the Isolated Plasma Membranes.

Endocytosis is a well-orchestrated cascade of lipid-protein and protein-protein interactions resulting in formation and internalization of vesicles. Membrane phospholipids have key regulatory functions in endocytosis and membrane traffic. I have previously described an in vitro assay based on the isolated, substrate-attached plasma membrane to study the spatial distribution and levels of phosphoinositides, in particular phosphatidylinositol-4,5-bisphospate [PI(4,5)P].

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration.

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.

Revisiting the Role of Clathrin-Mediated Endoytosis in Synaptic Vesicle Recycling.

Without robust mechanisms to efficiently form new synaptic vesicles (SVs), the tens to hundreds of SVs typically present at the neuronal synapse would be rapidly used up, even at modest levels of neuronal activity. SV recycling is thus critical for synaptic physiology and proper function of sensory and nervous systems. Yet, more than four decades after it was originally proposed that the SVs are formed and recycled locally at the presynaptic terminals, the mechanisms of endocytic processes at the synapse are heavily debated.

Endophilin-A Deficiency Induces the Foxo3a-Fbxo32 Network in the Brain and Causes Dysregulation of Autophagy and the Ubiquitin-Proteasome System.

Endophilin-A, a well-characterized endocytic adaptor essential for synaptic vesicle recycling, has recently been linked to neurodegeneration. We report here that endophilin-A deficiency results in impaired movement, age-dependent ataxia, and neurodegeneration in mice. Transcriptional analysis of endophilin-A mutant mice, complemented by proteomics, highlighted ataxia- and protein-homeostasis-related genes and revealed upregulation of the E3-ubiquitin ligase FBXO32/atrogin-1 and its transcription factor FOXO3A.

Loss of FBXO7 (PARK15) results in reduced proteasome activity and models a parkinsonism-like phenotype in mice.

Mutations in the FBXO7 (PARK15) gene have been implicated in a juvenile form of parkinsonism termed parkinsonian pyramidal syndrome (PPS), characterized by Parkinsonian symptoms and pyramidal tract signs. FBXO7 (F-box protein only 7) is a subunit of the SCF (SKP1/cullin-1/F-box protein) E3 ubiquitin ligase complex, but its relevance and function in neurons remain to be elucidated. Here, we report that the E3 ligase FBXO7-SCF binds to and ubiquitinates the proteasomal subunit PSMA2.

Molecular Remodeling of the Presynaptic Active Zone of Drosophila Photoreceptors via Activity-Dependent Feedback.

Neural activity contributes to the regulation of the properties of synapses in sensory systems, allowing for adjustment to a changing environment. Little is known about how synaptic molecular components are regulated to achieve activity-dependent plasticity at central synapses. Here, we found that after prolonged exposure to natural ambient light the presynaptic active zone in Drosophila photoreceptors undergoes reversible remodeling, including loss of Bruchpilot, DLiprin-α, and DRBP, but not of DSyd-1 or Cacophony.

Upregulation of Parkin in endophilin mutant mice.

Several proteins encoded by PD genes are implicated in synaptic vesicle traffic. Endophilin, a key factor in the endocytosis of synaptic vesicles, was shown to bind to, and be ubiquitinated by, the PD-linked E3 ubiquitin ligase Parkin. Here we report that Parkin's level is specifically upregulated in brain and fibroblasts of endophilin mutant mice due to increased transcriptional regulation.

Ethanol inhibits long-term potentiation in hippocampal CA1 neurons, irrespective of lamina and stimulus strength, through neurosteroidogenesis.

Ethanol inhibits memory encoding and the induction of long-term potentiation (LTP) in CA1 neurons of the hippocampus. Hippocampal LTP at Schaffer collateral synapses onto CA1 pyramidal neurons has been widely studied as a cellular model of learning and memory, but there is striking heterogeneity in the underlying molecular mechanisms in distinct regions and in response to distinct stimuli. Basal and apical dendrites differ in terms of innervation, input specificity, and molecular mechanisms of LTP induction and maintenance, and different stimuli determine distinct molecular pathways of potentiation.

MicroRNA-125b induces tau hyperphosphorylation and cognitive deficits in Alzheimer's disease.

Sporadic Alzheimer's disease (AD) is the most prevalent form of dementia, but no clear disease-initiating mechanism is known. Aβ deposits and neuronal tangles composed of hyperphosphorylated tau are characteristic for AD. Here, we analyze the contribution of microRNA-125b (miR-125b), which is elevated in AD.

A conserved axon type hierarchy governing peripheral nerve assembly.

In gnathostome vertebrates, including fish, birds and mammals, peripheral nerves link nervous system, body and immediate environment by integrating efferent pathways controlling movement apparatus or organ function and afferent pathways underlying somatosensation. Several lines of evidence suggest that peripheral nerve assembly involves instructive interactions between efferent and afferent axon types, but conflicting findings challenge this view. Using genetic modeling in zebrafish, chick and mouse we uncover here a conserved hierarchy of axon type-dependent extension and selective fasciculation events that govern peripheral nerve assembly, which recapitulates the successive phylogenetic emergence of peripheral axon types and circuits in the vertebrate lineage.

Dlk1 promotes a fast motor neuron biophysical signature required for peak force execution.

Motor neurons, which relay neural commands to drive skeletal muscle movements, encompass types ranging from "slow" to "fast," whose biophysical properties govern the timing, gradation, and amplitude of muscle force. Here we identify the noncanonical Notch ligand Delta-like homolog 1 (Dlk1) as a determinant of motor neuron functional diversification. Dlk1, expressed by ~30% of motor neurons, is necessary and sufficient to promote a fast biophysical signature in the mouse and chick.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons.

Direct live monitoring of heterotypic axon-axon interactions in vitro.

This protocol describes an optimized method for direct in vitro monitoring of homo- and heterotypic axon-axon interactions involved in the developmental assembly of neural circuits. The assay exploits a classical example of heterotypic axonal interactions by modeling the sequential extension of spinal motor and somatosensory neuron axons, but the procedure should be readily adaptable to other neuron types. The protocol is based on the rapid isolation and primary culture of genetically identified motor neurons combined with straightforward vital dye labeling and culture of dorsal root ganglion sensory neurons.

Anatomical coupling of sensory and motor nerve trajectory via axon tracking.

It is a long-standing question how developing motor and sensory neuron projections cooperatively form a common principal grid of peripheral nerve pathways relaying behavioral outputs and somatosensory inputs. Here, we explored this issue through targeted cell lineage and gene manipulation in mouse, combined with in vitro live axon imaging. In the absence of motor projections, dorsal (epaxial) and ventral (hypaxial) sensory projections form in a randomized manner, while removal of EphA3/4 receptor tyrosine kinases expressed by epaxial motor axons triggers selective failure to form epaxial sensory projections.

Caenorhabditits elegans LRK-1 and PINK-1 act antagonistically in stress response and neurite outgrowth.

Mutations in two genes encoding the putative kinases LRRK2 and PINK1 have been associated with inherited variants of Parkinson disease. The physiological role of both proteins is not known at present, but studies in model organisms have linked their mutants to distinct aspects of mitochondrial dysfunction, increased vulnerability to oxidative and endoplasmic reticulum stress, and intracellular protein sorting. Here, we show that a mutation in the Caenorhabditits elegans homologue of the PTEN-induced kinase pink-1 gene resulted in reduced mitochondrial cristae length and increased paraquat sensitivity of the nematode.