Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.

Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.

The LRRK2 mutation G2019S is the most common genetic cause of Parkinson's disease (PD). To better understand the link between mutant LRRK2 and PD pathology, we derived induced pluripotent stem cells from PD patients harboring LRRK2 G2019S and then specifically corrected the mutant LRRK2 allele. We demonstrate that gene correction resulted in phenotypic rescue in differentiated neurons and uncovered expression changes associated with LRRK2 G2019S. We found that LRRK2 G2019S induced dysregulation of CPNE8, MAP7, UHRF2, ANXA1, and CADPS2. Knockdown experiments demonstrated that four of these genes contribute to dopaminergic neurodegeneration. LRRK2 G2019S induced increased extracellular-signal-regulated kinase 1/2 (ERK) phosphorylation. Transcriptional dysregulation of CADPS2, CPNE8, and UHRF2 was dependent on ERK activity. We show that multiple PD-associated phenotypes were ameliorated by inhibition of ERK. Therefore, our results provide mechanistic insight into the pathogenesis induced by mutant LRRK2 and pointers for the development of potential new therapeutics.

Presynaptic nerve terminals often contain as few as a hundred vesicles and so must recycle them soon after exocytosis to preserve synaptic transmission and presynaptic morphology during repetitive firing. The kinetics and mechanisms of vesicular endocytosis and repriming have therefore been studied. Vesicles in hippocampal nerve terminals can become available to release their contents within approximately 40 s of the previous round of exocytosis. Studies using the styryl dye FM1-43 have estimated the time constant for endocytosis as approximately 20-30 s at least half of the total recycling time, which is much slower than endocytosis in other secretory systems. It seems paradoxical that the neurosecretory terminals that could benefit the most from rapid endocytosis do not use such a mechanism. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was much faster after exposure to staurosporine or elevated extracellular calcium. Thus hippocampal synapses take advantage of efficient mechanisms for endocytosis, and their vesicular retrieval is subject to modulatory control.

Upon exocytosis, synaptic vesicle proteins are released into the plasma membrane and have to be retrieved by compensatory endocytosis. When green fluorescent protein-labeled versions of the vesicle proteins synaptobrevin-2 and synaptotagmin-1 are overexpressed in rat hippocampal neurons, up to 30% are found on axonal membranes under resting conditions. To test whether and to what extent these plasma membrane-stranded proteins participate in exo-endocytic cycling, a new proteolytic approach was used to visualize the fate of newly exocytosed proteins separately from that of the plasma membrane-stranded ones. We found that both pools were mixed and that endocytosed vesicles were largely composed of previously stranded molecules. The degree of nonidentity of vesicular proteins exo- and endocytosed depended on stimulus duration. By using an antibody to the external domain of synaptotagmin-1, we estimated that under physiological conditions a few percent of vesicular proteins were located near the active zone, from where they were preferentially recycled upon stimulation.