Vti Proteins:Beyond Endolysosomal Trafficking

Vti proteins are conserved from yeast to humans and regulate intracellular membrane trafficking by providing one specific SNARE domain, the Qb SNARE, to the four helical SNARE bundle that drives membrane fusion. Two mammalian Vti genes, Vti1a and Vti1b are reported to regulate distinct aspects of endolysosomal trafficking and retrograde transport to the Golgi, but have also been implicated in synaptic vesicle secretion. In this review, we summarize the current evidence for the role of Vti proteins in intracellular trafficking in different cells. We propose that, despite some unique aspects, the two mammalian VTI genes have largely redundant functions in neurosecretory cells and recycle molecules required for the sorting of regulated cargo to the Golgi. Defects in this recycling also lead to defects in synaptic transmission and dense core vesicle secretion

Vti1a/b regulate synaptic vesicle and dense core vesicle secretion via protein sorting at the Golgi

The SNAREs Vti1a/1b are implicated in regulated secretion, but their role relative to canonical exocytic SNAREs remains elusive. Here, we show that synaptic vesicle and dense-core vesicle (DCV) secretion is indeed severely impaired in Vti1a/b-deficient neurons. The synaptic levels of proteins that mediate secretion were reduced, down to 50% for the exocytic SNARE SNAP25. The delivery of SNAP25 and DCV-cargo into axons was decreased and these molecules accumulated in the Golgi. These defects were rescued by either Vti1a or Vti1b expression. Distended Golgi cisternae and clear vacuoles were observed in Vti1a/b-deficient neurons. The normal non-homogeneous distribution of DCV-cargo inside the Golgi was lost. Cargo trafficking out of, but not into the Golgi, was impaired. Finally, retrograde Cholera Toxin trafficking, but not Sortilin/Sorcs1 distribution, was compromised. We conclude that Vti1a/b support regulated secretion by sorting secretory cargo and synaptic secretion machinery components at the Golgi

Differential Maturation of the Two Regulated Secretory Pathways in Human iPSC-Derived Neurons

Neurons communicate by regulated secretion of chemical signals from synaptic vesicles (SVs) and dense-core vesicles (DCVs). Here, we investigated the maturation of these two secretory pathways in micro-networks of human iPSC-derived neurons. These micro-networks abundantly expressed endogenous SV and DCV markers, including neuropeptides. DCV transport was microtubule dependent, preferentially anterograde in axons, and 2-fold faster in axons than in dendrites. SV and DCV secretion were strictly Ca2+ and SNARE dependent. DCV secretion capacity matured until day in vitro (DIV) 36, with intense stimulation releasing 6% of the total DCV pool, and then plateaued. This efficiency is comparable with mature mouse neurons. In contrast, SV secretion capacity continued to increase until DIV50, with substantial further increase in secretion efficiency and decrease in silent synapses. These data show that the two secretory pathways can be studied in human neurons and that they mature differentially, with DCV secretion reaching maximum efficiency when that of SVs is still low

PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure.

The active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid-liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function

Detection of silent cells, synchronization and modulatory activity in developing cellular networks

Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature "silent" cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity-based pixel correlations to identify cellular-based regions of interest (ROIs) and coincident cell activity. However, using activity-based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell-dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi-automatically detect cells within developing neuronal networks that were imaged using calcium-sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers

Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells

<div><p>Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.</p></div

Functional analysis of differentiated neurons in direct contact co-cultures.

<p>At day 49 we recorded Line A-derived (n = 2) and Line B-derived (n = 2) neurons, which show (A) fast inward Na⁺ currents followed by long-lasting outward K⁺ currents; current was evoked by voltage steps ranging from -90 mV to 50 mV with 10 mV increments. Inset shows fast Na⁺ currents in absence or presence of Na⁺ channel antagonist TTX (1 μM). (B) Quantification of peak Na⁺ and K⁺ currents evoked in (A) is shown. (C) We recorded action potentials evoked by a current injection of 100 pA and also (D) spontaneously generated action potentials in current clamp mode. (E) Spontaneous post-synaptic events from a single neuron (holding potential at -70 mV in voltage clamp mode) recorded in presence of 1μM TTX, TTX + 40 μM Bicuculline and TTX + 40 μM Bicuculline + 50 μM AP5 + 10 μM DNQX are shown. (F) Postsynaptic current evoked by local field stimulation (2 mA, 1 ms). We recorded calcium traces at day 49 of differentiation; (G, H) hiPSC-derived neurons loaded with Fluo-5 AM ester. (I) Raster plot showing onset and duration of intracellular calcium events from ROIs represented on G; upon TTX addition and 20 minutes after TTX was washed away (J) or traces represented in H upon bicuculline, bicuculline + AP5 + CNQX addition and 20 minutes after drugs were washed away (representative calcium traces for ROI 2 (G, I) and ROI 9 (H, J)) are shown. (K) We then analyzed activity dependent intracellular calcium traces (red dotted bars indicate beginning of electrical field stimulation and number of pulses applied). Scale bars are 10 μm. Data are represented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178533#pone.0178533.s004" target="_blank">S4 Fig</a> for hESC-derived neurons calcium imaging experiments.</p

Characterization of the differentiated neurons in indirect contact co-cultures.

<p>In indirect co-cultures we differentiated the human neurons in close proximity of rat astrocyte layers (Line B1 and B2). (A, B) Bright field images of indirect cultures during differentiation day 26 and 56 are shown. (C-F) We observed the expression of MAP2, VGAT, synaptophysin1 and (G-J) VGLUT1 and HOMER1 co-localizing puncta using immunocytochemistry at day 56. We then analyzed (K) spontaneous mEPSCs and mIPSCs (holding potential at -70mV in voltage clamp mode) recorded in 1 μM TTX supplemented with 40 μM Bicuculline or 50 μM AP5 + 10 μM DNQX respectively. (L) Quantification of amplitude, area, time to decay and frequency of mEPSCs and mIPSCs was also analyzed (n = 2). We then recorded calcium traces of (M, N) neurons loaded with Fluo-5 AM ester from n = 2. (O) Raster plot showing onset and duration of intracellular calcium events from ROIs represented on M and N respectively; upon TTX addition and 20 minutes after TTX was washed away (P) or upon bicuculline, bicuculline + AP5 + CNQX addition and 20 minutes after drugs were washed away (representative calcium traces for ROI 1(M, O) and ROI 8 (N, P)) are shown. (Q) Analysis of activity dependent intracellular calcium traces (red dotted bars indicate beginning of electrical field stimulation and number of pulses applied). Scale bars are 10 μm. Data are represented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178533#pone.0178533.s005" target="_blank">S5 Fig</a> for indirect contact plate preparation method.</p

Proteomic analysis of indirect contact co-culture neurons representing reproducible cultures.

<p>(A) Hierarchical clustering of protein groups based on expression intensity present in the day 56 differentiated neurons. Columns represent triplicates from 2 experiments (B1 and B2) normalized for loading and appear similar in highly expressed proteins (color key values > 4). (B) Average of the coefficient of variation normalized to loading for all the proteins. (C) Average and standard deviations of intensity based absolute quantification (iBAQ) values for pre-, post-synaptic and growth cone proteins in day 56 neuronal cultures.</p