Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells.

Stem cell transplantation shows enormous potential for treatment of incurable retinal degeneration (RD). To determine if and how grafts connect with the neural circuits of the advanced degenerative retina (ADR) and improve vision, we perform calcium imaging of GCaMP5-positive grafts in retinal slices. The organoid-derived C-Kit/SSEA1 (C-Kit) retinal progenitor cells (RPCs) become synaptically organized and build spontaneously active synaptic networks in three major layers of ADR.

Localization of the WD repeat-containing protein 5 to the Virion Assembly Compartment Facilitates Human Cytomegalovirus Assembly.

We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region, and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB).

Anterograde Viral Tracer Herpes Simplex Virus 1 Strain H129 Transports Primarily as Capsids in Cortical Neuron Axons.

The features of herpes simplex virus 1 (HSV-1) strain 129 (H129), including natural neurotropism and anterograde transneuronal trafficking, make it a potential tool for anterograde neural circuitry tracing. Recently anterograde polysynaptic and monosynaptic tracers were developed from H129 and have been applied for the identification of novel connections and functions of different neural circuitries. However, how H129 viral particles are transported in neurons, especially those of the central nervous system, remains unclear.

Human embryonic stem cell-derived retinal pigment epithelium transplants as a potential treatment for wet age-related macular degeneration.

Stem cell therapy may provide a safe and promising treatment for retinal diseases. Wet age-related macular degeneration (wet-AMD) is a leading cause of blindness in China. We developed a clinical-grade human embryonic stem cell (hESC) line, Q-CTS-hESC-2, under xeno-free conditions that differentiated into retinal pigment epithelial cells (Q-CTS-hESC-2-RPE).

Changes in intrinsic excitability of ganglion cells in degenerated retinas of RCS rats.

Aim: To evaluate the intrinsic excitability of retinal ganglion cells (RGCs) in degenerated retinas.

Methods: The intrinsic excitability of various morphologically defined RGC types using a combination of patch-clamp recording and the Lucifer yellow tracer in retinal whole-mount preparations harvested from Royal College of Surgeons (RCS) rats, a common retinitis pigmentosa (RP) model, in a relatively late stage of retinal degeneration (P90) were investigated. Several parameters of RGC morphologies and action potentials (APs) were measured and compared to those of non-dystrophic control rats, including dendritic stratification, dendritic field diameter, peak amplitude, half width, resting membrane potential, AP threshold, depolarization to threshold, and firing rates.

Proteomic profiling of early degenerative retina of RCS rats.

Aim: To identify the underlying cellular and molecular changes in retinitis pigmentosa (RP).

Methods: Label-free quantification-based proteomics analysis, with its advantages of being more economic and consisting of simpler procedures, has been used with increasing frequency in modern biological research. Dystrophic RCS rats, the first laboratory animal model for the study of RP, possess a similar pathological course as human beings with the diseases.

Human melanopsin-AAV2/8 transfection to retina transiently restores visual function in rd1 mice.

Aim: To explore whether ectopic expression of human melanopsin can effectively and safely restore visual function in rd1 mice.

Methods: Hematoxylin-eosin staining of retinal sections from rd1 mice was used to detect the thickness of the outer nuclear layer to determine the timing of surgery. We constructed a human melanopsin-AAV2/8 viral vector and injected it into the subretinal space of rd1 mice.

Connexin-based channels contribute to metabolic pathways in the oligodendroglial lineage.

Oligodendrocyte precursor cells (OPCs) undergo a series of energy-consuming developmental events; however, the uptake and trafficking pathways for their energy metabolites remain unknown. In the present study, we found that 2-NBDG, a fluorescent glucose analog, can be delivered between astrocytes and oligodendrocytes through connexin-based gap junction channels but cannot be transferred between astrocytes and OPCs. Instead, connexin hemichannel-mediated glucose uptake supports OPC proliferation, and ethidium bromide uptake or increase of 2-NBDG uptake rate is correlated with intracellular Ca(2+) elevation in OPCs, indicating a Ca(2+)-dependent activation of connexin hemichannels.

Synaptic vesicles are "primed" for fast clathrin-mediated endocytosis at the ribbon synapse.

Retrieval of synaptic vesicles can occur 1-10 s after fusion, but the role of clathrin during this process has been unclear because the classical mode of clathrin-mediated endocytosis (CME) is an order of magnitude slower, as during retrieval of surface receptors. Classical CME is thought to be rate-limited by the recruitment of clathrin, which raises the question: how is clathrin recruited during synaptic vesicle recycling? To investigate this question we applied total internal reflection fluorescence microscopy (TIRFM) to the synaptic terminal of retinal bipolar cells expressing fluorescent constructs of clathrin light-chain A. Upon calcium influx we observed a fast accumulation of clathrin within 100 ms at the periphery of the active zone.

Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells.

The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs.

Effect of optogenetic stimulus on the proliferation and cell cycle progression of neural stem cells.

Modulation of stem cell proliferation is a crucial aspect of neural developmental biology and regenerative medicine. To investigate the effect of optical stimulation on neural stem cell proliferation, cells transduced with channelrhodopsin-2 (ChR2) were used to analyze changes in cell proliferation and cell cycle distribution after light stimulation. Blue light significantly inhibited cell proliferation and affected the cell cycle, which increased the percentage of cells in G1 phase and reduced the percentage in S phase.

Homeostatic synaptic plasticity through changes in presynaptic calcium influx.

Chronic perturbations of electrical activity within neural circuits lead to compensatory changes in synaptic strength collectively termed homeostatic synaptic plasticity. The postsynaptic mechanisms underlying these modifications have been characterized in some detail, but the presynaptic mechanisms that alter the efficiency of evoked neurotransmitter release are less clear. To investigate the role of presynaptic calcium influx, we have combined the use of two fluorescent proteins in cultured hippocampal neurons: a calcium reporter localized to synaptic vesicles, SyGCaMP2, and a reporter of vesicle fusion, SypHy.

Nicotine-induced Ca2+-myristoyl switch of neuronal Ca2+ sensor VILIP-1 in hippocampal neurons: a possible crosstalk mechanism for nicotinic receptors.

Visinin-like protein (VILIP-1) belongs to the neuronal Ca2+ sensor family of EF-hand Ca2+-binding proteins that regulate a variety of Ca2+-dependent signal transduction processes in neurons. It is an interaction partner of alpha4beta2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca2+ concentration by Ca2+-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca2+-myristoyl switch.