A Model for Apoptotic-Cell-Mediated Adaptive Immune Evasion via CD80–CTLA-4 Signaling

Apoptotic cells carry a plethora of self-antigens but they suppress eliciting of innate and adaptive immune responses to them. How apoptotic cells evade and subvert adaptive immune responses has been elusive. Here, we propose a novel model to understand how apoptotic cells regulate T cell activation in different contexts, leading mostly to tolerogenic responses, mainly via taking control of the CD80–CTLA-4 coinhibitory signal delivered to T cells. This model may facilitate understanding of the molecular mechanisms of autoimmune diseases associated with dysregulation of apoptosis or apoptotic cell clearance, and it highlights potential therapeutic targets or strategies for treatment of multiple immunological disorders

Cerebral organoids exhibit mature neurons and astrocytes and recapitulate electrophysiological activity of the human brain

Multiple protocols have been devised to generate cerebral organoids that recapitulate features of the developing human brain, including the presence of a large, multi-layered, cortical-like neuronal zone. However, the central question is whether these organoids truly present mature, functional neurons and astrocytes, which may qualify the system for in-depth molecular neuroscience studies focused at neuronal and synaptic functions. Here, we demonstrate that cerebral organoids derived under optimal differentiation conditions exhibit mature, fully functional neurons and astrocytes, as validated by immunohistological, gene expression, and electrophysiological, analyses. Neurons in cerebral organoids showed gene expression profiles and electrophysiological properties similar to those reported for fetal human brain. These important findings indicate that cerebral organoids recapitulate the developing human brain and may enhance use of cerebral organoids in modeling human brain development or investigating neural deficits that underlie neurodevelopmental and neuropsychiatric conditions, such as autism or intellectual disorders

Optineurin Is a Universal Entry Receptor for Herpesviruses

Optineurin is a protein expressed in many human tissues. Mutations of optineurin were shown to be associated with major human diseases, such as primary open-angle glaucoma (a blinding disease) and amyotropic lateral sclerosis (a lethal condition). The functions of this protein, which was discovered relatively recently, are mostly unknown, or are not fully characterized. In this study, we investigated whether optineurin plays a role in regulating infections of multiple human herpesviruses (herpes simplex viruses-1 and -2, and human cytomegalovirus), and found that it functions to support the herpesvirus infection. After experimentally ruling out alternative hypotheses regarding its mechanism of action, we found that optineurin interacts with the viral envelope glycoprotein B (gB) and that such an interaction is required for membrane fusion and virus internalization into the cells. Using rigorous criteria of a viral entry receptor, we established that optineurin fulfills all these criteria and is qualified as the viral entry receptor for the three herpesviruses tested, and perhaps other herpesviruses that express the highly conserved ligand, gB. We also demonstrated that optineurin represents an essential component of the herpesvirus entry machinery that is required for infection in vitro and in vivo. For the first time, this study establishes the cell-surface receptor properties of optineurin, utilizing the virus as a biological probe. Discovery of the receptor optineurin will indubitably both advance the understanding of herpesvirus entry machinery and enlighten cell biology with a new receptor molecule that may perform similar receptor functions in non-infection conditions, or optineurin-regulated devastating human diseases

Analysis of Synapses in Cerebral Organoids

Cerebral organoids are an emerging cutting-edge technology to model human brain development and neurodevelopmental disorders, for which mouse models exhibit significant limitations. In the human brain, synaptic connections define neural circuits, and synaptic deficits account for various neurodevelopmental disorders. Thus, harnessing the full power of cerebral organoids for human brain modeling requires the ability to visualize and analyze synapses in cerebral organoids. Previously, we devised an optimized method to generate human cerebral organoids, and showed that optimal organoids express mature-neuron markers, including synaptic proteins and neurotransmitter receptors and transporters. Here, we give evidence for synaptogenesis in cerebral organoids, via microscopical visualization of synapses. We also describe multiple approaches to quantitatively analyze synapses in cerebral organoids. Collectively, our work provides sufficient evidence for the possibility of modeling synaptogenesis and synaptic disorders in cerebral organoids, and may help advance the use of cerebral organoids in molecular neuroscience and studies of neurodevelopmental disorders such as autism

Analysis of Autophagy in HSV-1 Infection of Corneal Epithelial Cells.

<p>A. HCE cells were uninfected or infected with HSV-1 at the indicated MOIs. SQSTM1/p62 and LC3 were immunoblotted at 2 hpi. Quantification of band intensities was performed using digital band quantification software (ImageQunatTL; GE). B. Uninfected or infected HCE cells were immunoblotted at 8 hpi.</p

Analysis of Autophagy in HSV-1 Infection of Retinal Ganglion Cells.

<p>A. Experimental layout. B. RGC5 cells were transfected with HcRed-LC3. After 24 hr, the cells were mock-treated, treated with MG132, or infected with HSV-1 for 24 hr. The cells were washed in presence of saponin, and analyzed cytofluorimetrically. C. Quantitation of the relative levels of autophagosomal LC3-II in (B), calculated using integrated MFI of the gated region R6. D. Quantification of autophagosomal LC3-II (from saponin-treated cells), shown as % of total LC3 (non saponin-treated cells), as a function of infection with various MOIs of HSV-1 in RGC5 cells.</p

Analysis of Autophagy in HSV-1 Infection of Fibroblast Cells.

<p>A. MEFs were transfected with LC3-GFP. After 24 hr, the cells were uninfected or infected with different HSV-1. At 2 or 24 hpi, the cells were imaged using confocal microscopy. B. Quantification of the number of LC3-GFP punctae per cell in (A); about 30 cells per sample were counted.</p

Analysis of Autophagy in HSV-1 Infection of HeLa Cells.

<p>A. HeLa cells were uninfected (mock), or infected with different strains of HSV-1 (KOS or McKrae). At 2 hpi, the cells were harvested, lysed, and the lysate was immunoblotted for LC3 levels. B. Uninfected or infected HeLa cells were harvested at 6 hpi, lysed, and immunoblotted. C. Immunoblotting of Uninfected or infected HeLa cells at 12 hpi. D. Immunoblotting of Uninfected or infected HeLa cells at 30 hpi. E. Quantification of LC3-II levels in (A-D), normalized to the housekeeping protein GAPDH.</p

Characterization of a proteolytically stable d-Peptide that suppresses herpes simplex virus 1 infection: implications for the development of entry-based antiviral therapy

Uncontrolled herpes simplex virus 1 (HSV-1) infection can advance to serious conditions, including corneal blindness or fatal encephalitis. Here, we describe a highly potent anti-HSV-1 peptide (DG2) that inhibits HSV-1 entry into host cells and blocks all aspects of infection. Importantly, DG2 is highly resistant to proteases and shows minimal toxicity, paving the way for prophylactic or therapeutic application of the peptide in vivo