CLN7 is an organellar chloride channel regulating lysosomal function.

Neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive lysosomal storage diseases. One variant form of late-infantile NCL (vLINCL) is caused by mutations of a lysosomal membrane protein CLN7, the function of which has remained unknown. Here, we identified CLN7 as a novel endolysosomal chloride channel. Overexpression of CLN7 increases endolysosomal chloride currents and enlarges endolysosomes through a Ca2+/calmodulin-dependent way. Human CLN7 and its yeast homolog exhibit characteristics of chloride channels and are sensitive to chloride channel blockers. Moreover, CLN7 regulates lysosomal chloride conductance, luminal pH, and lysosomal membrane potential and promotes the release of lysosomal Ca2+ through transient receptor potential mucolipin 1 (TRPML1). Knocking out CLN7 causes pathological features that are similar to those of patients with vLINCL, including retinal degeneration and autofluorescent lipofuscin. The pathogenic mutations in CLN7 lead to a decrease in chloride permeability, suggesting that reconstitution of lysosomal Cl− homeostasis may be an effective strategy for the treatment of vLINCL.

Electrophysiological characterization of photoreceptor-like cells in human inducible pluripotent stem cell-derived retinal organoids during in vitro maturation.

Retinal organoids (ROs) derived from human inducible pluripotent stem cells (hiPSCs) exhibit considerable therapeutic potential. However, current quality control of ROs during in vitro differentiation is largely limited to the detection of molecular markers, often by immunostaining, polymerase chain reaction (PCR) assays and sequencing, often without proper functional assessments. As such, in the current study, we systemically characterized the physiological maturation of photoreceptor-like cells in hiPSC-derived ROs. By performing patch-clamp recordings from photoreceptor-like cells in ROs at distinct differentiation stages (ie, Differentiation Day [D]90, D150, and D200), we determined the electrophysiological properties of the plasma membrane and several characteristic ion channels closely associated with the physiological functions of the photoreceptors. Ionic hallmarks, such as hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and cyclic nucleotide-gated (CNG) channels, matured progressively during differentiation. After D200 in culture, these characteristic currents closely resembled those in macaque or human native photoreceptors. Furthermore, we demonstrated that the hyperpolarization-activated inward current/depolarization-activated outward current ratio (I-120 /I+40 ), termed as the inward-outward current (IOC) ratio hereon, accurately represented the maturity of photoreceptors and could serve as a sensitive indicator of pathological state. Thus, this study provides a comprehensive dataset describing the electrophysiological maturation of photoreceptor-like cells in hiPSC-derived ROs for precise and sensitive quality control during RO differentiation.

Generation of nonhuman primate retinitis pigmentosa model by in situ knockout of RHO in rhesus macaque retina.

Retinitis pigmentosa (RP) is a form of inherited retinal degenerative diseases that ultimately involves the macula, which is present in primates but not in the rodents. Therefore, creating nonhuman primate (NHP) models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future. Here we applied adeno-associated virus (AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque (Macaca mulatta) to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP. Through a series of studies, we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression. More importantly, the mutant macaque retinae displayed clinical RP phenotypes, including photoreceptor degeneration, retinal thinning, abnormal rod subcellular structures, and reduced photoresponse. Therefore, we suggest somatic editing of the RHO gene is able to phenocopy RP, and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.

In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway.

Although Cas9-mediated genome editing has been widely used to engineer alleles in animal models of human inherited diseases, very few homology-directed repair (HDR)-based genetic editing systems have been established in postnatal mouse models for effective and lasting phenotypic rescue. Here, we developed an HDR-based Cas9/RecA system to precisely correct Pde6b mutation with increased HDR efficiency in postnatal rodless (rd1) mice, a retinitis pigmentosa (RP) mutant model characterized by photoreceptor degeneration and loss of vision. The Cas9/RecA system incorporated Cas9 endonuclease enzyme to generate double-strand breaks (DSBs) and bacterial recombinase A (RecA) to increase homologous recombination. Our data revealed that Cas9/RecA treatment significantly promoted the survival of both rod and cone photoreceptors, restored the expression of PDE6B in rod photoreceptors, and enhanced the visual functions of rd1 mice. Thus, this study provides a precise therapeutic strategy for RP and other genetic diseases.

Panton-Valentine leukocidin facilitates the escape of Staphylococcus aureus from human keratinocyte endosomes and induces apoptosis.

Skin and soft-tissue infections (SSTIs) caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have emerged as major health problems throughout the world. Most SSTI CA-MRSA strains produce Panton-Valentine leukocidin (PVL), but its contribution to CA-MRSA pathogenesis is poorly defined. Here, we used an endemic PVL-positive SSTI-causing CA-MRSA strain from Taiwan, together with an isogenic PVL-knockout mutant (Δpvl) and complemented PVL-positive derivative, to evaluate the role of PVL in the pathogenesis of CA-MRSA in the RHEK-1 human keratinocyte cell line and a rabbit skin infection model. We found that both PVL-positive CA-MRSA and isogenic Δpvl strains attached and were engulfed into endosomes of RHEK-1 cells within 1 hour following infection. However, by 2 hours after infection PVL-positive CA-MRSA more effectively disrupted endosomes, escaped into the cytoplasm, and replicated intracellularly. By 6 hours after infection, the PVL-positive strain caused significantly more caspase-dependent keratinocyte apoptosis than the isogenic Δpvl mutant. In the rabbit infection model, 1 week following infection the wild-type strain produced significantly more widespread lesions and cell apoptosis than the isogenic Δpvl mutant. These findings indicate that PVL is an important virulence factor that enables CA-MRSA to produce necrotizing skin infections by allowing the bacteria to escape from endosomes, replicate intracellularly, and induce apoptosis.

Conditioning vaccination site with irradiated MIP-3alpha-transfected tumor cells enhances efficacy of dendritic cell-based cancer vaccine.

Macrophage inflammation protein-3alpha (MIP-3alpha) is a chemokine expressed in inflamed tissue and capable of inducing migration of immature dendritic cells (DCs) or Langerhans cells. We postulated that conditioning vaccination sites with MIP-3alpha might enhance the efficacy of subsequently administered DC-based cancer vaccines. Our results demonstrate that subcutaneously injection of irradiated tumor cells expressing MIP-3alpha induces substantial cell infiltration to the injection site. Vaccination of irradiated tumor cells expressing MIP-3alpha followed by DCs pulsed with irradiated tumor cells can effectively suppress tumor growth in animals, which is significantly better than vaccination with irradiated MIP-3alpha-producing tumor cells or DCs pulsed with tumor cells alone. The protective effect was most evident when the MIP-3alpha-producing tumor cells and DC-based vaccines were injected at the same site. These results support the notion that this combination vaccination strategy might generate a more effective immune response to suppress the growth of tumor cells in animals.

Retrograde axonal transport: a major transmission route of enterovirus 71 in mice.

Inoculation of enterovirus 71 (EV71) by the oral (p.o.), intramuscular (i.m.), or intracranial route resulted in brain infection, flaccid paralysis, pulmonary dysfunction, and death of 7-day-old mice. The lag time of disease progression indicated that neuroinvasion from the inoculation sites was a prerequisite for the development of the clinical signs. Although EV71 p.o. inoculation led to a persistent viremia and a transient increase in blood-brain barrier permeability at the early stage of the infection, only low levels of virus, which led to neither severe infection nor clinical illness, could be detected in the brain, suggesting that hematogenous transport might not represent a major transmission route. In the spinal cord, following both p.o. and hind limb i.m. inoculation, the virus first appeared and increased rapidly in the lower segments, especially at the anterior horn areas, and then spread to the upper segments and brain in the presence of viremia. A reverse pattern, with the virus being first detected in the upper segment, was observed when the virus was i.m. inoculated in the forelimb. Colchicine, a fast axonal transport inhibitor, but not sciatic nerve transection reduced EV71 neuroinvasion in a dose-dependent manner, indicating a neuronal transmission of the virus.