Connexins Biology in the Pathophysiology of Retinal Diseases.

Connexins (Cx) are a family of transmembrane proteins that form gap junction intercellular channels that connect neighboring cells. These channels allow the passage of ions and other biomolecules smaller than 1 kDa, thereby synchronizing the cells both electrically and metabolically. Cxs are expressed in all retinal cell types and the diversity of Cx isoforms involved in the assembly of the channels provides a functional syncytium required for visual transduction. In this chapter, we summarize the status of current knowledge regarding Cx biology in retinal tissues and discuss how Cx dysfunction is associated with retinal disease pathophysiology. Although the contribution of Cx deficiency to retinal degeneration is not well understood, recent findings present Cx as a potential therapeutic target. Therefore, we will briefly discuss pharmacological approaches and gene therapies that are being explored to modulate Cx function and fight sight-threatening eye diseases.

Autophagy and Glycative Stress: A Bittersweet Relationship in Neurodegeneration.

Autophagy is a fine-tuned proteolytic pathway that moves dysfunctional/aged cellular components into the lysosomal compartment for degradation. Over the last 3 decades, global research has provided evidence for the protective role of autophagy in different brain cell components. Autophagic capacities decline with age, which contributes to the accumulation of obsolete/damaged organelles and proteins and, ultimately, leads to cellular aging in brain tissues. It is thus well-accepted that autophagy plays an essential role in brain homeostasis, and malfunction of this catabolic system is associated with major neurodegenerative disorders. Autophagy function can be modulated by different types of stress, including glycative stress. Glycative stress is defined as a cellular status with abnormal and accelerated accumulation of advanced glycation end products (AGEs). It occurs in hyperglycemic states, both through the consumption of high-sugar diets or under metabolic conditions such as diabetes. In recent years, glycative stress has gained attention for its adverse impact on brain pathology. This is because glycative stress stimulates insoluble, proteinaceous aggregation that is linked to the malfunction of different neuropathological proteins. Despite the emergence of new literature suggesting that autophagy plays a major role in fighting glycation-derived damage by removing cytosolic AGEs, excessive glycative stress might also negatively impact autophagic function. In this mini-review, we provide insight on the status of present knowledge regarding the role of autophagy in brain physiology and pathophysiology, with an emphasis on the cytoprotective role of autophagic function to ameliorate the adverse effects of glycation-derived damage in neurons, glia, and neuron-glia interactions.

Klotho Is Neuroprotective in the Superoxide Dismutase (SOD1G93A) Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of motor neurons in the brain and spinal cord. ALS neuropathology is associated with increased oxidative stress, excitotoxicity, and inflammation. We and others reported that the anti-aging and cognition-enhancing protein Klotho is a neuroprotective, antioxidative, anti-inflammatory, and promyelinating protein. In mice, its absence leads to an extremely shortened life span and to multiple phenotypes resembling human aging, including motor and hippocampal neurodegeneration and cognitive impairment. In contrast, its overexpression extends life span, enhances cognition, and confers resistance against oxidative stress; it also reduces premature mortality and cognitive and behavioral abnormalities in an animal model for Alzheimer's disease (AD). These pleiotropic beneficial properties of Klotho suggest that Klotho could be a potent therapeutic target for preventing neurodegeneration in ALS. Klotho overexpression in the SOD1 mouse model of ALS resulted in delayed onset and progression of the disease and extended survival that was more prominent in females than in males. Klotho reduced the expression of neuroinflammatory markers and prevented neuronal loss with the more profound effect in the spinal cord than in the motor cortex. The effect of Klotho was accompanied by reduced expression of proinflammatory cytokines and enhanced the expression of antioxidative and promyelinating factors in the motor cortex and spinal cord of Klotho × SOD1 compared to SOD1 mice. Our study provides evidence that increased levels of Klotho alleviate ALS-associated pathology in the SOD1 mouse model and may serve as a basis for developing Klotho-based therapeutic strategies for ALS.

Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging.

Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule-based minus-end-directed motor proteins, the well-characterized dynein, and the less-studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti-aging purposes.

Activation of the Anti-Aging and Cognition-Enhancing Gene Klotho by CRISPR-dCas9 Transcriptional Effector Complex.

Multiple lines of evidence show that the anti-aging and cognition-enhancing protein Klotho fosters neuronal survival, increases the anti-oxidative stress defense, and promotes remyelination of demyelinated axons. Thus, upregulation of the Klotho gene can potentially alleviate the symptoms and/or prevent the progression of age-associated neurodegenerative diseases such as Alzheimer's disease and demyelinating diseases such as multiple sclerosis. Here we used a CRISPR-dCas9 complex to investigate single-guide RNA (sgRNA) targeting the Klotho promoter region for efficient transcriptional activation of the Klotho gene. We tested the sgRNAs within the - 1 to - 300 bp of the Klotho promoter region and identified two sgRNAs that can effectively enhance Klotho gene transcription. We examined the transcriptional activation of the Klotho gene using three different systems: a Firefly luciferase (FLuc) and NanoLuc luciferase (NLuc) coincidence reporter system, a NLuc knock-in in Klotho 3'-UTR using CRISPR genomic editing, and two human cell lines: neuronal SY5Y cells and kidney HK-2 cells that express Klotho endogenously. The two sgRNAs enhanced Klotho expression at both the gene and protein levels. Our results show the feasibility of gene therapy for targeting Klotho using CRISPR technology. Enhancing Klotho levels has a therapeutic potential for increasing cognition and treating age-associated neurodegenerative, demyelinating and other diseases, such as chronic kidney disease and cancer.

SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing.

Mutations in the U2 snRNP component SF3B1 are prominent in myelodysplastic syndromes (MDSs) and other cancers and have been shown recently to alter branch site (BS) or 3' splice site selection in splicing. However, the molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles in Saccharomyces cerevisiae, counterparts of SF3B1 mutations frequently found in cancers, specifically change splicing of suboptimal BS pre-mRNA substrates. We found that Hsh155p interacts directly with Prp5p, the first ATPase that acts during spliceosome assembly, and localized the interacting regions to HEAT (Huntingtin, EF3, PP2A, and TOR1) motifs in SF3B1 associated with disease mutations. Furthermore, we show that mutations in these motifs from both human disease and yeast genetic screens alter the physical interaction with Prp5p, alter branch region specification, and phenocopy mutations in Prp5p. These and other data demonstrate that mutations in Hsh155p and Prp5p alter splicing because they change the direct physical interaction between Hsh155p and Prp5p. This altered physical interaction results in altered loading (i.e., "fidelity") of the BS-U2 duplex into the SF3B complex during prespliceosome formation. These results provide a mechanistic framework to explain the consequences of intron recognition and splicing of SF3B1 mutations found in disease.

Connexins modulate autophagosome biogenesis.

The plasma membrane contributes to the formation of autophagosomes, the double-membrane vesicles that sequester cytosolic cargo and deliver it to lysosomes for degradation during autophagy. In this study, we have identified a regulatory role for connexins (Cx), the main components of plasma membrane gap junctions, in autophagosome formation. We have found that plasma-membrane-localized Cx proteins constitutively downregulate autophagy through a direct interaction with several autophagy-related proteins involved in the initial steps of autophagosome formation, such as Atg16 and components of the PI(3)K autophagy initiation complex (Vps34, Beclin-1 and Vps15). On nutrient starvation, this inhibitory effect is released by the arrival of Atg14 to the Cx-Atg complex. This promotes the internalization of Cx-Atg along with Atg9, which is also recruited to the plasma membrane in response to starvation. Maturation of the Cx-containing pre-autophagosomes into autophagosomes leads to degradation of these endogenous inhibitors, allowing for sustained activation of autophagy.

Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner.

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.