Jason C. Lim

Neurons respond directly to mechanical deformation with pannexin-mediated ATP release and autostimulation of P2X7 receptors.

•  Neurons can be damaged when tissues are stretched or swollen; while astrocytes can contribute to this process, the mechanosensitive response from neurons is unclear. •  We show here that isolated retinal ganglion cell neurons respond to mechanical strain with a rapid, sustained release of the neurotransmitter ATP. •  The conduit for ATP release was through pannexin hemichannels, with probenicid, carbenoxelone and 10panx inhibiting release. •  Once released, this ATP acts back on the neurons to autostimulate lethal P2X7 receptors, as A438079, AZ 10606120 and zinc reduced currents in whole cell patch clamp recordings. •  Blocking release of ATP through pannexin channels, or activation of P2X7 receptors, might be neuroprotective for stretched or swollen neurons. •  Stretch‐dependent release of ATP through neuronal pannexins, combined with the autostimulation of the P2X7 receptors, provides a new pathway by which neuronal activity and health can be altered by mechanical strain independently of glial activity.

Mechanosensitive release of adenosine 5′-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: A mechanism for purinergic involvement in chronic strain

As adenosine 5′‐triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1–3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1−/− mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling‐induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg‐MYOCY437H mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma. GLIA 2014;62:1486–1501

Acidic Nanoparticles Are Trafficked to Lysosomes and Restore an Acidic Lysosomal pH and Degradative Function to Compromised ARPE-19 Cells

Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide) (PLGA) 502 H, PLGA 503 H and poly (DL-lactide) (PLA) colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity.

Approaches for detecting lysosomal alkalinization and impaired degradation in fresh and cultured RPE cells: evidence for a role in retinal degenerations.

Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TcFEB and of the vesicular proton pump vHATPase in the RPE/choroid of mice. An elevated lysosomal pH with upregulation of TcFEB and vHATPase resembles the pathology in fibroblasts of patients with mutant presenilin 1 (PS1), suggesting a common link between age-related macular degeneration (AMD) and Alzheimers disease. While the absolute rise in pH is often small in these disorders, elevations of only a few tenths of a pH unit can have a major impact on both lysosomal function and the accumulation of waste over decades. Accurate measurement of lysosomal pH can be complex, and imprecise measurements have clouded the field. Protocols to optimize pH measurement from fresh and cultured cells are discussed, and indirect measurements to confirm changes in lysosomal pH and degradative capacity are addressed. The ability of reacidifying treatments to restore degradative function confirms the central role of lysosomal pH in these disorders and identifies potential approaches to treat diseases of lysosomal accumulation like AMD and Alzheimers disease. In summary, various approaches to determine lysosomal pH in fresh and cultured cells, as well as the potential to restore pH levels to an optimal range, can help identify and repair pathologies associated with lysosomal defects in RPE cells and perhaps also suggest new approaches to treat lysosomal storage diseases throughout the body.

Stimulation of the D5 dopamine receptor acidifies the lysosomal pH of retinal pigmented epithelial cells and decreases accumulation of autofluorescent photoreceptor debris.

J. Neurochem. (2012) 122, 823–833.

Foxo1 Inhibits Diabetic Mucosal Wound Healing but Enhances Healing of Normoglycemic Wounds

Re-epithelialization is an important part in mucosal wound healing. Surprisingly little is known about the impact of diabetes on the molecular events of mucosal healing. We examined the role of the transcription factor forkhead box O1 (Foxo1) in oral wounds of diabetic and normoglycemic mice with keratinocyte-specific Foxo1 deletion. Diabetic mucosal wounds had significantly delayed healing with reduced cell migration and proliferation. Foxo1 deletion rescued the negative impact of diabetes on healing but had the opposite effect in normoglycemic mice. Diabetes in vivo and in high glucose conditions in vitro enhanced expression of chemokine (C-C motif) ligand 20 (CCL20) and interleukin-36γ (IL-36γ) in a Foxo1-dependent manner. High glucose–stimulated Foxo1 binding to CCL20 and IL-36γ promoters and CCL20 and IL-36γ significantly inhibited migration of these cells in high glucose conditions. In normal healing, Foxo1 was needed for transforming growth factor-β1 (TGF-β1) expression, and in standard glucose conditions, TGF-β1 rescued the negative effect of Foxo1 silencing on migration in vitro. We propose that Foxo1 under diabetic or high glucose conditions impairs healing by promoting high levels of CCL20 and IL-36γ expression but under normal conditions, enhances it by inducing TGF-β1. This finding provides mechanistic insight into how Foxo1 mediates the impact of diabetes on mucosal wound healing.

Osteoblast Lineage Cells Play an Essential Role in Periodontal Bone Loss Through Activation of Nuclear Factor-Kappa B.

Bacterial pathogens stimulate periodontitis, the most common osteolytic disease in humans and the most common cause of tooth loss in adults. Previous studies identified leukocytes and their products as key factors in this process. We demonstrate for the first time that osteoblast lineage cells play a critical role in periodontal disease. Oral infection stimulated nuclear localization of NF-κB in osteoblasts and osteocytes in the periodontium of wild type but not transgenic mice that expressed a lineage specific dominant negative mutant of IKK (IKK-DN) in osteoblast lineage cells. Wild-type mice were also susceptible to bacteria induced periodontal bone loss but transgenic mice were not. The lack of bone loss in the experimental group was linked to reduced RANKL expression by osteoblast lineage cells that led to diminished osteoclast mediated bone resorption and greater coupled new bone formation. The results demonstrate that osteoblast lineage cells are key contributors to periodontal bone loss through an NF-κB mediated mechanism.

TNFα contributes to diabetes impaired angiogenesis in fracture healing

Diabetes increases the likelihood of fracture, interferes with fracture healing and impairs angiogenesis. The latter may be significant due to the critical nature of angiogenesis in fracture healing. Although it is known that diabetes interferes with angiogenesis the mechanisms remain poorly defined. We examined fracture healing in normoglycemic and streptozotocin-induced diabetic mice and quantified the degree of angiogenesis with antibodies to three different vascular markers, CD34, CD31 and Factor VIII. The role of diabetes-enhanced inflammation was investigated by treatment of the TNFα-specific inhibitor, pegsunercept starting 10days after induction of fractures. Diabetes decreased both angiogenesis and VEGFA expression by chondrocytes. The reduced angiogenesis and VEGFA expression in diabetic fractures was rescued by specific inhibition of TNF in vivo. In addition, the TNF inhibitor rescued the negative effect of diabetes on endothelial cell proliferation and endothelial cell apoptosis. The effect of TNFα in vitro was enhanced by high glucose and an advanced glycation endproduct to impair microvascular endothelial cell proliferation and tube formation and to stimulate apoptosis. The effect of TNF, high glucose and an AGE was mediated by the transcription factor FOXO1, which increased expression of p21 and caspase-3. These studies indicate that inflammation plays a major role in diabetes-impaired angiogenesis in endochondral bone formation through its effect on microvascular endothelial cells and FOXO1.

Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis.

Background: Components of GABA catabolism feed into sleep and potential energy pathways. Results: We identified a metabolic phenotype in Drosophila mutants of GABA turnover and traced it to a limit in glutamate, which is not relevant for sleep. Conclusion: GABA regulates metabolic and sleep homeostasis through independent mechanisms. Significance: Neurological disorders involving GABA disruption may be associated with metabolic problems. Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD+/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.

The P2X7 Receptor Primes IL-1β and the NLRP3 Inflammasome in Astrocytes Exposed to Mechanical Strain

Inflammatory responses play a key role in many neural pathologies, with localized signaling from the non-immune cells making critical contributions. The NLRP3 inflammasome is an important component of innate immune signaling and can link neural insult to chronic inflammation. The NLRP3 inflammasome requires two stages to contribute: priming and activation. The priming stage involves upregulation of inflammasome components while the activation stage results in the assembly and activation of the inflammasome complex. The priming step can be rate limiting and can connect insult to chronic inflammation, but our knowledge of the signals that regulate NLRP3 inflammasome priming in sterile inflammation is limited. This study examined the link between mechanical strain and inflammasome priming in neural systems. Transient non-ischemic elevation of intraocular pressure increased mRNA for inflammasome components IL-1β, NLRP3, ASC, and CASP1 in rat and mouse retinas. The elevation was greater 1 day after the insult, with the rise in IL-1β most pronounced. The P2X7 receptor was implicated in the mechanosensitive priming of IL-1β mRNA in vivo, as the antagonist Brilliant Blue G (BBG) blocked the increased expression, the agonist BzATP mimicked the pressure-dependent rise in IL-1β, and the rise was absent in P2X7 knockout mice. In vitro measurements from optic nerve head astrocytes demonstrated an increased expression of IL-1β following stretch or swelling. This increase in IL-1β was eliminated by degradation of extracellular ATP with apyrase, or by the block of pannexin hemichannels with carbenoxolone, probenecid, or 10panx1 peptide. The rise in IL-1β expression was also blocked by P2X7 receptor antagonists BBG, A839977 or A740003. The rise in IL-1β was prevented by blocking transcription factor NFκB with Bay 11-7082, while the swelling-dependent fall in NFκB inhibitor IκB-α was reduced by A839977 and in P2X7 knockout mice. In summary, mechanical trauma to the retina primed NLRP3 inflammasome components, but only if there was ATP release through pannexin hemichannels, and autostimulation of the P2X7 receptor. As the P2X7 receptor can also trigger stage two of inflammasome assembly and activation, the P2X7 receptor may have a central role in linking mechanical strain to neuroinflammation.