Wennan Lu

Elevated Pressure Triggers a Physiological Release of ATP from the Retina: Possible Role for Pannexin Hemichannels

Increased hydrostatic pressure can damage neurons, although the mechanisms linking pressure to neurochemical imbalance or cell injury are not fully established. Throughout the body, mechanical perturbations such as shear stress, cell stretching, or changes in pressure can lead to excessive release of ATP. It is thus possible that increased pressure across neural tissues triggers an elevated release of ATP into extracellular space. As stimulation of the P2X(7) receptor for ATP on retinal ganglion cells leads to elevation of intracellular calcium and excitotoxic death, we asked whether increased levels of extracellular ATP accompanied an elevation in pressure across the retina. The hydrostatic pressure surrounding bovine retinal eyecups was increased and the ATP content of the vitreal compartment adjacent to the retina was determined. A step increase of only 20 mm Hg induced a threefold increase in the vitreal ATP concentration. The ATP levels correlated closely with the degree of pressure increase over 20-100 mm Hg. The increase was transient at lower pressures but sustained at higher pressures. The rise in vitreal ATP was the same regardless of whether nitrogen or air was used to increase pressure, implying changes in oxygen partial pressure did not contribute. Lactate dehydrogenase activity was not affected by pressure, ruling out a substantial contribution from cell lysis. The ATP increase was largely inhibited by either 30 muM 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or 10 muM carbenoxolone (CBX). While this pharmacological profile is consistent with physiological release of ATP through pannexins hemichannels, a contribution from anion channels, vesicular release or other mechanisms cannot be ruled out. In conclusion, a step elevation in pressure leads to a physiologic increase in the levels of extracellular ATP bathing retinal neurons. This excess extracellular ATP may link increased pressure to the death of ganglion cells in acute glaucoma, and suggests a possible role for ATP in the neuronal damage accompanying increased intracranial pressure.

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.

Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4-/-Mice : Pharmacologic Approaches and Functional Recovery

PURPOSE Degradation of engulfed material is primarily mediated by lysosomal enzymes that function optimally within a narrow range of acidic pH values. RPE cells are responsible for daily degradation of photoreceptor outer segments and are thus particularly susceptible to perturbations in lysosomal pH. The authors hypothesized that elevated lysosomal pH levels could slow enzyme activity and encourage accumulation of partially digested material. Consequently, treatment to lower perturbed lysosomal pH levels may enhance degradative activity. METHODS A high-throughput screening assay was developed to quantify the lysosomal pH of fresh mouse and cultured ARPE-19 cells. The effect of lysosomal pH on outer segment clearance was determined. RESULTS Lysosomal pH is elevated in RPE cells from ABCA4 knockout mice and in cultured human ARPE-19 cells exposed to N-retinylidene-N-retinylethanolamine (A2E), tamoxifen, or chloroquine. The lysosomal pH of fresh RPE cells from ABCA4(-/-) mice and of chemically compromised RPE cells was reacidified by elevating intracellular cAMP directly. Compromised lysosomal pH was also restored by stimulating A(2A) adenosine or beta-adrenergic receptors, consistent with G(s)-protein coupling of these receptors. Restoring lysosomal pH with these treatments enhanced photoreceptor outer segment clearance, demonstrating functional relevance consistent with an enhancement of degradative enzyme activity. CONCLUSIONS Elevation of lysosomal pH in RPE cells interferes with the degradation of outer segments and may contribute to the pathologies associated with A2E. Pharmacologic elevation of cAMP can restore an acid pH and improve degradative function.

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.

Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP

The photoreceptors lie between the inner retina and the retinal pigment epithelium (RPE). The release of glutamate by the phototoreceptors can signal changes in light levels to inner retinal neurons, but the role of glutamate in communicating with the RPE is unknown. Since RPE cells are known to release ATP, we asked whether glutamate could trigger ATP release from RPE cells and whether this altered cell signalling. Stimulation of the apical face of fresh bovine RPE eyecups with 100 μm NMDA increased ATP levels more than threefold, indicating that both receptors for NMDA and release of ATP occurred across the apical membrane of fresh RPE cells. NMDA increased ATP levels bathing cultured human ARPE‐19 cells more than twofold, with NMDA receptor inhibitors MK‐801 and d‐AP5 preventing this release. Blocking the glycine site of the NMDA receptor with 5,7‐dichlorokynurenic acid prevented ATP release from ARPE‐19 cells. Release was also blocked by channel blocker NPPB and Ca2+ chelator BAPTA, but not by cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide or vesicular release inhibitor brefeldin A. Glutamate produced a dose‐dependent release of ATP from ARPE‐19 cells that was substantially inhibited by MK‐801. NMDA triggered a rise in cell Ca2+ that was blocked by MK‐801, by the ATPase apyrase, by the P2Y1 receptor antagonist MRS2179 and by depletion of intracellular Ca2+ stores with thapsigargin. These results suggest that glutamate stimulates NMDA receptors on the apical membrane of RPE cells to release ATP. This secondary release can amplify the glutaminergic signal by increasing Ca2+ inside RPE cells, and might activate Ca2+‐dependent conductances. The interplay between glutaminergic and purinergic systems may thus be important for light‐dependent interactions between photoreceptors and the RPE.

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.

The P2X7 receptor in retinal ganglion cells: A neuronal model of pressure-induced damage and protection by a shifting purinergic balance

Retinal ganglion cells process the visual signal and transmit it along their axons in the optic nerve to the brain. Molecular, immunohistochemical, and functional analyses indicate that the majority of retinal ganglion cells express the ionotropic P2X7 receptor. Stimulation of the receptor can lead to a rise in intracellular calcium and cell death, although death does not involve the opening of a large diameter pore. Adenosine acting at A3 receptors can attenuate the rise in calcium and death accompanying P2X7 receptor activation, suggesting that dephosphorylation of ATP into adenosine is neuroprotective and that the balance of extracellular purines can influence neuronal survival. Increased intraocular pressure can lead to release of excessive extracellular ATP in the retina and damage ganglion cells by acting on P2X7 receptors, implicating a role for the receptor in the loss of ganglion cell activity in glaucoma. In summary, the activation of P2X7 receptors has both physiologic and pathophysiologic implications for ganglion cell function. These characteristics may also provide an insight into the contributions the P2X7 receptor makes to neurons elsewhere.

Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardts disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.

Stimulation of the P2Y1 Receptor Up-Regulates Nucleoside-Triphosphate Diphosphohydrolase-1 in Human Retinal Pigment Epithelial Cells

Stimulation of receptors for either ATP or adenosine leads to physiologic changes in retinal pigment epithelial (RPE) cells that may influence their relationship with the adjacent photoreceptors. The ectoenzyme nucleoside-triphosphate diphosphohydrolase-1 (NTPDase1) catalyzes the dual dephosphorylation of ATP and ADP to AMP. Although NTPDase1 can consequently control the balance between ATP and adenosine, it is unclear how its expression and activity are regulated. Classic negative feedback theory predicts an increase in enzyme activity in response to enhanced exposure to substrate. This study asked whether exposure to ATP increases NTPDase1 activity in RPE cells. Although levels of NTPDase1 mRNA and protein in cultured human ARPE-19 cells were generally low under control conditions, exposure to slowly hydrolyzable ATPγS led to a time-dependent increase in NTPDase1 mRNA that was accompanied by a rise in levels of the functional 78-kDa protein. Neither NTPDase2 nor NTPDase3 mRNA message was elevated by ATPγS. The ATPase activity of cells increased in parallel, indicating the up-regulation of NTPDase1 was functionally relevant. The up-regulation of NTPDase1 protein was partially blocked by P2Y1 receptor inhibitors MRS2179 (N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate) and MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate] and increased by P2Y1 receptor agonist MRS2365 [(N)-methanocarba-2MeSADP]. In conclusion, prolonged exposure to extracellular ATPγS increased NTPDase1 message and protein levels and increased ecto-ATPase activity. This up-regulation reflects a feedback circuit, mediated at least in part by the P2Y1 receptor, to regulate levels of extracellular purines in subretinal space. NTPDase1 levels may thus serve as an index for increased extracellular ATP levels under certain pathologic conditions, although other mechanisms could also contribute.