Solving neurodegeneration: common mechanisms and strategies for new treatments.

Across neurodegenerative diseases, common mechanisms may reveal novel therapeutic targets based on neuronal protection, repair, or regeneration, independent of etiology or site of disease pathology. To address these mechanisms and discuss emerging treatments, in April, 2021, Glaucoma Research Foundation, BrightFocus Foundation, and the Melza M. and Frank Theodore Barr Foundation collaborated to bring together key opinion leaders and experts in the field of neurodegenerative disease for a virtual meeting titled "Solving Neurodegeneration". This "think-tank" style meeting focused on uncovering common mechanistic roots of neurodegenerative disease and promising targets for new treatments, catalyzed by the goal of finding new treatments for glaucoma, the world's leading cause of irreversible blindness and the common interest of the three hosting foundations. Glaucoma, which causes vision loss through degeneration of the optic nerve, likely shares early cellular and molecular events with other neurodegenerative diseases of the central nervous system. Here we discuss major areas of mechanistic overlap between neurodegenerative diseases of the central nervous system: neuroinflammation, bioenergetics and metabolism, genetic contributions, and neurovascular interactions. We summarize important discussion points with emphasis on the research areas that are most innovative and promising in the treatment of neurodegeneration yet require further development. The research that is highlighted provides unique opportunities for collaboration that will lead to efforts in preventing neurodegeneration and ultimately vision loss.

Role of the PNPLA2 Gene in the Regulation of Oxidative Stress Damage of RPE.

Oxidative stress-mediated injury of the retinal pigment epithelium (RPE) can precede progressive retinal degeneration and ultimately lead to blindness (e.g., age-related macular degeneration (AMD)). The RPE expresses the PNPLA2 gene and produces its protein product PEDF-R that exhibits lipase activity. We have shown that transient PNPLA2 overexpression decreases dead-cell proteolytic activity and that synthetic peptides derived from a central region of PEDF-R efficiently protect ARPE-19 and pig primary RPE cells from oxidative stress. This study aims to evaluate the effect of loss of PNPLA2 in RPE cells undergoing oxidative stress. Loss of PNPLA2 conferred increased resistance to cells when subjected to oxidative stress.

PEDF peptides promote photoreceptor survival in rd10 retina models.

The purpose of the study is to evaluate the protective properties of PEDF peptide fragments on rd10 mouse models of retinal degeneration ex vivo. Human recombinant PEDF and synthetic peptides were used. Rd10 retinal explants as well as wild-type retinal explants treated with zaprinast to mimic the rd10 photoreceptor cell death were employed. PEDF protein was intravitreally administered into rd10 mice. Outer nuclear layer thickness measurements in retinal sections, TUNEL labeling in retinal explants, western blots and immunofluorescence with retinal samples were performed. PEDF protein levels in the RPE of rd10 mice decreased with age (P15 - P25). Levels of PEDF receptor PEDF-R declined in the photoreceptor inner segments from rd10 relative to wild-type mice at P25. PEDF administration increased the outer nuclear layer thickness of rd10 retinas in vivo and decreased the number of TUNEL+ nuclei of photoreceptors in rd10 retinal explant cultures, both relative to untreated controls. Peptides containing the PEDF neurotrophic region decreased the number of TUNEL+ photoreceptors in both rd10 and zaprinast-induced cell death ex vivo models, while peptides without the neurotrophic region and/or lacking affinity for PEDF-R were ineffective in protecting photoreceptors. Thus, retinal explants are a valuable system to evaluate PEDF activity. Short peptides with the photoreceptor-protective property of PEDF may prove useful for the development of therapeutic agents for photoreceptor protection in retinal degenerations.

Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina.

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.

Comparison of two neurotrophic serpins reveals a small fragment with cell survival activity.

PURPOSE: Protease nexin-1 (PN-1), a serpin encoded by the SERPINE2 gene, has serine protease inhibitory activity and neurotrophic properties in the brain. PN-1 inhibits retinal angiogenesis; however, PN-1's neurotrophic capacities in the retina have not yet been evaluated. Pigment epithelium-derived factor (PEDF) is a serpin that exhibits neurotrophic and antiangiogenic activities but lacks protease inhibitory properties. The aim of this study is to compare PN-1 and PEDF. METHODS: Sequence comparisons were performed using computer bioinformatics programs. Mouse and bovine eyes, human retina tissue, and ARPE-19 cells were used to prepare RNA and protein samples. Interphotoreceptor matrix lavage was obtained from bovine eyes. Gene expression and protein levels were evaluated with reverse-transcription PCR (RT-PCR) and western blotting, respectively. Recombinant human PN-1, a version of PN-1 referred to as PN-1[R346A] lacking serine protease inhibitory activity, and PEDF proteins were used, as well as synthetic peptides designed from PEDF and PN-1 sequences. Survival activity in serum-starved, rat-derived retinal precursor (R28) cells was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) cell death assays. Bcl2 levels were measured with RT-PCR. RESULTS: PN-1 is analogous in primary and tertiary structure to PEDF. A region in PN-1 shares homology with the neurotrophic active region of PEDF, a 17-residue region within alpha helix C. The native human retina, ARPE-19 cells, and murine RPE and retina expressed the gene for PN-1 (SERPINE2 and Serpine2 mRNA). The retina, ARPE-19 cell lysates, and bovine interphotoreceptor matrix contained PN-1 protein. The addition of PN-1, PN-1[R346A], or the 17mer peptide of PN-1 to serum-starved retina cells decreased the number of TUNEL-positive nuclei relative to the untreated cells, such as PEDF. PN-1, PN-1[R346A], and PN-1-17mer treatments increased the Bcl2 transcript levels in serum-starved cells, as seen with PEDF. CONCLUSIONS: PN-1 and PEDF share structural and functional features, and expression patterns in the retina. These serpins' mechanisms of action as cell survival factors are independent of serine protease inhibition. We have identified PN-1 as a novel factor for the retina that may play a neuroprotective role in vivo, and small peptides as relevant candidates for preventing retinal degeneration.

Ventilator Associated Pneumonia in an Infant Caused by Stenotrophomonas maltophila - A Case Report.

Stenotrophomonas maltophila (S.maltophila) is an aerobic Gram-negative bacillus that is a frequent colonizer of fluids used in the hospital setting. The organism is known to cause life threatening infections in immuno-compromised patients especially in those who are neutropenic, on chemotherapy or on broad spectrum antibiotics. We report a case of ventilator associated pneumonia caused by Stenotrophomonas maltophila in a two-month old infant who later developed multi organ dysfunction syndrome. In seriously ill paediatric patients, S.maltophila should also be considered as a possible pathogen for Ventilator-Associated Pneumonia (VAP), hence empiric antibiotic choice should include antimicrobials that are active against S. maltophila. An early identification and treatment of VAP with Multi-Drug Resistant (MDR) strains with appropriate antibiotics has a significant impact on morbidity and mortality.

A Novel Inhibitor of 5-Lipoxygenase (5-LOX) Prevents Oxidative Stress-Induced Cell Death of Retinal Pigment Epithelium (RPE) Cells.

PURPOSE: 5-Lipoxygenase (5-LOX) oxygenates arachidonic acid to form 5-hydroperoxyeicosatetraenoic acid, which is further converted into biologically detrimental leukotrienes, such as leukotriene B4 (LTB4). The RPE and retina express the PNPLA2 gene for pigment epithelium-derived factor receptor (PEDF-R), a lipase involved in cell survival. The purpose here was to investigate the role of PEDF-R on the 5-LOX pathway in oxidative stress of RPE. METHODS: Lipoxygenase activity assays were performed with soybean and potato lipoxygenase. Binding was evaluated by peptide-affinity chromatography and pull-down assays with PEDF-R-derived synthetic peptides or recombinant protein. Oxidative stress was induced in human ARPE-19 and primary pig RPE cells with indicated concentrations of H2O2/TNF-α. Reverse transcription-PCR of ALOX5 and PNPLA2 genes was performed. Cell viability and death rates were determined using respective biomarkers. Leukotriene B4 levels were measured by ELISA. RESULTS: Among five peptides spanning between positions Leu159 and Met325 of human PEDF-R polypeptide, only two overlapping peptides, E5b and P1, bound and inhibited lipoxygenase activity. Human recombinant 5-LOX bound specifically to peptide P1 and to His6/Xpress-tagged PEDF-R via ionic interactions. The two inhibitor peptides E5b and P1 promoted cell viability and decreased cell death of RPE cells undergoing oxidative stress. Oxidative stress decreased the levels of PNPLA2 transcripts with no effect on ALOX5 expression. Exogenous additions of P1 peptide or overexpression of the PNPLA2 gene decreased both LTB4 levels and death of RPE cells undergoing oxidative stress. CONCLUSIONS: A novel peptide region of PEDF-R inhibits 5-LOX, which intersects with RPE cell death pathways induced by oxidative stress.

Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor.

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78-121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98-114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98-114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.

Pigment epithelium-derived factor protects cone photoreceptor-derived 661W cells from light damage through Akt activation.

Pigment epithelium-derived factor (PEDF) can delay and prevent the death of photoreceptors in vivo. We investigated the survival activity of PEDF on cone photoreceptor-derived 661W cells in culture, the presence of PEDF receptor (PEDF-R) in these cells and the activation of prosurvival Akt. Cell death was induced by light exposure in the presence of 9-cis retinal. Cell viability assays showed that PEDF increased the number of 661W cells exposed to these conditions. Western blots showed that PEDF-treated 661W cells had a higher ratio of phosphorylated Akt to total Akt than untreated cells. The PEDF receptor PEDF-R was immunodetected in the plasma membrane fractions of 661W cells. The results demonstrated that PEDF can protect 661W cells against light-induced cell death and suggest that the binding of PEDF to cell surface PEDF-R triggers a prosurvival signaling pathway.

Pigment epithelium-derived factor reduces apoptosis and pro-inflammatory cytokine gene expression in a murine model of focal retinal degeneration.

AMD (age-related macular degeneration) is a neurodegenerative disease causing irreversible central blindness in the elderly. Apoptosis and inflammation play important roles in AMD pathogenesis. PEDF (pigment epithelium-derived factor) is a potent neurotrophic and anti-inflammatory glycoprotein that protects the retinal neurons and photoreceptors against cell death caused by pathological insults. We studied the effects of PEDF on focal retinal lesions in DKO rd8 (Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)]) mice, a model for progressive, focal rd (retinal degeneration). First, we found a significant decrease in PEDF transcript expression in DKO rd8 mouse retina and RPE (retinal pigment epithelium) than WT (wild-type, C57BL/6N). Next, cultured DKO rd8 RPE cells secreted lower levels of PEDF protein in the media than WT. Then the right eyes of DKO rd8 mice were injected intravitreously with recombinant human PEDF protein (1 µg), followed by a subconjunctival injection of PEDF (3 µg) 4 weeks later. The untreated left eyes served as controls. The effect of PEDF was assessed by fundoscopy, ocular histopathology and A2E {[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium} levels, as well as apoptotic and inflammatory molecules. The PEDF-treated eyes showed slower progression or attenuation of the focal retinal lesions, fewer and/or smaller photoreceptor and RPE degeneration, and significantly lower A2E, relative to the untreated eyes. In addition, lower expression of apoptotic and inflammatory molecules were detected in the PEDF-treated than untreated eyes. Our results establish that PEDF potently stabilizes photoreceptor degeneration via suppression of both apoptotic and inflammatory pathways. The multiple beneficial effects of PEDF represent a novel approach for potential AMD treatment.

Pigment epithelium-derived factor (PEDF) prevents retinal cell death via PEDF Receptor (PEDF-R): identification of a functional ligand binding site.

The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu(159)-Met(325)) with affinity similar to the full-length PEDF-R (Met(1)-Leu(504)). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile(193)-Leu(232)) and P1 (Thr(210)-Leu(249)) peptides. Recombinant C-terminal truncated PEDF-R4 (Met(1)-Leu(232)) and internally truncated PEDF-R and PEDF-R4 (ΔHis(203)-Leu(232)) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His(203)-Leu(232) region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.

Searching for Alternatively Spliced Variants of Phospholipase Domain-Containing 2 (Pnpla2), a Novel Gene in the Retina.

PURPOSE: Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for Pnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for Pnpla2 splice variants in mouse. MATERIALS AND METHODS: Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-length PNPLA2 cDNA or a PNPLA2 cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots. RESULTS: PCR products for Pnpla2 transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein. CONCLUSIONS: The data provide evidence for the existence of a single, full-length Pnpla2 transcript that could give rise to a single protein product that undergoes posttranslational processing.

Inhibition of tumor cell surface ATP synthesis by pigment epithelium-derived factor: implications for antitumor activity.

Recently, we have shown that the antiangiogenic pigment epithelium-derived factor (PEDF) can bind the catalytic ß-subunit of F1-ATP synthase and inhibit endothelial cell surface ATP synthase activity. This factor can additionally restrict tumor growth, invasion and metastasis, and can directly induce death on several tumor cell types. Active cell surface ATP synthase is also present in certain tumor cells and its ATP product is considered a stimulus for tumor growth. The present study aimed to elucidate the biological implications of the interactions between the extracellular PEDF and tumor cell surface ATP synthase. Incubation of T24 human urinary bladder carcinoma cells in media containing human recombinant PEDF protein for 48-96 h dramatically decreased cell viability in a concentration-dependent fashion as monitored by real-time cell impedance with a microelectronic system, microscopic imaging and biomarkers of live cells. Intact tumor cells exhibited cell surface ATP synthesis activity, which was inhibited by piceatannol, a specific inhibitor of F1/F0-ATP synthase. Immunoblotting revealed that the ß subunit of F1-ATP synthase was present in plasma membrane fractions of these cells. Interestingly, pre-incubation of tumor cells with PEDF inhibited the activity of cell surface ATP synthase in a concentration-dependent fashion. The PEDF-derived peptide 34-mer decreased tumor cell viability and inhibited extracellular ATP synthesis to the same extent as full-length PEDF. Moreover, ATP additions attenuated both the PEDF-mediated decrease in tumor cell viability and the inhibition of endothelial cell tube formation. The results lead to conclude that PEDF is a novel inhibitor of tumor cell surface ATP synthase activity that exhibits a cytotoxic effect on tumor cells, and that the structural determinants for these properties are within the peptide region 34-mer of the PEDF polypeptide. The data strongly suggest a role for the interaction between the 34-mer region of PEDF and tumor cell-surface ATP synthase in promoting tumor cell death.

Opposing effects of pigment epithelium-derived factor on breast cancer cell versus neuronal survival: implication for brain metastasis and metastasis-induced brain damage.

Brain metastases are a significant cause of morbidity and mortality for patients with cancer, yet preventative and therapeutic options remain an unmet need. The cytokine pigment epithelium-derived factor (PEDF) is downregulated in resected human brain metastases of breast cancer compared with primary breast tumors, suggesting that restoring its expression might limit metastatic spread. Here, we show that outgrowth of large experimental brain metastases from human 231-BR or murine 4T1-BR breast cancer cells was suppressed by PEDF expression, as supported by in vitro analyses as well as direct intracranial implantation. Notably, the suppressive effects of PEDF were not only rapid but independent of the effects of this factor on angiogenesis. Paralleling its cytotoxic effects on breast cancer cells, PEDF also exerted a prosurvival effect on neurons that shielded the brain from tumor-induced damage, as indicated by a relative 3.5-fold reduction in the number of dying neurons adjacent to tumors expressing PEDF. Our findings establish PEDF as both a metastatic suppressor and a neuroprotectant in the brain, highlighting its role as a double agent in limiting brain metastasis and its local consequences.

Assays for the antiangiogenic and neurotrophic serpin pigment epithelium-derived factor.

Pigment epithelium-derived factor (PEDF) is a secreted serpin that exhibits a variety of interesting biological activities. The multifunctional PEDF has neurotrophic and antiangiogenic properties, and acts in retinal differentiation, survival, and maintenance. It is also antitumorigenic and antimetastatic, and has stem cell self-renewal properties. It is widely distributed in the human body and exists in abundance in the eye as a soluble extracellular glycoprotein. Its levels are altered in diseases characterized by retinopathies and angiogenesis. Its mechanisms of neuroprotection and angiogenesis are associated with receptor interactions at cell-surface interfaces and changes in protein expression. This serpin lacks demonstrable serine protease inhibitory activity, but has binding affinity to extracellular matrix components and cell-surface receptors. Here we describe purification protocols, methods to quantify PEDF, and determine interactions with specific molecules, as well as neurotrophic and angiogenesis assays for this multifunctional protein.

Pigment epithelium-derived factor receptor (PEDF-R): a plasma membrane-linked phospholipase with PEDF binding affinity.

Pigment epithelium-derived factor (PEDF), a multifunctional protein, acts in retinal differentiation, survival and maintenance by interacting with high affinity receptors on the surface of target cells. We have recently identified PEDF-R, a new member of the patatin-like phospholipase domain-containing 2 (PNPLA2) family with characteristics of a PEDF receptor. The PEDF-R sequence reveals a patatin-like phospholipase domain toward its amino-end, and four transmembrane domains interrupted by two extracellular loops and three intracellular regions along its polypeptide sequence. This newly identified protein is present on the surface of retina and RPE cells, and has the expected transmembrane topology. It has specific and high binding affinity for PEDF, and exhibits a potent phospholipase A(2) activity that liberates fatty acids. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. In summary, PEDF-R is a novel component of the retina that is a phospholipase-linked membrane protein with high affinity for PEDF. The results suggest a molecular pathway by which PEDF ligand/receptor interactions on the cell surface could generate a cellular signal. These conclusions enhance our understanding of the role of PEDF as a neurotrophic survival factor.