Lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) lncRNA differentially regulates gene and protein expression, signaling and morphology of human ocular cells.

Pseudoexfoliation glaucoma (PEXG) is characterized by dysregulated extracellular matrix (ECM) homeostasis that disrupts conventional outflow function and increases intraocular pressure (IOP). Prolonged IOP elevation results in optic nerve head damage and vision loss. Uniquely, PEXG is a form of open angle glaucoma that has variable penetrance, is difficult to treat and does not respond well to common IOP-lowering pharmaceuticals. Therefore, understanding modulators of disease severity will aid in targeted therapies for PEXG. Genome-wide association studies have identified polymorphisms in the long non-coding RNA lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) as a risk factor for PEXG. Risk alleles, oxidative stress and mechanical stretch all alter LOXL1-AS1 expression. As a long non-coding RNA, LOXL1-AS1 binds hnRNPL and regulates global gene expression. In this study, we focus on the role of LOXL1-AS1 in the ocular cells (trabecular meshwork and Schlemm's canal) that regulate IOP. We show that selective knockdown of LOXL1-AS1 leads to cell-type-specific changes in gene expression, ECM homeostasis, signaling and morphology. These results implicate LOXL1-AS1 as a modulator of cellular homeostasis, altering cell contractility and ECM turnover, both of which are well-known contributors to PEXG. These findings support LOXL1-AS1 as a key target for modifying the disease.

Integral role for lysyl oxidase-like-1 in conventional outflow tissue function and behavior.

Lysyl oxidase-like-1 (LOXL1), a vital crosslinking enzyme in elastin fiber maintenance, is essential for the stability and strength of elastic vessels and tissues. Variants in the LOXL1 locus associate with a dramatic increase in risk of exfoliation syndrome (XFS), a systemic fibrillopathy, which often presents with ocular hypertension and exfoliation glaucoma (XFG). We examined the role of LOXL1 in conventional outflow function, the prime regulator of intraocular pressure (IOP). Using Loxl1-/- , Loxl1+/- , and Loxl1+/+ mice, we observed an inverse relationship between LOXL1 expression and IOP, which worsened with age. Elevated IOP in Loxl1-/- mice was associated with a larger globe, decreased ocular compliance, increased outflow facility, extracellular matrix (ECM) abnormalities, and dilated intrascleral veins, yet, no dilation of arteries or capillaries. Interestingly, in living Loxl1-/- mouse eyes, Schlemm's canal (SC) was less susceptible to collapse when challenged with acute elevations in IOP, suggesting elevated episcleral venous pressure (EVP). Thus, LOXL1 expression is required for normal IOP control, while ablation results in altered ECM repair/homeostasis and conventional outflow physiology. Dilation of SC and distal veins, but not arteries, is consistent with key structural and functional roles for elastin in low-pressure vessels subjected to cyclical mechanical stress.

Expression of mRNAs, miRNAs, and lncRNAs in Human Trabecular Meshwork Cells Upon Mechanical Stretch.

Purpose: Intraocular pressure (IOP), the primary risk factor for primary open-angle glaucoma, is determined by resistance to aqueous outflow through the trabecular meshwork (TM). IOP homeostasis relies on TM responses to mechanical stretch. To model the effects of elevated IOP on the TM, this study sought to identify coding and non-coding RNAs differentially expressed in response to mechanical stretch. Methods: Monolayers of TM cells from non-glaucomatous donors (n = 5) were cultured in the presence or absence of 15% mechanical stretch, 1 cycle/second, for 24 hours using a computer-controlled Flexcell unit. We profiled mRNAs and lncRNAs with stranded total RNA sequencing and microRNA (miRNA) expression with NanoString-based miRNA assays. We used two-tailed paired t-tests for mRNAs and long non-coding RNAs (lncRNAs) and the Bioconductor limma package for miRNAs. Gene ontology and pathway analyses were performed with WebGestalt. miRNA-mRNA interactions were identified using Ingenuity Pathway Analysis Integrative miRNA Target Finder software. Validation of differential expression was conducted using droplet digital PCR. Results: We identified 219 mRNAs, 42 miRNAs, and 387 lncRNAs with differential expression in TM cells upon cyclic mechanical stretch. Pathway analysis indicated significant enrichment of genes involved in steroid biosynthesis, glycerolipid metabolism, and extracellular matrix-receptor interaction. We also identified several miRNA master regulators (miR-125a-5p, miR-30a-5p, and miR-1275) that regulate several mechanoresponsive genes. Conclusions: To our knowledge, this is the first demonstration of the differential expression of coding and non-coding RNAs in a single set of cells subjected to cyclic mechanical stretch. Our results validate previously identified, as well as novel, genes and pathways.

Identification and activity of the functional complex between hnRNPL and the pseudoexfoliation syndrome-associated lncRNA, LOXL1-AS1.

Individuals with pseudoexfoliation (PEX) syndrome exhibit various connective tissue pathologies associated with dysregulated extracellular matrix homeostasis. PEX glaucoma is a common, aggressive form of open-angle glaucoma resulting from the deposition of fibrillary material in the conventional outflow pathway. However, the molecular mechanisms that drive pathogenesis and genetic risk remain poorly understood. PEX glaucoma-associated single-nucleotide polymorphisms are located in and affect activity of the promoter of LOXL1-AS1, a long non-coding RNA (lncRNA). Nuclear and non-nuclear lncRNAs regulate a host of biological processes, and when dysregulated, contribute to disease. Here we report that LOXL1-AS1 localizes to the nucleus where it selectively binds to the mRNA processing protein, heterogeneous nuclear ribonucleoprotein-L (hnRNPL). Both components of this complex are critical for the regulation of global gene expression in ocular cells, making LOXL1-AS1 a prime target for investigation in PEX syndrome and glaucoma.

Prion protein modulates endothelial to mesenchyme-like transition in trabecular meshwork cells: Implications for primary open angle glaucoma.

Endothelial-to-mesenchyme-like transition (Endo-MT) of trabecular meshwork (TM) cells is known to be associated with primary open angle glaucoma (POAG). Here, we investigated whether the prion protein (PrPC), a neuronal protein known to modulate epithelial-to-mesenchymal transition in a variety of cell types, is expressed in the TM, and plays a similar role at this site. Using a combination of primary human TM cells and human, bovine, and PrP-knock-out (PrP-/-) mouse models, we demonstrate that PrPC is expressed in the TM of all three species, including endothelial cells lining the Schlemm's canal. Silencing of PrPC in primary human TM cells induces aggregation of ß1-integrin and upregulation of α-smooth muscle actin, fibronectin, collagen 1A, vimentin, and laminin, suggestive of transition to a mesenchyme-like phenotype. Remarkably, intraocular pressure is significantly elevated in PrP-/- mice relative to wild-type controls, suggesting reduced pliability of the extracellular matrix and increased resistance to aqueous outflow in the absence of PrPC. Since PrPC is cleaved by members of the disintegrin and matrix-metalloprotease family that are increased in the aqueous humor of POAG arising from a variety of conditions, it is likely that concomitant cleavage of PrPC exaggerates and confounds the pathology by inducing Endo-MT-like changes in the TM.

How to treat complicated grief.

Several DSM-5 criteria lasting 6 months or longer can help identify complicated grief, as can available assessment tools. Treatment has a unique focus.

Composition of Exfoliation Material.

Exfoliation glaucoma (XFG) is the most common identifiable cause of open-angle glaucoma worldwide, and results from the accumulation of extracellular fibrillary material (XFM) within the trabecular meshwork and the Schlemm canal leading to increased intraocular pressure and potential blindness. Immunohistochemical and mass spectrometry analyses have revealed that XFM is a highly glycosylated proteinaceous complex that is extremely resistant to degradation both within the body and under experimental conditions. The protein core contains a wide variety of proteins, including basement membrane proteins, elastic fiber proteins, latent TGFß proteins, metalloproteinases, chaperone proteins, complement proteins, lysyl oxidase-like 1 (LOXL1), and apolipoprotein E (ApoE). This supplemental section identifies the advances in knowledge and current understanding of the components within XFM with a specific focus on the most recent work defining proteins within XFM and to pose several biological questions that remain unanswered.

Motor and non-motor features of Parkinson's disease in LRRK2 G2019S carriers versus matched controls.

INTRODUCTION: LRRK2 G2019S mutation carriers with Parkinson's disease (PD) have been generally indistinguishable from those with idiopathic PD, with the exception of variable differences in some motor and non-motor domains, including cognition, gait, and balance. LRRK2 G2019S is amongst the most common genetic etiologies for PD, particularly in Ashkenazi Jewish (AJ) populations. METHODS: This cross-sectional data collection study sought to clarify the phenotype of LRRK2 G2019S mutation carriers with PD. Primary endpoints were the Movement Disorder Society Unified Parkinson Disease Rating Scale (MDS-UPDRS) and Montreal Cognitive Assessment (MoCA). Other motor and non-motor data were also assessed. The Mann-Whitney U Test was utilized to compare LRRK2 G2019S carriers with PD (LRRK2+) with non-carrier PD controls who were matched for age, gender, education, and PD duration. Survival analyses and log rank tests were utilized to compare interval from onset of PD to development of motor and non-motor complications. RESULTS: We screened 251 subjects and 231 completed the study, of whom 9 were LRRK2+, including 7 AJ subjects. 22.73% of AJ subjects with a family history of PD (FH) and 12.96% of AJ subjects without a FH were LRRK2+. There were no significant differences between the 9 LRRK2+ subjects and 19 matched PD controls in MDS-UPDRS, MoCA, or other motor and non-motor endpoints. CONCLUSION: Prevalence of the LRRK2 G2019S mutation in AJ and non-AJ subjects in our study population in Cleveland, Ohio was comparable to other clinical studies. There were no significant motor or non-motor differences between LRRK2+ PD and matched PD controls.

lncRNAs, DNA Methylation, and the Pathobiology of Exfoliation Glaucoma.

Exfoliation glaucoma (XFG) is a clinically aggressive and genetically distinct form of glaucoma that results in neuronal death and irreversible blindness. Gene variants associate with many neurodegenerative diseases including XFG, Parkinson's disease (PD) and Alzheimer's disease (AD). Intriguingly, variants found within the same gene can either confer risk for or provide protection against all 3 of these diseases, complicating the genetic component of pathology. Unfortunately, studies that examine proteins encoded by genes having relevant variants have failed to produce therapeutic interventions that slow or stop the progression of XFG, PD, or AD in patients. This roadblock has researchers focusing on alternative pathways that may be dysregulated and potentially lead to the development of disease. Two emerging areas of research in PD and AD are the pathobiology of long noncoding RNAs and DNA methylation. This review briefly introduces the roles of long noncoding RNAs and DNA methylation in disease pathogenesis, and highlights some of the cutting edge work that has been carried out in PD and AD, along with the limited but important studies in XFG. Finally, we propose a new direction for XFG research that may explain apparently conflicting genetic data and lead to the discovery of novel dysregulated pathways that will allow for targeted therapeutic development.

Major review: Exfoliation syndrome; advances in disease genetics, molecular biology, and epidemiology.

Exfoliation syndrome (XFS) is a common age-related disorder that leads to deposition of extracellular fibrillar material throughout the body. The most recognized disease manifestation is exfoliation glaucoma (XFG), which is a common cause of blindness worldwide. Recent developments in XFS genetics, cell biology and epidemiology have greatly improved our understanding of the etiology of this complex inherited disease. This review summarizes current knowledge of XFS pathogenesis, identifies gaps in knowledge, and discusses areas for future research.

Regulation of DJ-1 by Glutaredoxin 1 in Vivo: Implications for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH, and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in Caenorhabditis elegans; however many of the functional downstream targets of Grx1 in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivo and characterize its glutathionylation in vitro. Here, with Grx(-/-) mice we provide show that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents, and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homologue in a C. elegans in vivo model of PD. Therefore, our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo.

Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons.

Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca(2+)-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca(2+)-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca(2+) signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca(2+) signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function.

Serum levels of lipid metabolites in age-related macular degeneration.

Age-related macular degeneration (AMD) is a neurodegenerative disease that causes adult-onset blindness. There are 2 forms of this progressive disease: wet and dry. Currently there is no cure for AMD, but several treatment options have started to emerge making early detection critical for therapeutic success. Analysis of the eyes of Abca4(-/-)Rdh8(-/-) mice that display light-induced retinal degeneration indicates that 11-cis-retinal and docosahexaenoic acid (DHA) levels were significantly decreased as compared with the eyes of control dark-adapted C57BL/6J mice. In addition, exposure to intense light correlated with higher levels of prostaglandin G2 in the eyes of Abca4(-/-)Rdh8(-/-) mice. Intense light exposure also lowered DHA levels in the eyes of wild-type C57BL/6J mice without discernible retinal degeneration. Analysis of human serum from patients with AMD recapitulated these dysregulated DHA levels and revealed dysregulation of arachidonic acid (AA) levels as well (∼32% increase in patients with AMD compared with average levels in healthy individuals). From these observations, we then built a statistical model that included levels of DHA and AA from human serum. This model had a 74% probability of correctly identifying patients with AMD from controls. Addition of a genetic analysis for one of the most prevalent amino acid substitutions in the age-related maculopathy susceptibility 2 gene linked to AMD, Ala(69)→Ser, did not improve the statistical model. Thus, we have characterized a reliable method with the potential to detect AMD without a genetic component, paving the way for a larger-scale clinical evaluation. Our studies on mouse models along with the analysis of human serum suggest that our small molecule-based model may serve as an effective tool to estimate the risk of developing AMD.

The roles of redox enzymes in Parkinson's disease: Focus on glutaredoxin.

Parkinson's disease (PD) results from the loss of dopaminergic neurons in the substantia nigra portion of the midbrain, and represents the second most common neurodegenerative disease in the world. Although the etiology of PD is currently unclear, oxidative stress and redox dysfunction are generally understood to play key roles in PD pathogenesis and progression. Aging and environmental factors predispose cells to adverse effects of redox changes. In addition to these factors, genetic mutations linked to PD have been observed to disrupt the redox balance. Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with autosomal dominant PD, and several of these mutations have also been shown to increase the levels of reactive oxygen species in cells. Anti-oxidant proteins are necessary to restore the redox balance and maintain cell viability. Over the past decade studies have started to demonstrate the critical importance for redox proteins mediating neuronal protection in models of PD. This commentary briefly describes some of the factors hypothesized to contribute to PD, specifically regarding the redox changes that occur in PD. Dysregulation of redox proteins in PD is highlighted by some of the work detailing the roles of peroxiredoxin-3 and thioredoxin-1 in models of PD. In an attempt to generate novel therapies for PD, several potent inhibitors of LRRK2 have been developed. The use of these compounds, both as tools to understand the biology of LRRK2 and as potential therapeutic strategies is also discussed. This mini-review then provides a historical prospective on the discovery and characterization of glutaredoxin (Grx1), and briefly describes current understanding of the role of Grx1 in PD. The review concludes by highlighting our recent publication describing the novel role for Grx1 in mediating dopaminergic neuronal protection both in vitro and in vivo.

Glutaredoxin deficiency exacerbates neurodegeneration in C. elegans models of Parkinson's disease.

Parkinson's disease (PD) is characterized by selective degeneration of dopaminergic neurons. Although the etiology of PD remains incompletely understood, oxidative stress has been implicated as an important contributor in the development of PD. Oxidative stress can lead to oxidation and functional perturbation of proteins critical to neuronal survival. Glutaredoxin 1 (Grx1) is an evolutionally conserved antioxidant enzyme that repairs protein oxidation by reversing the oxidative modification of cysteine known as S-glutathionylation. We aimed to explore the regulatory role of Grx1 in PD. We first examined the levels of Grx1 in postmortem midbrain samples from PD patients, and observed that Grx1 content is decreased in PD, specifically within the dopaminergic neurons. We subsequently investigated the potential role of Grx1 deficiency in PD pathogenesis by examining the consequences of loss of the Caenorhabditis elegans Grx1 homolog in well-established worm models of familial PD caused by overexpression of pathogenic human LRRK2 mutants G2019S or R1441C. We found that loss of the Grx1 homolog led to significant exacerbation of the neurodegenerative phenotype in C. elegans overexpressing the human LRRK2 mutants. Re-expression in the dopaminergic neurons of the active, but not a catalytically inactive form of the Grx1 homolog rescued the exacerbated phenotype. Loss of the Grx1 homolog also exacerbated the neurodegenerative phenotype in other C. elegans models, including overexpression of human α-synuclein and overexpression of tyrosine hydroxylase (a model of sporadic PD). Therefore, our results reveal a novel neuroprotective role of glutaredoxin against dopaminergic neurodegeneration in models of familial and sporadic PD.

Kinase inhibitors arrest neurodegeneration in cell and C. elegans models of LRRK2 toxicity.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent known cause of late-onset Parkinson's disease (PD). To explore the therapeutic potential of small molecules targeting the LRRK2 kinase domain, we characterized two LRRK2 kinase inhibitors, TTT-3002 and LRRK2-IN1, for their effects against LRRK2 activity in vitro and in Caenorhabditis elegans models of LRRK2-linked neurodegeneration. TTT-3002 and LRRK2-IN1 potently inhibited in vitro kinase activity of LRRK2 wild-type and mutant proteins, attenuated phosphorylation of cellular LRRK2 and rescued neurotoxicity of mutant LRRK2 in transfected cells. To establish whether LRRK2 kinase inhibitors can mitigate pathogenesis caused by different mutations including G2019S and R1441C located within and outside of the LRRK2 kinase domain, respectively, we evaluated effects of TTT-3002 and LRRK2-IN1 against R1441C- and G2019S-induced neurodegeneration in C. elegans models. TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment in transgenic C. elegans expressing human R1441C- and G2019S-LRRK2. The inhibitors displayed nanomolar to low micromolar rescue potency when administered either pre-symptomatically or post-symptomatically, indicating both prevention and reversal of the dopaminergic deficit. The same treatments also led to long-lasting prevention and rescue of neurodegeneration. In contrast, TTT-3002 and LRRK2-IN1 were ineffective against the neurodegenerative phenotype in transgenic worms carrying the inhibitor-resistant A2016T mutation of LRRK2, suggesting that they elicit neuroprotective effects in vivo by targeting LRRK2 specifically. Our findings indicate that the LRRK2 kinase activity is critical for neurodegeneration caused by R1441C and G2019S mutations, suggesting that kinase inhibition of LRRK2 may represent a promising therapeutic strategy for PD.

Dysregulation of glutathione homeostasis in neurodegenerative diseases.

Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are increasingly implicated in the induction and progression of neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, and Friedreich's ataxia. In this review background is provided on the steady-state synthesis, regulation, and transport of glutathione, with primary focus on the brain. A brief overview is presented on the distinct but vital roles of glutathione in cellular maintenance and survival, and on the functions of key glutathione-dependent enzymes. Major contributors to initiation and progression of neurodegenerative diseases are considered, including oxidative stress, protein misfolding, and protein aggregation. In each case examples of key regulatory mechanisms are identified that are sensitive to changes in glutathione redox status and/or in the activities of glutathione-dependent enzymes. Mechanisms of dysregulation of glutathione and/or glutathione-dependent enzymes are discussed that are implicated in pathogenesis of each neurodegenerative disease. Limitations in information or interpretation are identified, and possible avenues for further research are described with an aim to elucidating novel targets for therapeutic interventions. The pros and cons of administration of N-acetylcysteine or glutathione as therapeutic agents for neurodegenerative diseases, as well as the potential utility of serum glutathione as a biomarker, are critically evaluated.

Primary lung large cell carcinoma metastatic to the vulva: a case report and review of the literature.

BACKGROUND: Metastatic cancer to the vulva is a rare diagnosis and is estimated to account for 5-8% of all vulvar cancers. CASE: A 57-year-old postmenopausal woman was referred for the evaluation of a new vulvar mass. CT scan of the pelvis demonstrated a 4 x 6 cm lobular mass of the left labia. An outpatient excisional biopsy revealed a poorly differentiated large cell carcinoma with prominent necrosis and focal perineural invasion consistent with a bronchogenic carcinoma. A subsequent PET scan of the chest revealed a large primary lung carcinoma confirmed by CT-guided biopsy that was identical to the vulvar tumor. CONCLUSION: This case represents the only literature described primary lung carcinoma presenting as a vulvar metastasis.