Preservation of endoplasmic reticulum (ER) Ca2+ stores by deletion of inositol-1,4,5-trisphosphate receptor type 1 promotes ER retrotranslocation, proteostasis, and protein outer segment localization in cyclic nucleotide-gated channel-deficient cone photoreceptors.

Endoplasmic reticulum (ER) Ca2+ homeostasis relies on an appropriate balance between efflux- and influx-channel activity responding to dynamic changes of intracellular Ca2+ levels. Dysregulation of this complex signaling network has been shown to contribute to neuronal and photoreceptor death in neuro- and retinal degenerative diseases, respectively. In mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model of achromatopsia/cone dystrophy, cones display early-onset ER stress-associated apoptosis and protein mislocalization. Cones in these mice also show reduced cytosolic Ca2+ level and subsequent elevation in the ER Ca2+ -efflux-channel activity, specifically the inositol-1,4,5-trisphosphate receptor type 1 (IP3 R1), and deletion of IP3 R1 results in preservation of cones. This work investigated how preservation of ER Ca2+ stores leads to cone protection. We examined the effects of cone specific deletion of IP3 R1 on ER stress responses/cone death, protein localization, and ER proteostasis/ER-associated degradation. We demonstrated that deletion of IP3 R1 improves trafficking of cone-specific proteins M-/S-opsin and phosphodiesterase 6C to cone outer segments and reduces localization to cone inner segments. Consistent with the improved protein localization, deletion of IP3 R1 results in increased ER retrotranslocation protein expression, reduced proteasome subunit expression, reduced ER stress/cone death, and reduced retinal remodeling. We also observed the enhanced ER retrotranslocation in mice that have been treated with a chemical chaperone, supporting the connection between improved ER retrotranslocation/proteostasis and alleviation of ER stress. Findings from this work demonstrate the importance of ER Ca2+ stores in ER proteostasis and protein trafficking/localization in photoreceptors, strengthen the link between dysregulation of ER Ca2+ homeostasis and ER stress/cone degeneration, and support an involvement of improved ER proteostasis in ER Ca2+ preservation-induced cone protection; thereby identifying IP3 R1 as a critical mediator of ER stress and protein mislocalization and as a potential target to preserve cones in CNG channel deficiency.

Potential contribution of ryanodine receptor 2 upregulation to cGMP/PKG signaling-induced cone degeneration in cyclic nucleotide-gated channel deficiency.

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca2+ influx in rod and cone photoreceptors. Mutations in cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. Mice lacking functional cone CNG channel show endoplasmic reticulum (ER) stress-associated cone degeneration. The elevated cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) signaling and upregulation of the ER Ca2+ channel ryanodine receptor 2 (RyR2) have been implicated in cone degeneration. This work investigates the potential contribution of RyR2 to cGMP/PKG signaling-induced ER stress and cone degeneration. We demonstrated that the expression and activity of RyR2 were highly regulated by cGMP/PKG signaling. Depletion of cGMP by deleting retinal guanylate cyclase 1 or inhibition of PKG using chemical inhibitors suppressed the upregulation of RyR2 in CNG channel deficiency. Depletion of cGMP or deletion of Ryr2 equivalently inhibited unfolded protein response/ER stress, activation of the CCAAT-enhancer-binding protein homologous protein, and activation of the cyclic adenosine monophosphate response element-binding protein, leading to early-onset cone protection. In addition, treatment with cGMP significantly enhanced Ryr2 expression in cultured photoreceptor-derived Weri-Rb1 cells. Findings from this work demonstrate the regulation of cGMP/PKG signaling on RyR2 in the retina and support the role of RyR2 upregulation in cGMP/PKG signaling-induced ER stress and photoreceptor degeneration.

Effects of Procedural Discomfort and Expectation of Benefit on Therapy Continuation in Chronic Migraine Patients Treated With OnabotulinumtoxinA.

BACKGROUND: OnabotulinumtoxinA (BTX) has become a mainstream treatment for chronic migraine (CM). Patients often have varied expectations for treatment success but little is known about how these initial impressions influence continuation of therapy. OBJECTIVE: To record expectations of benefit and procedural discomfort (PD) from initial BTX treatment and to investigate their association with treatment success, defined as continuation of treatment for >3 sessions within a 2-year period. METHODS: A retrospective chart review of CM patients receiving initial treatment with BTX was performed. Patients were questioned about their expectations of benefit and PD as rated on a 0-10 scale. Responses were then compared with continuation of therapy beyond 3 sessions to identify the presence of significant association. RESULTS: Responses from patients (N = 297) were analyzed. About 173 subjects continued with BTX therapy for more than 3 sessions (173/297, 58.3%). Unadjusted odds ratios (OR) for expectation of benefit (EOB) (OR 1.11, 95% CI 0.99-1.24, P = .087) and PD (OR 1.02, 95% CI 0.90-1.16, P = .780) were not significantly predictive of continuing treatment. After considering sex, age, year of treatment, and previous headache preventative trials, only female sex (OR 2.02, 95% CI 1.09-3.74, P = .025) was found to be significantly associated with treatment continuation. CONCLUSIONS: In the usual care setting, PD and EOB are not significantly associated with therapy continuation in patients receiving initial treatment with BTX for the prevention of CM. However, after considering sex, age, year of treatment, and number of previous headache preventives attempted, we found that female patients had twice the likelihood of continuing with BTX therapy compared to male patients with CM.

Deficiency of type 2 iodothyronine deiodinase reduces necroptosis activity and oxidative stress responses in retinas of Leber congenital amaurosis model mice.

Thyroid hormone (TH) signaling has been shown to regulate cone photoreceptor viability. Suppression of TH signaling with antithyroid drug treatment or by targeting iodothyronine deiodinases and TH receptors preserves cones in mouse models of retinal degeneration, including the Leber congenital amaurosis Rpe65-deficient mice. This work investigates the cellular mechanisms underlying how suppressing TH signaling preserves cones in Rpe65-deficient mice, using mice deficient in type 2 iodothyronine deiodinase (Dio2), the enzyme that converts the prohormone thyroxine to the active hormone triiodothyronine (T3). Deficiency of Dio2 improved cone survival and function in Rpe65-/- and Rpe65-deficiency on a cone dominant background ( Rpe65-/-/ Nrl-/-) mice. Analysis of cell death pathways revealed that receptor-interacting serine/threonine-protein kinase (RIPK)/necroptosis activity was increased in Rpe65-/-/ Nrl-/- retinas, and Dio2 deficiency reversed the alterations. Cell-stress analysis showed that the cellular oxidative stress responses were increased in Rpe65-/-/ Nrl-/- retinas, and Dio2 deficiency abolished the elevations. Similarly, antithyroid drug treatment resulted in reduced RIPK/necroptosis activity and oxidative stress responses in Rpe65-/-/ Nrl-/- retinas. Moreover, treatment with T3 significantly induced RIPK/necroptosis activity and oxidative stress responses in the retina. This work shows that suppression of TH signaling reduces cellular RIPK/necroptosis activity and oxidative stress responses in degenerating retinas, suggesting a mechanism underlying the observed cone preservation.-Yang, F., Ma, H., Butler, M. R., Ding, X.-Q. Deficiency of type 2 iodothyronine deiodinase reduces necroptosis activity and oxidative stress responses in retinas of Leber congenital amaurosis model mice.

Endoplasmic reticulum (ER) Ca2+-channel activity contributes to ER stress and cone death in cyclic nucleotide-gated channel deficiency.

Endoplasmic reticulum (ER) stress and mislocalization of improperly folded proteins have been shown to contribute to photoreceptor death in models of inherited retinal degenerative diseases. In particular, mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model for achromatopsia, display both early-onset ER stress and opsin mistrafficking. By 2 weeks of age, these mice show elevated signaling from all three arms of the ER-stress pathway, and by 1 month, cone opsin is improperly distributed away from its normal outer segment location to other retinal layers. This work investigated the role of Ca2+-release channels in ER stress, protein mislocalization, and cone death in a mouse model of CNG-channel deficiency. We examined whether preservation of luminal Ca2+ stores through pharmacological and genetic suppression of ER Ca2+ efflux protects cones by attenuating ER stress. We demonstrated that the inhibition of ER Ca2+-efflux channels reduced all three arms of ER-stress signaling while improving opsin trafficking to cone outer segments and decreasing cone death by 20-35%. Cone-specific gene deletion of the inositol-1,4,5-trisphosphate receptor type I (IP3R1) also significantly increased cone density in the CNG-channel-deficient mice, suggesting that IP3R1 signaling contributes to Ca2+ homeostasis and cone survival. Consistent with the important contribution of organellar Ca2+ signaling in this achromatopsia mouse model, significant differences in dynamic intraorganellar Ca2+ levels were detected in CNG-channel-deficient cones. These results thus identify a novel molecular link between Ca2+ homeostasis and cone degeneration, thereby revealing novel therapeutic targets to preserve cones in inherited retinal degenerative diseases.

Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, THRA and THRB, encode TRs; THRB2 has been associated with cone viability. Using TR antagonists and Thrb2 deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the Rpe65-/- mouse model of Leber congenital amaurosis and reduced the number of TUNEL+ cells. Cone survival was significantly improved in Rpe65-/- and Cpfl1 (a model of achromatopsia with Pde6c defect) mice with Thrb2 deletion. Ventral cone density in Cpfl1/Thrb2-/- and Rpe65-/- /Thrb2-/- mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Targeting iodothyronine deiodinases locally in the retina is a therapeutic strategy for retinal degeneration.

Recent studies have implicated thyroid hormone (TH) signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we found that antithyroid treatment preserves cones. This work investigates the significance of targeting intracellular TH components locally in the retina. The cellular TH level is mainly regulated by deiodinase iodothyronine (DIO)-2 and -3. DIO2 converts thyroxine (T4) to triiodothyronine (T3), which binds to the TH receptor, whereas DIO3 degrades T3 and T4. We examined cone survival after overexpression of DIO3 and inhibition of DIO2 and demonstrated the benefits of these manipulations. Subretinal delivery of AAV5-IRBP/GNAT2-DIO3, which directs expression of human DIO3 specifically in cones, increased cone density by 30-40% in a Rpe65-/- mouse model of Lebers congenital amaurosis (LCA) and in a Cpfl1 mouse with Pde6c defect model of achromatopsia, compared with their respective untreated controls. Intravitreal and topical delivery of the DIO2 inhibitor iopanoic acid also significantly improved cone survival in the LCA model mice. Moreover, the expression levels of DIO2 and Slc16a2 were significantly higher in the diseased retinas, suggesting locally elevated TH signaling. We show that targeting DIOs protects cones, and intracellular inhibition of TH components locally in the retina may represent a novel strategy for retinal degeneration management.-Yang, F., Ma, H., Belcher, J., Butler, M. R., Redmond, T. M., Boye, S. L., Hauswirth, W. W., Ding, X.-Q. Targeting iodothyronine deiodinases locally in the retina is a therapeutic strategy for retinal degeneration.

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice.

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca(2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3(-/-)/Nrl(-/-) mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca(2+) channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.

Ryanodine Receptor 2 Contributes to Impaired Protein Localization in Cyclic Nucleotide-Gated Channel Deficiency.

The photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransduction and cellular calcium homeostasis. Mutations in the cone photoreceptor CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. CNG channel deficiency leads to endoplasmic reticulum (ER) stress-associated cone apoptosis, protein mislocalization, and ER calcium dysregulation. This work investigated the potential mechanisms of protein mislocalization associated with ER calcium dysregulation using Cnga3-/- mice lacking ER Ca2+ channel ryanodine receptor 2 (RyR2) specifically in cones. Deletion of Ryr2 improved outer segment (OS) localization of the cone proteins M-opsin, S-opsin, and cone phosphodiesterase subunit α' (PDE6C) and decreased inner segment localization. One-month-old Cnga3-/- mice showed ∼30% of M-opsin, 55% of S-opsin, and 50% of PDE6C localized to the OS. Cnga3-/- mice with Ryr2 deletion at the same age showed almost 60% of M-opsin, 70% of S-opsin, and 70% of PDE6C localized to the OS. Deletion of Ryr2 nearly completely reversed elevations of the ER stress markers phospho-IRE1α and phospho-eIF2α and suppressed cone apoptosis. Consistent with the improved cone protein localization and reduced ER stress/cone apoptosis, cone survival was improved by deletion of Ryr2 The number of cones was increased by ∼28% in 2- to 4-month-old Cnga3-/- mice with Ryr2 deletion compared with age-matched Cnga3-/- mice. This work demonstrates a role of RyR2/ER calcium dysregulation in protein mislocalization, ER stress, and cone death. The findings provide novel insights into the mechanisms of photoreceptor degeneration and support strategies targeting ER calcium regulation to manage retinal degeneration.

Public awareness of testis cancer and the prevalence of testicular self-examination-changing patterns over 20 years.

OBJECTIVES: Delay in treatment of testis cancer (TC) has a proven negative impact on disease stage, treatment outcome, and mortality. Poor public awareness of the disease and lack of testis self-examination (TSE) may account for late presentation. The aim of this study was to examine the knowledge of TC and performance of TSE in a group of men over 2 time periods 20 years apart. METHODS: In the current study, 677 men from a banking institution were surveyed on their knowledge of TC and their performance of TSE. Comparisons were made from the current data and those from the original study in 1986. RESULTS: This study demonstrates an increase in public awareness and modest concomitant increase in TSE since first studied in this country in 1986. There was no difference in knowledge across age groups in this study. Furthermore, men who demonstrate a superior degree of knowledge were more likely to perform TSE. Limitations included possible selection bias in the 2 studies conducted in a banking institution. CONCLUSIONS: Increased testicular cancer knowledge combined with TSE may have a role in improving detection of significant testicular pathology.

Keratinising squamous metaplasia of the bladder: natural history and rationalization of management based on review of 54 years experience.

OBJECTIVE: To review our experience of keratinising squamous metaplasia of the bladder as a predictor for the development of cancer and other complications, and formulate a policy for its management. MATERIALS AND METHODS: A retrospective review (1945-1999) identified 34 patients with histologically proven keratinising squamous metaplasia (27 males and 7 females, average age 50 years, range 13-80 years). The histological criteria used to diagnose keratinising squamous metaplasia were squamous metaplasia of the urothelium with keratinisation and/or hyperkeratosis and/or acanthosis. Female patients with non-keratinising squamous metaplasia (vaginal metaplasia) were excluded. RESULTS: Four patients had synchronous bladder carcinoma (three advanced with early death; one localised, cured by cystectomy). Another 14 patients had extensive metaplasia (Group A, >50% of mucosal involvement). Three cases had cystectomy and cure. Six cases (out of 11) developed subsequent cancer (4 advanced and early death, two localised and cured by cystectomy). One other case died of obstructive uropathy secondary to squamous metaplasia. Two cases died of unrelated causes. Sixteen patients had limited squamous metaplasia (Group B, <50% involvement mucosal surface). Twelve patients had endoscopic resection, extraction bladder calculus etc. with no further complications. Another two patients underwent urinary diversion. Two patients (out of 16) developed subsequent cancer both with advanced disease and early death. CONCLUSION: Keratinising squamous metaplasia of the bladder is a significant risk factor for vesical carcinoma and complications, such as bladder contracture and ureteral obstruction. This risk of complications increases with more extensive bladder mucosal involvement. The wide variation in lag time to the development of complications necessitates indefinite follow-up. Selected patients with extensive bladder involvement and long life expectancy should be offered cystectomy.