RESUMEN
Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest benefits. To design new and more effective therapies for ALS, particularly for sporadic ALS of unknown and diverse etiologies, we must identify key, convergent mechanisms of disease pathogenesis. This review focuses on the origin and effects of glutamate-mediated excitotoxicity in ALS (the cortical hyperexcitability hypothesis), in which increased glutamatergic signaling causes motor neurons to become hyperexcitable and eventually die. We characterize both primary and secondary contributions to excitotoxicity, referring to processes taking place at the synapse and within the cell, respectively. 'Primary pathways' include upregulation of calcium-permeable AMPA receptors, dysfunction of the EAAT2 astrocytic glutamate transporter, increased release of glutamate from the presynaptic terminal, and reduced inhibition by cortical interneurons-all of which have been observed in ALS patients and model systems. 'Secondary pathways' include changes to mitochondrial morphology and function, increased production of reactive oxygen species, and endoplasmic reticulum (ER) stress. By identifying key targets in the excitotoxicity cascade, we emphasize the importance of this pathway in the pathogenesis of ALS and suggest that intervening in this pathway could be effective for developing therapies for this disease.
Asunto(s)
Esclerosis Amiotrófica Lateral , Ácido Glutámico , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Humanos , Ácido Glutámico/metabolismo , Animales , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Envejecimiento/metabolismo , Receptores AMPA/metabolismo , Estrés del Retículo Endoplásmico , Mitocondrias/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Astrocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Glaucoma is a vision threatening optic neuropathy that affects millions of people worldwide. In primary open angle, increased intraocular pressure (IOP) is the main risk factor for the development of this disease. Studies investigating the causes and mechanisms of increased IOP show fibrotic changes in the trabecular meshwork (TM) that are different from those of age-matched controls. Tissue transglutaminase (TGM2), an extracellular matrix (ECM) crosslinking enzyme, covalently crosslinks ECM proteins and causes excessive ECM protein deposition in the TM that could cause increased IOP. Previous literature reports increased expression of TGM2 in glaucomatous eyes compared to controls. We recently have shown that overexpression of TGM2 causes increased ECM crosslinking in the TM, increases IOP, and decreases aqueous humor (AH) outflow facility in mouse eyes. Therefore, we wanted to study the effect of TGM2 knockout (KO) on IOP in TGM2 floxed mice. Ad5.Cre transduction caused partial KO of TGM2, which decreased TGM2 expression in the TM region of mouse eyes. TGM2 KO significantly decreased IOP by itself and also in TGFß2 induced ocular hypertensive mice. TGM2 KO also restores the outflow facility in TGFß2 transduced eyes. Overall, TGM2 KO rescued the TGFß2-induced ocular hypertensive phenotype. Thus, TGM2 may offer potential as a new therapeutic target for glaucoma.
Asunto(s)
Proteínas de Unión al GTP/genética , Presión Intraocular , Hipertensión Ocular/prevención & control , Malla Trabecular/enzimología , Transglutaminasas/genética , Adenoviridae/genética , Animales , Regulación Enzimológica de la Expresión Génica/fisiología , Técnicas de Inactivación de Genes , Presión Intraocular/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Hipertensión Ocular/inducido químicamente , Hipertensión Ocular/enzimología , Proteína Glutamina Gamma Glutamiltransferasa 2 , Reacción en Cadena en Tiempo Real de la Polimerasa , Tonometría Ocular , Transfección , Factor de Crecimiento Transformador beta2/toxicidadRESUMEN
Glaucoma is a leading cause of irreversible vision loss and is associated with fibrotic changes in two ocular tissues-the optic nerve head (ONH) and trabecular meshwork (TM). We investigated the differences in extracellular matrix components (ECM) including collagen, elastin, transforming growth factor beta-2, type-II receptor (TGFßRII) and Galectin3 (Gal3) in the glaucomatous human eyes to quantify fibrotic changes in ONH and TM. Glaucomatous and control human donor eyes were prepared for chemical and immunological staining to quantify ECM protein expression in the TM and ONH. Chemical staining included: Trichrome (collagen), Vernhoeff-Van Giesen (elastin) and Sirius Red (collagen). Immunohistochemistry was used to determine levels of Gal3 and TGFß2RII. Quantitative analyses were performed using Image J software. Student's t-test was used to compare groups and Pearson's test was used to determine correlations P-values of 0.05 (or less) were considered statistically significant. Deposition of ECM proteins was elevated in glaucomatous tissues. There was increased collagen (P = 0.0469), Gal3 (P < 0.0001) and TGFß2RII (P = 0.0005) in the TM of glaucomatous eyes. Likewise, collagen (P = 0.0517) and Galectin3 (P = 0.041) were increased in the ONH glaucomatous eyes. There was a correlation of TGFßRII with Gal3 in the TM (P < 0.0001) and optic nerve (P = 0.0003). The TM and ONH of glaucomatous eyes showed increased expression of ECM proteins supporting a fibrotic pathology. Galectin3 and TGFß-2R II showed a positive correlation in TM and optic nerve supporting co-localization and suggesting their potential role in the glaucoma fibrotic process. Clin. Anat. 31:1031-1049, 2018. © 2018 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.
Asunto(s)
Galectina 3/metabolismo , Glaucoma/metabolismo , Disco Óptico/metabolismo , Malla Trabecular/metabolismo , Anciano , Anciano de 80 o más Años , Proteínas Sanguíneas , Estudios de Casos y Controles , Proteínas de la Matriz Extracelular/metabolismo , Colágenos Fibrilares/metabolismo , Fibrosis , Galectinas , Glaucoma/patología , Humanos , Disco Óptico/patología , Malla Trabecular/patología , Factor de Crecimiento Transformador beta2/metabolismoRESUMEN
Mice are now routinely utilized in studies of aqueous humor outflow dynamics. In particular, conventional aqueous outflow facility (C) is routinely measured via perfusion of the aqueous chamber by a number of laboratories. However, in mouse eyes perfused ex-vivo, values for C are variable depending upon whether the perfusate is introduced into the posterior chamber (PC) versus the anterior chamber (AC). Perfusion via the AC leads to posterior bowing of the iris, and traction on the iris root/scleral spur, which may increase C. Perfusion via the PC does not yield this effect. But the equivalent situation in living mice has not been investigated. We sought to determine whether AC versus PC perfusion of the living mouse eye may lead to different values for C. All experiments were conducted in C57BL/6J mice (all â) between the ages of 20 and 30 weeks. Mice were divided into groups of 3-4 animals each. In all groups, both eyes were perfused. C was measured in groups 1 and 2 by constant flow infusion (from a 50 µL microsyringe) via needle placement in the AC, and in the PC, respectively. To investigate the effect of ciliary muscle (CM) tone on C, groups 3 and 4 were perfused live via the AC or PC with tropicamide (muscarinic receptor antagonist) added to the perfusate at a concentration of 100 µM. To investigate immediate effect of euthanasia, groups 5 and 6 were perfused 15-30 min after death via the AC or PC. To investigate the effect of CM tone on C immediately following euthanasia, groups 7 and 8 were perfused 15-30 min after death via the AC or PC with tropicamide added to the perfusate at a concentration of 100 µM. C in Groups 1 (AC perfusion) and 2 (PC perfusion) was computed to be 19.5 ± 0.8 versus 21.0 ± 2.1 nL/min/mmHg, respectively (mean ± SEM, p > 0.4, not significantly different). In live animals in which tropicamide was present in the perfusate, C in Group 3 (AC perfusion) was significantly greater than C in Group 4 (PC perfusion) (22.0 ± 4.0 versus 14.0 ± 2.0 nL/min/mmHg, respectively, p = 0.0021). In animals immediately following death, C in groups 5 (AC perfusion) and 6 (PC perfusion) was computed to be 21.2 ± 2.0 versus 22.8 ± 1.4 nL/min/mmHg, respectively (mean ± SEM, p = 0.1196, not significantly different). In dead animals in which tropicamide was present in the perfusate, C in group 7 (AC perfusion) was greater than C in group 8 (PC perfusion) (20.6 ± 1.4 versus 14.2 ± 2.6 nL/min/mmHg, respectively, p < 0.0001). C in eyes in situ in living mice or euthanized animals within 15-30 min post mortem is not significantly different when measured via AC perfusion or PC perfusion. In eyes of live or freshly euthanized mice, C is greater when measured via AC versus PC perfusion when tropicamide (a mydriatic and cycloplegic agent) is present in the perfusate.
Asunto(s)
Cámara Anterior/fisiología , Humor Acuoso/fisiología , Presión Intraocular/fisiología , Segmento Posterior del Ojo/fisiología , Animales , Cámara Anterior/efectos de los fármacos , Cámara Anterior/metabolismo , Humor Acuoso/metabolismo , Modelos Animales de Enfermedad , Femenino , Presión Intraocular/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Antagonistas Muscarínicos/farmacología , Segmento Posterior del Ojo/efectos de los fármacos , Segmento Posterior del Ojo/metabolismo , Malla Trabecular/metabolismo , Tropicamida/farmacologíaRESUMEN
Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFß/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.
RESUMEN
Purpose: Tissue transglutaminase (TGM2) is elevated in glaucomatous trabecular meshwork (TM) tissues. We investigated whether increased expression of TGM2 increases extracellular matrix crosslinking in the TM, thereby increasing aqueous humor outflow resistance and elevating intraocular pressure (IOP) in mouse eyes. Methods: GTM3, primary human GTM 125-05, and cultured mouse TM cells were transduced with adenovirus serotype 5 expressing human transglutaminase 2 (Ad5.TGM2; multiplicity of infection [MOI]-75) and fixed for immunocytochemistry. To test the effect on IOP in living eyes, Ad5.TGM2 was injected intravitreally into one eye of BALB/cJ (n = 18) or C57BL/6J mice (n = 9). The uninjected contralateral eye and Ad5.GFP served as controls. Daytime conscious IOPs were measured twice per week. Aqueous outflow facility (C) was measured by constant flow infusion on completion of IOP measurements. Immunohistochemistry was performed on BALB/cJ mouse eyes to study TGM2 expression and activity. Results: The treatment of cultured TM cells with Ad5.TGM2 increased immunostaining of N-ε(γ-glutamyl) lysine crosslinks. Ad5.TGM2 injection significantly increased IOP in BALB/cJ (15.86 mm Hg [injected] vs. 10.70 mm Hg [control]) and in C57BL/6J mice (17.09 mm Hg [injected] vs. 12.01 mm Hg [control]). Mean aqueous outflow facility in the injected eyes of BALB/cJ (0.013 µL/min/mm Hg) and C57BL/6J mice (0.012 µL/min/mm Hg) was significantly lower than in the uninjected control eyes (BALB/cJ, 0.021 µL/min/mm Hg; C57BL/6J, 0.019 µL/min/mm Hg). The Ad5.TGM2 transduction of mouse eyes increased TGM2 expression in the TM region and increased N-ε(γ-glutamyl) lysine crosslinks. Conclusions: The increased expression of TGM2 in the TM increases N-ε(γ-glutamyl) lysine crosslinking in the TM, increases aqueous outflow resistance, and elevates IOP in mice. TGM2 may be at least partially responsible for ocular hypertension in POAG.