RESUMO
Farber disease (FD) is a debilitating lysosomal storage disorder characterized by severe inflammation and neurodegeneration. FD is caused by mutations in the ASAH1 gene, resulting in deficient acid ceramidase (ACDase) activity. Patients with ACDase deficiency exhibit a broad clinical spectrum. In classic cases, patients develop hepatosplenomegaly, nervous system involvement, and childhood mortality. Ocular manifestations include decreased vision, a grayish appearance to the retina with a cherry red spot, and nystagmus. That said, the full effect of ACDase deficiency on the visual system has not been studied in detail. We previously developed a mouse model that is orthologous for a known patient mutation in Asah1 that recapitulates human FD. Herein, we report evidence of a severe ocular pathology in Asah1P361R/P361R mice. Asah1P361R/P361R mice exhibit progressive retinal and optic nerve pathology. Through noninvasive ocular imaging and histopathological analyses of these Asah1P361R/P361R animals, we revealed progressive inflammation, the presence of retinal dysplasia, and significant storage pathology in various cell types in both the retina and optic nerves. Lipidomic analyses of retinal tissues revealed an abnormal accumulation of ceramides and other sphingolipids. Electroretinograms and behavioral tests showed decreased retinal and visual responses. Taken together, these data suggest that ACDase deficiency leads to sphingolipid imbalance, inflammation, dysmorphic retinal and optic nerve pathology, and severe visual impairment.
Assuntos
Ceramidase Ácida/genética , Lipogranulomatose de Farber , Mutação de Sentido Incorreto , Nervo Óptico , Retina , Transtornos da Visão , Ceramidase Ácida/metabolismo , Substituição de Aminoácidos , Animais , Ceramidas/genética , Ceramidas/metabolismo , Modelos Animais de Doenças , Lipogranulomatose de Farber/enzimologia , Lipogranulomatose de Farber/genética , Lipogranulomatose de Farber/patologia , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Camundongos , Camundongos Mutantes , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Retina/enzimologia , Retina/patologia , Esfingolipídeos/genética , Esfingolipídeos/metabolismo , Transtornos da Visão/enzimologia , Transtornos da Visão/genética , Transtornos da Visão/patologiaRESUMO
Recombinant adeno associated viruses (AAV) are the most commonly used vectors in animal model studies of gene therapy for retinal diseases. The ability of a vector to localize and remain in the target tissue, and in this manner to avoid off-target effects beyond the site of delivery, is critical to the efficacy and safety of the treatment. The in vivo imaging system (IVIS) is a non-invasive imaging tool used for detection and quantification of bioluminescence activity in rodents. Our aim was to investigate whether IVIS can detect localization and biodistribution of AAV5 vector in mice following subretinal (SR) and intravitreal (IVT) injections. AAV5 carrying firefly luciferase DNA under control of the ubiquitous cytomegalovirus (CMV) promoter was injected unilaterally IVT or SR (in the central or peripheral retina) of forty-one mice. Luciferase activity was tracked for up to 60 weeks in the longest surviving animals, using repeated (up to 12 times) IVIS bioluminescence imaging. Luciferase presence was also confirmed immunohistochemically (IHC) and by PCR in representative animals. In the SR group, IVIS readings demonstrated luciferase activity in all (32/32) eyes, and luciferase presence was confirmed by IHC (4/4 eyes) and PCR (12/12 eyes). In the IVT group, IVIS readings demonstrated luciferase activity in 7/9 eyes, and luciferase presence was confirmed by PCR in 5/5 eyes and by IHC (2/2 eyes). In two SR-injected animals (one each from the central and peripheral injection sites), PCR detected luciferase presence in the ipsilateral optic nerves, a finding that was not detected by IVIS or IHC. Our results show that when evaluating SR delivery, IVIS has a sensitivity and specificity of 100% compared with the gold standard PCR. When evaluating IVT delivery, IVIS has a sensitivity of 78% and specificity of 100%. These finding confirm the ability of IVIS to detect in-vivo localized expression of AAV following SR delivery in the retina up to 60 weeks post-treatment, using repeated imaging for longitudinal evaluation, without fading of the biological signal, thereby replacing the need for post mortem processing in order to confirm vector expression. However, IVIS is probably not sensitive enough, compared with genome detection, to demonstrate biodistribution to the optic nerve, as it could not detect luciferase activity in ipsilateral optic nerves following SR delivery in mice.
Assuntos
Dependovirus/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Vetores Genéticos , Luciferases de Vaga-Lume/genética , Nervo Óptico/enzimologia , Retina/enzimologia , Corpo Vítreo/enzimologia , Animais , Técnicas de Transferência de Genes , Imuno-Histoquímica , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nervo Óptico/diagnóstico por imagem , Reação em Cadeia da Polimerase , Retina/diagnóstico por imagem , Corpo Vítreo/diagnóstico por imagemRESUMO
The cJun N-terminal kinases (JNKs; JNK1, JNK2, and JNK3) promote degenerative processes after neuronal injury and in disease. JNK2 and JNK3 have been shown to promote retinal ganglion cell (RGC) death after optic nerve injury. In their absence, long-term survival of RGC somas is significantly increased after mechanical optic nerve injury. In glaucoma, because optic nerve damage is thought to be a major cause of RGC death, JNKs are an important potential target for therapeutic intervention. To assess the role of JNK2 and JNK3 in an ocular hypertensive model of glaucoma, null alleles of Jnk2 and Jnk3 were backcrossed into the DBA/2J (D2) mouse. JNK activation occurred in RGCs following increased intraocular pressure in D2 mice. However, deficiency of both Jnk2 and Jnk3 together did not lessen optic nerve damage or RGC death. These results differentiate the molecular pathways controlling cell death in ocular hypertensive glaucoma compared with mechanical optic nerve injury. It is further shown that JUN, a pro-death component of the JNK pathway in RGCs, can be activated in glaucoma in the absence of JNK2 and JNK3. This implicates JNK1 in glaucomatous RGC death. Unexpectedly, at younger ages, Jnk2-deficient mice were more likely to develop features of glaucomatous neurodegeneration than D2 mice expressing Jnk2. This appears to be due to a neuroprotective effect of JNK2 and not due to a change in intraocular pressure. The Jnk2-deficient context also unmasked a lesser role for Jnk3 in glaucoma. Jnk2 and Jnk3 double knockout mice had a modestly increased risk of neurodegeneration compared with mice only deficient in Jnk2. Overall, these findings are consistent with pleiotropic effects of JNK isoforms in glaucoma and suggest caution is warranted when using JNK inhibitors to treat chronic neurodegenerative conditions.
Assuntos
Glaucoma/enzimologia , Glaucoma/patologia , Proteína Quinase 9 Ativada por Mitógeno/deficiência , Degeneração Neural/enzimologia , Degeneração Neural/patologia , Hipertensão Ocular/enzimologia , Hipertensão Ocular/patologia , Animais , Axônios/metabolismo , Morte Celular , Ativação Enzimática , Regulação da Expressão Gênica , Glaucoma/fisiopatologia , Pressão Intraocular , Camundongos Endogâmicos DBA , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Degeneração Neural/fisiopatologia , Hipertensão Ocular/fisiopatologia , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Nervo Óptico/fisiopatologia , Retina/enzimologia , Retina/patologia , Retina/fisiopatologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologiaRESUMO
Myelinating glial cells (MGCs), oligodendrocytes (OLs) in the central nervous system (CNS) and Schwann cells (SCs) in the peripheral nervous system (PNS), generate myelin sheaths that insulate axons. After myelination is completed in adulthood, MGC functions independent from myelin are required to support axon survival, but the underlying mechanisms are still unclear. Dicer is a key enzyme that is responsible for generating functional micro-RNAs (miRNAs). Despite the importance of Dicer in initiating myelination, the role of Dicer in mature MGCs is still unclear. Here, Dicer was specifically deleted in mature MGCs in 2-month old mice (PLP-CreERT; Dicer fl/fl) by tamoxifen administration. Progressive motor dysfunction was observed in the Dicer conditional knockout mice, which displayed hind limb ataxia at 3 months post recombination that deteriorated into paralysis within 5 months. Massive axonal degeneration/atrophy in peripheral nerves was responsible for this phenomenon, but overt demyelination was not observed in either the CNS or PNS. In contrast to the PNS, signs of axonal degeneration were not observed in the CNS of these animals. We induced a Dicer deletion in oligodendroglia at postnatal day 5 in NG2-CreERT; Dicer fl/fl mice to evaluate whether Dicer expression in OLs is essential for axonal survival. Dicer deletion in oligodendroglia did not cause motor dysfunction at the age of 7 months. Neither axonal atrophy nor demyelination was observed in the CNS. Based on our results, Dicer expression in SCs is required to maintain axon integrity in adult PNS, and Dicer is dispensable for maintaining myelin sheaths in MGCs.
Assuntos
Axônios/enzimologia , RNA Helicases DEAD-box/deficiência , Bainha de Mielina/enzimologia , Degeneração Neural/enzimologia , Ribonuclease III/deficiência , Animais , Ataxia/enzimologia , Ataxia/patologia , Atrofia , Axônios/patologia , RNA Helicases DEAD-box/genética , Progressão da Doença , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Bainha de Mielina/patologia , Degeneração Neural/patologia , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Paralisia/enzimologia , Paralisia/patologia , Ribonuclease III/genética , Nervo Isquiático/enzimologia , Nervo Isquiático/patologia , Medula Espinal/enzimologia , Medula Espinal/patologia , Substância Branca/enzimologia , Substância Branca/patologiaRESUMO
When axons of the mammalian central nervous system (CNS) are injured, they fail to regenerate, while those of lower vertebrates undergo regeneration after injury. Wingless-type MMTV integration site family (Wnt) proteins play important roles in the CNS, and are reported to be activated after mammalian spinal cord or brain injury. Moreover, for axon growth to proceed, it is thought that small G-proteins, such as CDC42 and Rac1, need to be activated, whereas RhoA must be inactivated. However, the cell and molecular mechanisms involved in optic nerve regeneration remain unclear. In this study, we investigated axonal regeneration after injury using the zebrafish optic nerve as a model system. We sought to clarify the role of Wnt proteins and the mechanisms involved in the activation and inactivation of small G-proteins in nerve regeneration. After optic nerve injury, mRNA levels of Wnt5b, TAX1BP3 and ICAT increased in the retina, while those of Wnt10a decreased. These changes were associated with a reduction in ß-catenin in nuclei. We found that Wnt5b activated CDC42 and Rac1, leading to the inactivation of RhoA, which appeared to be dependent on increased TAX1BP3 mRNA levels. Furthermore, we found that mRNA levels of Daam1a and ARHGEF16 decreased. We speculate that the decrease in ß-catenin levels, which also further reduces levels of active RhoA, might contribute to regeneration in the zebrafish. Collectively, our novel results suggest that Wnt5b, Wnt10a, ICAT and TAX1BP3 participate in the activation and inactivation of small G-proteins, such as CDC42, Rac1 and RhoA, during the early stage of optic nerve regeneration in the zebrafish.
Assuntos
Regeneração Nervosa/fisiologia , Nervo Óptico/enzimologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Nervo Óptico/efeitos dos fármacos , Peixe-Zebra , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease that we identified as a nerve regeneration-associated molecule. The expression of DINE is upregulated in response to nerve injury in both the peripheral and central nervous systems, while its transcription is regulated by the activating transcription factor 3 (ATF3), a potent hub-transcription factor for nerve regeneration. Despite its unique hallmark of injury-induced upregulation, the physiological relevance of DINE in injured neurons has been unclear. In this study, we have demonstrated that the expression of DINE is upregulated in injured retinal ganglion cells (RGCs) in a coordinated manner with that of ATF3 after optic nerve injury, whereas DINE and ATF3 are not observed in any normal retinal cells. Recently, we have generated a mature DINE-deficient (KOTg) mouse, in which exogenous DINE is overexpressed specifically in embryonic motor neurons to avoid aberrant arborization of motor nerves and lethality after birth that occurs in the conventional DINE KO mouse. The DINE KOTg mice did not show any difference in retinal structure and the projection to brain from that of wild-type (wild type) mice under normal conditions. However, injured RGCs of DINE KOTg mice failed to regenerate even after the zymosan treatment, which is a well-known regeneration-promoting reagent. Furthermore, a DINE KOTg mouse crossed with a Atf3:BAC Tg mouse, in which green fluorescent protein (GFP) is visualized specifically in injured RGCs and optic nerves, has verified that DINE deficiency leads to regeneration failure. These findings suggest that injury-induced DINE is a crucial endopeptidase for injured RGCs to promote axonal regeneration after optic nerve injury. Thus, a DINE-mediated proteolytic mechanism would provide us with a new therapeutic strategy for nerve regeneration.
Assuntos
Fator 3 Ativador da Transcrição/genética , Metaloendopeptidases/genética , Regeneração Nervosa/genética , Traumatismos do Nervo Óptico/genética , Células Ganglionares da Retina/enzimologia , Fator 3 Ativador da Transcrição/metabolismo , Animais , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Metaloendopeptidases/deficiência , Camundongos , Camundongos Knockout , Fármacos Neuroprotetores/farmacologia , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Traumatismos do Nervo Óptico/enzimologia , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Transdução de Sinais , Zimosan/farmacologiaRESUMO
PURPOSE: The defining feature of glaucoma is excavation of the optic nerve head; however, the mechanism of this loss of tissue is not well understood. We recently discovered a copy number variation upstream of matrix metalloproteinase 19 (MMP19) in a large, autosomal dominant pedigree with a congenital malformation of the optic disc called cavitary optic disc anomaly (CODA). Patients with CODA have abnormal optic discs that exhibit an excavated shape similar to cupping seen in glaucoma. The goal of this study is to characterize the localization of MMP19 within the human optic nerve. METHODS: The MMP19 protein in the optic nerve was evaluated with western blot analysis and with immunohistochemistry in sagittal and en face/cross sections of optic nerves obtained from healthy human donor eyes. RESULTS: The MMP19 protein was detected in the human optic nerve, retina, and RPE/choroid with western blot analysis, with highest expression in the retina and the optic nerve. Using immunohistochemistry, MMP19 was localized within the optic nerve to the extracellular space within the septa that separate bundles of optic nerve axons into fascicles. The presence of MMP19 within the optic nerve septa was further confirmed by the colocalization of MMP19 to this structure with type IV collagen. Strong labeling of MMP19 was also detected in the arachnoid layer of the optic nerve sheath. Finally, immunohistochemistry of the optic nerve cross sections demonstrated that MMP19 shows a peripheral to central gradient, with more abundant labeling along the edges of the optic nerve and in the arachnoid layer than in the center of the nerve. CONCLUSIONS: Abundant MMP19 was detected in the optic nerve head, the primary site of pathology in patients with CODA. The localization of MMP19 to the optic nerve septa is consistent with its predicted secretion and accumulation within the extracellular spaces of this tissue. Moreover, the lateral localization of MMP19 observed in the optic nerve cross sections suggests that it might have a role in regulating adhesion to the optic nerve to the scleral canal and remodeling the extracellular matrix that provides the structural integrity of the optic disc. Dysregulation of MMP19 production might, therefore, undermine the connections between the optic nerve and the scleral canal and cause a collapse of the optic disc and the development of CODA. Similar processes might also be at work in the formation of optic disc cupping in glaucoma.
Assuntos
Metaloproteinases da Matriz Secretadas/metabolismo , Disco Óptico/enzimologia , Nervo Óptico/enzimologia , Western Blotting , Técnica Indireta de Fluorescência para Anticorpo , Voluntários Saudáveis , Humanos , Doadores de TecidosRESUMO
The aim of this paper is to study the morphology and the distribution of the monoamine oxidase enzymatic system in the optic nerve of 4 month-old Wistar (young) and 28 month-old Wistar (old) rats. The optic nerve was harvested from 20 young and old rats. The segment of optic nerve was divided longitudinally into two pieces, each 0.1 mm in length. The first piece was used for transmission electron microscopy. The second piece was stained with histochemical reaction for monoamine oxidase. The agerelated changes in the optic nerve of rats include micro-anatomical details, ultrastructure and monoamine oxidase histochemical staining. A strong decrease of the thin nerve fibers and a swelling of the thick ones can be observed in optic nerve fibers of old rats. Increased monoamine oxidase histochemical staining of the optic nerve of aged rats is well demonstrated. The increase of meningeal shealth and the decrease of thin nerve fibers of the optic nerve in old rats are well documented. Morphological, ultrastructural and histochemical changes observed in optic nerve fibers of the old rats show a close relation with aging.
Assuntos
Envelhecimento/patologia , Monoaminoxidase/análise , Proteínas do Tecido Nervoso/análise , Nervo Óptico/ultraestrutura , Envelhecimento/metabolismo , Animais , Axônios/ultraestrutura , Microscopia Eletrônica , Bainha de Mielina/enzimologia , Fibras Nervosas/enzimologia , Fibras Nervosas/ultraestrutura , Nervo Óptico/enzimologia , Ratos , Ratos WistarRESUMO
PURPOSE: Glaucoma is one of the most important reason causes of the blindness, associated with retinal ganglion cells (RGC) death. This process is not fully understood, however apoptosis due to hypoxia is one of the most important processes leading to RGC death. Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix due to metalloproteinase activation, which leads to loss of RGC and axons at the optic nerve head. The aim of the study was to evaluate metalloproteinase 9 (MMP-9) and tissue metalloproteinase inhibitor-1 (TIMP-1) expression in the retinal ganglion cells and optic nerve axons in 33 eyes with absolute primary glaucoma. MATERIAL/METHODS: To evaluate MMP-9 and TIMP-1 expression primary polyclonal goat antibodies against MMP-9 and TIMP-1 were used. The control group was composed of 8 cases of eyes enucleated and fixed in the first day after trauma. RESULTS: MMP-9 expression was observed in retinal ganglion cells and in the inner nuclear layer of the retina in all the examined cases. In 28 out of 33 glaucomatous eyes, MMP-9 expression was observed in the proliferating glial cells surrounding the optic nerve axons. TIMP-1 expression was observed in 10 out of 33 glaucomatous eyes, only in retinal ganglion cells. None of the examined injured eyes showed MMP-9 and TIMP-1 expression. CONCLUSIONS: MMP-9 activation rather than TIMP-1 may by associated with the pathomechanism of retinal ganglion cell and optic nerve damage in absolute glaucoma.
Assuntos
Glaucoma de Ângulo Fechado/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Nervo Óptico/enzimologia , Retina/enzimologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Glaucoma de Ângulo Fechado/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Disco Óptico/enzimologia , Disco Óptico/patologia , Nervo Óptico/patologia , Retina/patologia , Células Ganglionares da Retina/enzimologia , Células Ganglionares da Retina/patologiaRESUMO
The toxicity of the class of chemicals known as the organophosphates (OP) is most commonly attributed to the inhibition of the enzyme acetylcholinesterase. However, there is significant evidence that this mechanism may not account for all of the deleterious neurologic and neurobehavioral symptoms of OP exposure, especially those associated with levels that produce no overt signs of acute toxicity. In the study described here we evaluated the effects of the commonly used OP-pesticide, chlorpyrifos (CPF) on axonal transport in the brains of living rats using manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI) of the optic nerve (ON) projections from the retina to the superior colliculus (SC). T1-weighted MEMRI scans were evaluated at 6 and 24h after intravitreal injection of Mn(2+). As a positive control for axonal transport deficits, initial studies were conducted with the tropolone alkaloid colchicine administered by intravitreal injection. In subsequent studies both single and repeated exposures to CPF were evaluated for effects on axonal transport using MEMRI. As expected, intravitreal injection of colchicine (2.5µg) produced a robust decrease in transport of Mn(2+) along the optic nerve (ON) and to the superior colliculus (SC) (as indicated by the reduced MEMRI contrast). A single subcutaneous (s.c.) injection of CPF (18.0mg/kg) was not associated with significant alterations in the transport of Mn(2+). Conversely, 14-days of repeated s.c. exposure to CPF (18.0mg/kg/day) was associated with decreased transport of Mn(2+) along the ONs and to the SC, an effect that was also present after a 30-day (CPF-free) washout period. These results indicate that repeated exposures to a commonly used pesticide, CPF can result in persistent alterations in axonal transport in the living mammalian brain. Given the fundamental importance of axonal transport to neuronal function, these observations may (at least in part) explain some of the long term neurological deficits that have been observed in humans who have been repeatedly exposed to doses of OPs not associated with acute toxicity.
Assuntos
Transporte Axonal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Clorpirifos/toxicidade , Inseticidas/toxicidade , Acetilcolinesterase/análise , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Meios de Contraste , Imageamento por Ressonância Magnética , Masculino , Manganês , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/enzimologia , Nervo Óptico/metabolismo , Ratos , Ratos Wistar , Vias Visuais/efeitos dos fármacos , Vias Visuais/enzimologia , Vias Visuais/metabolismoRESUMO
The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.
Assuntos
Degeneração Neural , Regeneração Nervosa , Traumatismos do Nervo Óptico/enzimologia , Nervo Óptico/enzimologia , Células Ganglionares da Retina/enzimologia , Quinases Associadas a rho/metabolismo , Animais , Apoptose , Autofagia , Axônios/enzimologia , Axônios/patologia , Calpaína/metabolismo , Caspase 3/metabolismo , Morte Celular , Células Cultivadas , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes , Vetores Genéticos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Quinases Lim/genética , Quinases Lim/metabolismo , Compressão Nervosa , Proteínas do Tecido Nervoso/metabolismo , Neuritos/enzimologia , Neuritos/patologia , Nervo Óptico/patologia , Nervo Óptico/fisiopatologia , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/patologia , Traumatismos do Nervo Óptico/fisiopatologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Ratos , Ratos Wistar , Células Ganglionares da Retina/patologia , Transdução de Sinais , Fatores de Tempo , Transfecção , Quinases Associadas a rho/genéticaRESUMO
Multiple studies in glaucoma patients and in animal models of spontaneous and experimentally-induced glaucoma, reported changes in the expression and activity of several matrix metalloproteinases (MMPs) in the retina, optic nerve, aqueous humor, and trabecular meshwork. These data have led to the hypothesis that MMPs might be involved in glaucoma onset and/or disease progression. However, reports are conflicting and research aiming at providing a clear definition of their causative role is lacking. In glaucoma, MMPs are thought to act at two different levels. In the trabecular meshwork, they fine-tune the aqueous humor outflow rate and intraocular pressure, in the neuroretina and optic nerve, however, their role during glaucoma disease progression is much less clear. This review provides a comprehensive overview of the research conducted on the expression and function of MMPs in the retina and optic nerve, and on the elucidation of their potential involvement during glaucoma pathogenesis. Additionally, we describe the insecure balance between detrimental and potential beneficial MMP activities during central nervous system recovery and how MMP-based therapies could help to overcome the current pitfalls in the development of retinal ganglion cell neuroprotection and axon regeneration approaches for the treatment of glaucoma.
Assuntos
Glaucoma , Pressão Intraocular , Metaloproteinases da Matriz/biossíntese , Nervo Óptico/enzimologia , Células Ganglionares da Retina/enzimologia , Glaucoma/enzimologia , Glaucoma/patologia , Glaucoma/fisiopatologia , Humanos , Nervo Óptico/patologia , Células Ganglionares da Retina/patologiaRESUMO
PURPOSE: We determined the presence and levels of gelatinase matrix metalloproteinases (MMPs) in the optic nerve and surrounding rim region of the human fundus. METHODS: Samples of optic nerve, rim region, and Bruch's membrane-choroid from macular and peripheral regions were isolated from 9 pairs of human donor eyes. The MMPs were extracted and separated by gelatin zymography. Individual gelatinase species were identified by their respective molecular weights and levels quantified by standard densitometric techniques. Ratios of active/latent MMPs were calculated as representative indicators of the degree of proteolytic activity at each of the locations examined. RESULTS: All of the gelatinase species normally found in Bruch's membrane also were present in the optic nerve region. The presence of the high molecular weight MMP species (HMW1 and HMW2) was indicative of the age-related accumulation of polymerized MMPs 2 and 9. Level of activated MMPs was considerably raised in comparison with their latent forms at the optic nerve and surrounding region indicative of greater ongoing turnover of the matrix (P < 0.005). CONCLUSIONS: The components of the gelatinase pathway mediating matrix turnover in Bruch's membrane also were present in the optic nerve region. The presence of high levels of active MMPs 2 and 9 in comparison with the latent forms in the optic nerve and rim area is indicative of a high rate of matrix remodeling in these regions. Enhanced matrix turnover within the optic nerve region may represent an important mechanism for maintaining the plasticity of the lamina cribrosa.
Assuntos
Envelhecimento/fisiologia , Lâmina Basilar da Corioide/enzimologia , Corioide/enzimologia , Gelatinases/metabolismo , Nervo Óptico/enzimologia , Esclera/enzimologia , Idoso , Eletroforese em Gel de Poliacrilamida , Humanos , Pessoa de Meia-IdadeRESUMO
Nogo-A is a myelin-derived inhibitor playing a pivotal role in the prevention of axonal regeneration. A functional domain of Nogo-A, Amino-Nogo, exerts an inhibitory effect on axonal regeneration, although the mechanism is unclear. The present study investigated the role of the Amino-Nogo-integrin signaling pathway in primary retinal ganglion cells (RGCs) with respect to axonal outgrowth, which is required for axonal regeneration. Immunohistochemistry showed that integrin αv, integrin α5 and FAK were widely expressed in the visual system. Thy-1 and GAP-43 immunofluorescence showed that axonal outgrowth of RGCs was promoted by Nogo-A siRNA and a peptide antagonist of the Nogo-66 functional domain of Nogo-A (Nep1-40), and inhibited by a recombinant rat Nogo-A-Fc chimeric protein (Δ20). Western blotting revealed increased integrin αv and p-FAK expression in Nogo-A siRNA group, decreased integrin αv expression in Δ20 group and decreased p-FAK expression in Nep1-40 group. Integrin α5 expression was not changed in any group. RhoA G-LISA showed that RhoA activation was inhibited by Nogo-A siRNA and Δ20, but increased by Nep1-40 treatment. These results suggest that Amino-Nogo inhibits RGC axonal outgrowth primarily through the integrin αv signaling pathway.
Assuntos
Axônios , Proteínas da Mielina/metabolismo , Células Ganglionares da Retina/citologia , Transdução de Sinais , Animais , Sequência de Bases , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Integrina alfa5/metabolismo , Proteínas da Mielina/genética , Proteínas Nogo , Nervo Óptico/enzimologia , Fosforilação , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/metabolismo , Córtex Visual/enzimologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
This work was conducted to evaluate the efficacy of a treatment on retinal ganglion cells (RGC) and on astrocytes of the optic nerve of glaucomatous eyes, using a combination of alpha-lipoic acid (ALA) and superoxide dismutase (SOD). Thirty-two male Wistar rats were fed with a diet supplemented with ALA, SOD, ALA and SOD or with no product for 8 weeks. Ocular hypertension was induced with 2% methylcellulose (MTC) and then rats were sacrificed. TUNEL assay showed a marked fluorescence in the ganglion cells and astrocytes of MTC-treated rats evidencing induction of apoptosis. In contrast, sections of eyes pretreated with ALA and SOD showed a lack of fluorescence quite similar to that of the controls. Similarly, eyes sections from rats pre-treated with ALA and SOD showed reduced differential expression of inducible nitric oxide synthase (iNOS) and of caspase-3 in compared to normally-fed/MTC-inoculated cases. An increase of ALA and SOD exerts an antiapoptotic effect and protects against oxidative stress and hence against the structural remodelling of the RGCs and astrocytes of the optic nerve in the presence of an ischemic and pressure stress.
Assuntos
Antioxidantes/farmacologia , Olho/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Nervo Óptico/efeitos dos fármacos , Ácido Tióctico/farmacologia , Animais , Western Blotting , Caspase 3/metabolismo , Forma Celular/efeitos dos fármacos , Dano ao DNA , Ativação Enzimática/efeitos dos fármacos , Olho/enzimologia , Olho/patologia , Fluorescência , Marcação In Situ das Extremidades Cortadas , Masculino , Modelos Animais , Óxido Nítrico Sintase Tipo II/metabolismo , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Ratos , Ratos Wistar , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/enzimologia , Células Ganglionares da Retina/patologia , Superóxido Dismutase/farmacologiaRESUMO
PURPOSE: The Wlds mutation affords protection of retinal ganglion cell (RGC) axons in retinal ischemia and in inducible and hereditary preclinical models of glaucoma. We undertook the present study to determine whether the Nmnat1 portion of the chimeric protein provides axonal and somatic protection of RGCs in models of ischemia and glaucoma, particularly when localized to nonnuclear regions of the cell. METHODS: The survival and integrity of RGC axons and soma from transgenic mice with confirmed cytoplasmic overexpression of Nmnat1 in retina and optic nerve (cytNmnat1-Tg mice) were examined in the retina and postlaminar optic nerve 4 days following acute retinal ischemia, and 3 weeks following the chronic elevation of intraocular pressure. RESULTS: Ischemia- and glaucoma-induced disruptions of proximal segments of RGC axons that comprise the nerve fiber layer in wild-type mice were both robustly abrogated in cytNmnat1-Tg mice. More distal portions of RGC axons within the optic nerve were also protected from glaucomatous disruption in the transgenic mice. In both disease models, Nmnat1 overexpression in extranuclear locations significantly enhanced the survival of RGC soma. CONCLUSIONS: Overexpression of Nmnat1 in the cytoplasm and axons of RGCs robustly protected against both ischemic and glaucomatous loss of RGC axonal integrity, as well as loss of RGC soma. These findings reflect the more pan-cellular protection of CNS neurons that is realized by cytoplasmic Nmnat1 expression, and thus provide a therapeutic strategy for protecting against retinal neurodegeneration, and perhaps other CNS neurodegenerative diseases as well.
Assuntos
Axônios/patologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glaucoma/prevenção & controle , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Neuropatia Óptica Isquêmica/prevenção & controle , Doenças Retinianas/prevenção & controle , Células Ganglionares da Retina/patologia , Animais , Axônios/enzimologia , Sobrevivência Celular , Citoplasma/enzimologia , Citoproteção , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Glaucoma/enzimologia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microscopia Confocal , Nervo Óptico/enzimologia , Neuropatia Óptica Isquêmica/enzimologia , Neuropatia Óptica Isquêmica/patologia , Retina/enzimologia , Doenças Retinianas/enzimologia , Doenças Retinianas/patologia , Células Ganglionares da Retina/enzimologiaRESUMO
PURPOSE: We previously reported that calcineurin, a Ca(2+)/calmodulin-dependent serine/threonine phosphatase, is activated and proposed that it participates in retinal ganglion cell (RGC) apoptosis in two rodent ocular hypertension models. In this study, we tested whether calcineurin activation by itself, even in the absence of ocular hypertension, is sufficient to cause RGC degeneration. METHODS: We compared RGC and optic nerve morphology after adeno-associated virus serotype 2 (AAV2)-mediated transduction of RGCs with constitutively active calcineurin (CaNCA) or unactivated, wild-type calcineurin (CaNwt). Retinas and optic nerves were harvested 7-16 weeks after injection of the AAV into mouse vitreous. In flatmounted retinas, the transduced RGCs were identified with immunohistochemistry. The morphology of the RGCs was revealed by immunostaining for neurofilament SMI32 or by using GFP-M transgenic mice. A modified Sholl analysis was applied to analyze the RGC dendritic morphology. Optic nerve damage was assessed with optic nerve grading according to the Morrison standard. RESULTS: CaNwt and CaNCA were highly expressed in the injected eyes. Compared to the CaNwt-expressing RGCs, the CaNCA-expressing RGCs had smaller somas, smaller dendritic field areas, shorter total dendrite lengths, and simpler dendritic branching patterns. At 16 weeks, the CaNCA-expressing eyes had greater optic nerve damage than the CaNwt-expressing eyes. CONCLUSIONS: Calcineurin activation is sufficient to cause RGC dendritic degeneration and optic nerve damage. These data support the hypothesis that calcineurin activation is an important mediator of RGC degeneration, and are consistent with the hypothesis that calcineurin activation may contribute to RGC neurodegeneration in glaucoma.
Assuntos
Axônios/enzimologia , Calcineurina/genética , Dendritos/enzimologia , Degeneração Neural/enzimologia , Nervo Óptico/enzimologia , Degeneração Retiniana/enzimologia , Células Ganglionares da Retina/enzimologia , Animais , Axônios/patologia , Calcineurina/metabolismo , Dendritos/patologia , Dependovirus/genética , Ativação Enzimática , Vetores Genéticos , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Injeções Intravítreas , Camundongos , Camundongos Transgênicos , Degeneração Neural/patologia , Nervo Óptico/patologia , Degeneração Retiniana/patologia , Células Ganglionares da Retina/patologia , Transdução Genética , TransgenesRESUMO
In this study, age-related changes in the monoamine oxidases (MAO) were studied in the optic nerve (ON) of both young and aged male rats. The aim of the study was to assess the role of MAO in age-related changes in the rat ON and explain the mechanisms of neuroprotection mediated by MAO-B-specific inhibitors. Fifteen three month old and fifteen 26 month old Sprague-Dawley rats were used. The animals were killed by terminal anaesthesia. Staining of MAO, quantitative analysis of images, biochemical assays and statistical analysis of data were carried out. Samples of the ON were washed in water, fixed in Bowen fluid, dehydrated and embedded in Entellan. Histological sections were stained for MAO-enzymatic activities. The specificity of the reaction was evaluated by incubating control sections in a medium either without substrate or without dye. The quantitative analysis of images was carried out at the same magnification and the same lighting using a Zeiss photomicroscope. The histochemical findings were compared with the biochemical results. After enzymatic staining, MAO could be demonstrated in the ON fibres of both young and aged animals; however, MAO were increased in the nerve fibres of the elderly rats. These morphological findings were confirmed biochemically. The possibility that age-related changes in MAO levels may be attributed to impaired energy production mechanisms and/or represent the consequence of reduced energy needs is discussed.
Assuntos
Envelhecimento/fisiologia , Monoaminoxidase/metabolismo , Fibras Nervosas/enzimologia , Nervo Óptico/enzimologia , Estresse Oxidativo/fisiologia , Fatores Etários , Animais , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Fibras Nervosas/patologia , Nervo Óptico/patologia , Ratos , Ratos Sprague-DawleyRESUMO
PURPOSE: Although mutated G11778A NADH ubiquinone oxidoreductase subunit 4 (ND4) mitochondrial DNA (mtDNA) is firmly linked to the blindness of Leber hereditary optic neuropathy (LHON), a bona fide animal model system with mutated mtDNA complex I subunits that would enable probing the pathogenesis of optic neuropathy and testing potential avenues for therapy has yet to be developed. METHODS: The mutant human ND4 gene with a guanine to adenine transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc) adeno-associated virus (AAV) backbone. The HSP-ND4FLAG was directed toward the mitochondria by adding the 23 amino acid cytochrome oxidase subunit 8 (COX8) presequence fused in frame to the N-terminus of green fluorescent protein (GFP) into the AAV2 capsid open reading frame. The packaged scAAV-HSP mutant ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV-GFP (OS). Translocation and integration of mutant human ND4 in mouse mitochondria were assessed with PCR, reverse transcription-polymerase chain reaction (RT-PCR), sequencing, immunoblotting, and immunohistochemistry. Visual function was monitored with serial pattern electroretinography (PERG) and in vivo structure with spectral domain optical coherence tomography (OCT). Animals were euthanized at 1 year and processed for light and transmission electron microscopy. RESULTS: The PCR products of the mitochondrial and nuclear DNA extracted from infected retinas and optic nerves gave the expected 500 base pair bands. RT-PCR confirmed transcription of the mutant human ND4 DNA in mice. DNA sequencing confirmed that the PCR and RT-PCR products were mutant human ND4 (OD only). Immunoblotting revealed the expression of mutant ND4FLAG (OD only). Pattern electroretinograms showed a significant decrement in retinal ganglion cell function OD relative to OS at 1 month and 6 months after AAV injections. Spectral domain optical coherence tomography showed optic disc edema starting at 1 month post injection followed by optic nerve head atrophy with marked thinning of the inner retina at 1 year. Histopathology of optic nerve cross sections revealed reductions in the optic nerve diameters of OD versus OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes where some remaining axons were still in various stages of irreversible degeneration with electron dense aggregation. Electron lucent mitochondria accumulated in swollen axons where fusion of mitochondria was also evident. CONCLUSIONS: Due to the UGA codon at amino acid 16, mutant G11778A ND4 was translated only in the mitochondria where its expression led to significant loss of visual function, loss of retinal ganglion cells, and optic nerve degeneration recapitulating the hallmarks of human LHON.
Assuntos
Cegueira/genética , Dependovirus/genética , Mitocôndrias/genética , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica/genética , Animais , Cegueira/enzimologia , Cegueira/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Complexo IV da Cadeia de Transporte de Elétrons/genética , Eletrorretinografia , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Injeções Intravítreas , Camundongos , Mitocôndrias/enzimologia , NADH Desidrogenase/metabolismo , Atrofia Óptica/enzimologia , Atrofia Óptica/patologia , Atrofia Óptica Hereditária de Leber/enzimologia , Atrofia Óptica Hereditária de Leber/patologia , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Mutação Puntual , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Ganglionares da Retina/enzimologia , Células Ganglionares da Retina/patologiaRESUMO
BACKGROUND: Corpora amylacea (CAm) are a hallmark of aging and neurodegeneration. The presence of kynurenine aminotransferases I and II (KAT I and II) in CAm in the human retina and optic nerve has been already shown. The present study aimed to examine kynurenine aminotransferase III (KAT III) immunoreactivity in CAm in the human retina and optic nerve. MATERIAL AND METHODS: Polyclonal antibody against KAT III was used on sections of human eyes enucleated due to malignant uveal melanoma. PAS-stained sections of CAm were compared with KAT III stained ones. RESULTS: KAT III immunoreactivity was observed in CAm in the retina, prelaminar, laminar and retrolaminar region of the optic nerve with similar location to PAS-stained sections. The most intense staining was observed in the retrolaminar part of the optic nerve. KAT III immunoreactivity was also present in the cytoplasm of retinal ganglion cells. CONCLUSIONS: Expression of KAT III in CAm in the human retina and optic nerve indicates that this enzyme may be relevant in mechanisms of neurodegeneration leading to CAm formation.