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1.
Molecules ; 26(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361825

RESUMO

Lipid-based nanocarriers (LNs) have made it possible to prolong corneal residence time and improve the ocular bioavailability of ophthalmic drugs. In order to investigate how the LNs interact with the ocular mucosa and reach the posterior eye segment, we have formulated lipid nanocarriers that were designed to bear a traceable fluorescent probe in the present work. The chosen fluorescent probe was obtained by a conjugation reaction between fluoresceinamine and the solid lipid excipient stearic acid, forming a chemically synthesized adduct (ODAF, N-(3',6'-dihydroxy-3-oxospiro [isobenzofuran-1(3H),9'-[9H] xanthen]-5-yl)-octadecanamide). The novel formulation (LN-ODAF) has been formulated and characterized in terms of its technological parameters (polydispersity index, mean particle size and zeta potential), while an in vivo study was carried out to assess the ability of LN-ODAF to diffuse through different ocular compartments. LN-ODAF were in nanometric range (112.7 nm ± 0.4), showing a good homogeneity and long-term stability. A TEM (transmission electron microscopy) study corroborated these results of characterization. In vivo results pointed out that after ocular instillation, LN ODAF were concentrated in the cornea (two hours), while at a longer time (from the second hour to the eighth hour), the fluorescent signals extended gradually towards the back of the eye. From the results obtained, LN-ODAF demonstrated a potential use of lipid-based nanoparticles as efficient carriers of an active pharmaceutical ingredient (API) involved in the management of retinal diseases.


Assuntos
Córnea/metabolismo , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Lipídeos/química , Nanopartículas/administração & dosagem , Segmento Posterior do Olho/metabolismo , Compostos de Espiro/administração & dosagem , Animais , Córnea/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Segmento Posterior do Olho/efeitos dos fármacos , Coelhos , Compostos de Espiro/química
2.
Sci Rep ; 11(1): 15146, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312410

RESUMO

Retinitis Pigmentosa is a genetically heterogeneous, degenerative retinal disorder characterized by gradual dysfunction and death of photoreceptors, first rods and later cones, and progressive blindness. Studies suggested that application of L-type calcium channel blockers rescues photoreceptors in paradigms related to Ca2+ overflow. To investigate whether Cav1.3 L-type channels have protective effects in the retina, we established a new mouse model by crossing rd10, modeling autosomal-recessive RP, with Cav1.3 deficient mice (rd10/Cav1.3KO). Our immunohistochemical analyses revealed an influence of Cav1.3 channels on the degenerative process of photoreceptors. The absence of Cav1.3 delayed the centre-to-periphery degeneration of rods indicated by a significantly higher number of photoreceptor rows and, consequently, of cones. In accordance with a preserved number of cones we observed a regular row of cone somas in rd10/Cav1.3-KO retinas. Surviving rod photoreceptors maintained synaptic contacts with rod bipolar cells. However, the delay in degeneration was only observed up to postnatal day 45. Although we observed a reduction in the spontaneous oscillatory retinal activity during multielectrode array analyses, measurable functional preservation was lacking in behavioural tests. In conclusion, Cav1.3 channels contribute to photoreceptor degeneration in rd10 retinas but photoreceptor temporary rescue might rather be achieved indirectly through other retinal cell layers.

3.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065385

RESUMO

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.


Assuntos
Retina/fisiopatologia , Epitélio Pigmentado da Retina/fisiopatologia , Retinite Pigmentosa/fisiopatologia , Animais , Dexametasona/farmacologia , Modelos Animais de Doenças , Estudos de Avaliação como Assunto , Inflamação/metabolismo , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Retina/efeitos dos fármacos , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Retinite Pigmentosa/metabolismo , Rodopsina/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
4.
Front Neurosci ; 14: 372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32435178

RESUMO

Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary pre-requisites for translation into human therapy to slow down RP.

5.
J Comp Neurol ; 528(9): 1502-1522, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31811649

RESUMO

Rod-cone degenerations, for example, retinitis pigmentosa are leading causes of blindness worldwide. Despite slow disease progression in humans, vision loss is inevitable; therefore, development of vision restoration strategies is crucial. Among others, promising approaches include optogenetics and prosthetic implants, which aim to bypass lost photoreceptors (PRs). Naturally, the efficacy of these therapeutic strategies will depend on inner retinal structural and functional preservation. The present study shows that in photoinducible I307N rhodopsin mice (Translational Vision Research Model 4 [Tvrm4]), a 12k lux light exposure eliminates PRs in the central retina in 1 week, but interneurons and their synapses are maintained for as long as 9 weeks postinduction. Despite bipolar cell dendritic retraction and moderate loss of horizontal cells, the survival rate of various cell types is very high. Significant preservation of conventional synapses and gap junctions in the inner plexiform layer is also observed. We found the number of synaptic ribbons to gradually decline and their ultrastructure to become transiently abnormal, although based on our findings intrinsic retinal architecture is maintained despite complete loss of PRs. Unlike common rodent models of PR degeneration, where the disease phenotype often interferes with retinal development, in Tvrm4 mice, the degenerative process can be induced after retinal development is complete. This time course more closely mimics the timing of disease onset in affected patients. Stability of the inner retina found in these mutants 2 months after PR degeneration suggests moderate, stereotyped remodeling in the early stages of the human disease and represents a promising finding for prompt approaches of vision restoration.

6.
Thyroid ; 30(1): 147-160, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31709926

RESUMO

Background: A novel form of thyroid hormone (TH) signaling is represented by 3-iodothyronamine (T1AM), an endogenous TH derivative that interacts with specific molecular targets, including trace amine-associated receptor 1 (TAAR1), and induces pro-learning and anti-amnestic effects in mice. Dysregulation of TH signaling has long been hypothesized to play a role in Alzheimer's disease (AD). In the present investigation, we explored the neuroprotective role of T1AM in beta amyloid (Aß)-induced synaptic and behavioral impairment, focusing on the entorhinal cortex (EC), an area that is affected early by AD pathology. Methods: Field potentials were evoked in EC layer II, and long-term potentiation (LTP) was elicited by high frequency stimulation (HFS). T1AM (5 µM) and/or Aß(1-42) (200 nM), were administered for 10 minutes, starting 5 minutes before HFS. Selective TAAR1 agonist RO5166017 (250 nM) and TAAR1 antagonist EPPTB (5 nM) were also used. The electrophysiological experiments were repeated in EC-slices taken from a mouse model of AD (mutant human amyloid precursor protein [mhAPP], J20 line). We also assessed the in vivo effects of T1AM on EC-dependent associative memory deficits, which were detected in mhAPP mice by behavioral evaluations based on the novel-object recognition paradigm. TAAR1 expression was determined by Western blot, whereas T1AM and its metabolite 3-iodothyroacetic acid (TA1) were assayed by high-performance liquid chromatography coupled to mass spectrometry. Results: We demonstrate the presence of endogenous T1AM and TAAR1 in the EC of wild-type and mhAPP mice. Exposure to Aß(1-42) inhibited LTP, and T1AM perfusion (at a concentration of 5 µM, leading to an actual concentration in the perfusion buffer ranging from 44 to 298 nM) restored it, whereas equimolar amounts of 3,5,3'-triiodo-L-thyronine (T3) and TA1 were ineffective. The response to T1AM was abolished by the TAAR1 antagonist EPPTB, whereas it was mimicked by the TAAR1 agonist RO5166017. In the EC of APPJ20 mice, LTP could not be elicited, but it was rescued by T1AM. The intra-cerebro-ventricular administration of T1AM (0.89 µg/kg) also restored recognition memory that was impaired in mhAPP mice. Conclusions: Our results suggest that T1AM and TAAR1 are part of an endogenous system that can be modulated to prevent synaptic and behavioral deficits associated with Aß-related toxicity.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Córtex Entorrinal/efeitos dos fármacos , Potenciais Evocados/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Tironinas/farmacologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Córtex Entorrinal/fisiologia , Potenciais Evocados/fisiologia , Camundongos , Camundongos Transgênicos
7.
Front Neurosci ; 13: 991, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31607844

RESUMO

Retinal degeneration 9 (rd9) mice carry a mutation in the retina specific "Retinitis Pigmentosa GTPase Regulator (RPGR)" Open Reading Frame (ORF) 15 gene, located on the X chromosome and represent a rare model of X-linked Retinitis Pigmentosa (XLRP), a common and severe form of retinal degeneration (Wright et al., 2010; Tsang and Sharma, 2018). The rd9 RPGR-ORF15 mutation in mice causes lack of the protein in photoreceptors and a slow degeneration of these cells with consequent decrease in Outer Nuclear Layer (ONL) thickness and amplitude of ERG responses, as previously described (Thompson et al., 2012). However, relative rates of rod and cone photoreceptor loss, as well as secondary alterations occurring in neuronal and non-neuronal retinal cell types of rd9 mutants remain to be assessed. Aim of this study is to extend phenotype analysis of the rd9 mouse retina focusing on changes occurring in cells directly interacting with photoreceptors. To this purpose, first we estimated rod and cone survival and its degree of intraretinal variation over time; then, we studied the morphology of horizontal and bipolar cells and of the retinal pigment epithelium (RPE), extending our observations to glial cell reactivity. We found that in rd9 retinas rod (but not cone) death is the main cause of decrease in ONL thickness and that degeneration shows a high degree of intraretinal variation. Rod loss drives remodeling in the outer retina, with sprouting of second-order neurons of the rod-pathway and relative sparing of cone pathway elements. Remarkably, despite cone survival, functional defects can be clearly detected in ERG recordings in both scotopic and photopic conditions. Moderate levels of Muller cells and microglial reactivity are sided by striking attenuation of staining for RPE tight junctions, suggesting altered integrity of the outer Blood Retina Barrier (BRB). Because of many features resembling slowly progressing photoreceptor degeneration paradigms or early stages of more aggressive forms of RP, the rd9 mouse model can be considered a rare and useful tool to investigate retinal changes associated to a process of photoreceptor death sustained throughout life and to reveal disease biomarkers (e.g., BRB alterations) of human XLRP.

8.
FASEB J ; 33(9): 10177-10192, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31199887

RESUMO

Hallmark of retinitis pigmentosa (RP) is the primary, genetic degeneration of rods followed by secondary loss of cones, caused by still elusive biologic mechanisms. We previously shown that exposure of rd10 mutant mice, modeling autosomal recessive RP, to environmental enrichment (EE), with enhanced motor, sensorial and social stimuli, results into a sensible delay of retinal degeneration and vision loss. Searching for effectors of EE-mediated retinal protection, we performed transcriptome analysis of the retina of rd10 enriched and control mice and found that gene expression at the peaks of rod and cone degeneration is characterized by a strong inflammatory/immune response, which is however measurably lower in enrichment conditions. Treating rd10 mice with dexamethasone during the period of maximum photoreceptors death lowered retinal inflammation and caused a preservation of cones and cone-mediated vision. Our findings indicate a link between retinal inflammation and bystander cone degeneration, reinforcing the notion that cone vision in RP can be preserved using anti-inflammatory approaches.-Guadagni, V., Biagioni, M., Novelli, E., Aretini, P., Mazzanti, C. M., Strettoi, E. Rescuing cones and daylight vision in retinitis pigmentosa mice.


Assuntos
Anti-Inflamatórios/uso terapêutico , Visão de Cores/fisiologia , Dexametasona/uso terapêutico , Células Fotorreceptoras Retinianas Cones/fisiologia , Retinite Pigmentosa/tratamento farmacológico , Animais , Sobrevivência Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/deficiência , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Feminino , Regulação da Expressão Gênica , Ativação de Macrófagos , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/genética , Retinite Pigmentosa/patologia , Transcriptoma , Acuidade Visual
9.
J Comp Neurol ; 527(1): 187-211, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27391320

RESUMO

Ganglion cells (GCs), the retinal output neurons, receive synaptic inputs from bipolar and amacrine cells in the inner plexiform layer (IPL) and send information to the brain nuclei via the optic nerve. Although GCs constitute less than 1% of the total retinal cells, they occur in numerous types and are the first neurons formed during retinal development. Using Brn3a and Brn3b mutant mice in which the alkaline phosphatase gene was knocked-in (Badea et al. [Neuron] 2009;61:852-864; Badea and Nathans [Vision Res] 2011;51:269-279), we studied the general effects after gene removal on the retinal neuropil together with the consequences of lack of development of large numbers of GCs onto the remaining retinal neurons of the same class. We analyzed the morphology, number, and general architecture of various neuronal types presynaptic to GCs, searching for changes secondary to the decrement in the number of their postsynaptic partners, as well as the morphology and distribution of retinal astrocytes, for their strong topographical relation to GCs. We found that, despite GC losses, retinal organization in Brn3 null mice is remarkably similar to that of wild-type controls. J. Comp. Neurol. 527:187-211, 2019. © 2016 Wiley Periodicals, Inc.


Assuntos
Retina/citologia , Células Ganglionares da Retina/citologia , Fator de Transcrição Brn-3A/deficiência , Fator de Transcrição Brn-3B/deficiência , Animais , Proteínas de Homeodomínio , Camundongos , Camundongos Knockout , Retina/metabolismo , Retina/ultraestrutura , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/ultraestrutura
10.
Sci Rep ; 7(1): 5730, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28720880

RESUMO

Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision, followed by decreased daylight sight and progressive visual acuity loss up to legal blindness. Great heterogeneity in nature and function of mutated genes, variety of mutations for each of them, variability in phenotypic appearance and transmission modality contribute to make RP a still incurable disease. Translational research relies on appropriate animal models mimicking the genetic and phenotypic diversity of the human pathology. Here, we provide a systematic, morphological and functional analysis of RhoTvrm4/Rho+ rhodopsin mutant mice, originally described in 2010 and portraying several features of common forms of autosomal dominant RP caused by gain-of-function mutations. These mice undergo photoreceptor degeneration only when exposed briefly to strong, white light and allow controlled timing of induction of rod and cone death, which therefore can be elicited in adult animals, as observed in human RP. The option to control severity and retinal extent of the phenotype by regulating intensity and duration of the inducing light opens possibilities to exploit this model for multiple experimental purposes. Altogether, the unique features of this mutant make it an excellent resource for retinal degeneration research.


Assuntos
Modelos Animais de Doenças , Retina/patologia , Retina/fisiologia , Retinite Pigmentosa/patologia , Rodopsina/genética , Rodopsina/metabolismo , Animais , Sobrevivência Celular/efeitos da radiação , Luz , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação
11.
Biomed Chromatogr ; 31(12)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28621883

RESUMO

Myriocin is a potent inhibitor of serine-palmitoyl-transferase, the first and rate-determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC-MS/MS method to measure myriocin in minute specimens of animal tissue. The chemical analog 14-OH-myriocin was used as the internal standard. The two molecules were extracted from the tissue homogenate by solid-phase extraction, separated by gradient reversed-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection was accomplished by multiple reaction monitoring, employing the most representative transitions, 400@104 and 402@104 for myriocin and 14-OH-myriocin, respectively. The typical limit of detection and lower limit of quantitation of the optimized method were 0.9 pmol/mL (~0.016 pmol injected) and 2.3 pmol/mL, respectively, and the method was linear up to 250 pmol/mL range (r2 = 0.9996). The intra- and between-day repeatability afforded a coefficient of variation ≤7.0%. Applications included quantification of myriocin in mouse lungs after 24 h from administration of ~4 nmol by intra-tracheal delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n = 4) to 11.7 (median; 7.6-22.7 interquartile range (IQR), n = 6) pmol/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.


Assuntos
Ácidos Graxos Monoinsaturados/análise , Ácidos Graxos Monoinsaturados/farmacocinética , Serina C-Palmitoiltransferase/antagonistas & inibidores , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia de Fase Reversa , Ácidos Graxos Monoinsaturados/química , Feminino , Limite de Detecção , Modelos Lineares , Pulmão/química , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Retina/química , Retina/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Distribuição Tecidual
13.
Sci Rep ; 6: 35919, 2016 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-27775019

RESUMO

Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive photoreceptor degeneration. In rodent models of RP, expression of defective genes and retinal degeneration usually manifest during the first weeks of postnatal life, making it difficult to distinguish consequences of primary genetic defects from abnormalities in retinal development. Moreover, mouse eyes are small and not always adequate to test pharmacological and surgical treatments. An inducible paradigm of retinal degeneration potentially extensible to large animals is therefore desirable. Starting from the serendipitous observation that intraocular injections of a Rho GTPase activator, the bacterial toxin Cytotoxic Necrotizing Factor 1 (CNF1), lead to retinal degeneration, we implemented an inducible model recapitulating most of the key features of Retinitis Pigmentosa. The model also unmasks an intrinsic vulnerability of photoreceptors to the mechanism of CNF1 action, indicating still unexplored molecular pathways potentially leading to the death of these cells in inherited forms of retinal degeneration.


Assuntos
Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/toxicidade , Modelos Animais de Doenças , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/toxicidade , Retinite Pigmentosa/induzido quimicamente , Retinite Pigmentosa/patologia , Animais , Camundongos , Retina/patologia
14.
Front Cell Neurosci ; 10: 42, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26924963

RESUMO

The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis.

15.
PLoS One ; 11(2): e0148874, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26881841

RESUMO

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken ß-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.


Assuntos
Terapia Genética , Proteínas de Membrana/biossíntese , Degeneração Retiniana/genética , Síndromes de Usher/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Camundongos , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/patologia , Degeneração Retiniana/terapia , Síndromes de Usher/patologia , Síndromes de Usher/terapia
16.
Prog Retin Eye Res ; 48: 62-81, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26113212

RESUMO

Retinal photoreceptors are highly specialized and performing neurons. Their cellular architecture is exquisitely designed to host a high concentration of molecules involved in light capture, phototransduction, electric and chemical signaling, membrane and molecular turnover, light and dark adaption, network activities etc. Such high efficiency and molecular complexity require a great metabolic demand, altogether conferring to photoreceptors particular susceptibility to external and internal insults, whose occurrence usually precipitate into degeneration of these cells and blindness. In Retinitis Pigmentosa, an impressive number of mutations in genes expressed in the retina and coding for a large varieties of proteins leads to the progressive death of photoreceptors and blindness. Recent advances in molecular tools have greatly facilitated the identification of the underlying genetics and molecular bases of RP leading to the successful implementation of gene therapy for some types of mutations, with visual restoration in human patients. Yet, genetic heterogeneity of RP makes mutation-independent approaches highly desirable, although many obstacles pave the way to general strategies for treating this complex disease, which remains orphan. The review will focus on treatments for RP based on pharmacological tools, choosing, among the many ongoing studies, approaches which rely on strong experimental evidence or rationale. For perspective treatments, new concepts are foreseen to emerge from basic studies elucidating the pathways connecting the primary mutations to photoreceptor death, possibly revealing common molecular targets for drug intervention.


Assuntos
Fatores de Crescimento Neural/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Retinite Pigmentosa/tratamento farmacológico , Animais , Fator Neurotrófico Ciliar/uso terapêutico , Modelos Animais de Doenças , Humanos , Tiorredoxinas/uso terapêutico
17.
Mol Vis ; 20: 1545-56, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25489227

RESUMO

PURPOSE: In human patients and animal models of retinitis pigmentosa (RP), a gradual loss of rod photoreceptors and decline in scotopic vision are the primary manifestations of the disease. Secondary death of cones and gradual, regressive remodeling of the inner retina follow and progress at different speeds according to the underlying genetic defect. In any case, the final outcome is near-blindness without a conclusive cure yet. We recently reported that environmental enrichment (EE), an experimental manipulation based on exposure to enhanced motor, sensory, and social stimulation, when started at birth, exerts clear beneficial effects on a mouse model of RP, by slowing vision loss. The purpose of this study was to investigate in the same mouse the long-term effects of chronic exposure to an EE and assess the outcome of this manipulation on cone survival, inner retinal preservation, and visual behavior. METHODS: Two groups of rd10 mutant mice were maintained in an EE or standard (ST) laboratory conditions up to 1 year of age. Then, retinal preservation was assessed with immunocytochemistry, confocal microscopy examination, cone counts, and electron microscopy of the photoreceptor layer, while visual acuity was tested behaviorally with a Prusky water maze. RESULTS: rd10 mice are a model of autosomal recessive RP with a typical rod-cone, center to the periphery pattern of photoreceptor degeneration. They carry a mutation of the rod-specific phosphodiesterase gene and undergo rod death that peaks at around P24, while cone electroretinogram (ERG) is extinct by P60. We previously showed that early exposure to an EE efficiently delays photoreceptor degeneration in these mutants, extending the time window of cone viability and cone-mediated vision well beyond the phase of maximum rod death. Here we find that a maintained EE can delay the degeneration of cones even in the long term. Confocal and electron microscopy examination of the retinas of the rd10 EE and ST mice at 1 year of age showed major degeneration of the photoreceptor layer in both experimental groups, with small clusters of photoreceptors persisting in the peripheral retina. These vestigial cells were positive for L and M opsins and cone arrestin and represented the residual population of cones. In the retinas of the EE mice, cones were more numerous and less remodeled than in the ST counterparts, albeit virtually devoid of outer segments, as confirmed with electron microscopy (EM) observations. Cone counting in retinal whole mounts showed that rd10 EE mice at 1 year had almost three times as many surviving cones (34,000±4,000) as the ST control mice (12,700±1,800), t test p=0.003. Accordingly, the rd10 EE mice at 1 year of age were still capable of performing the visual water task in photopic conditions, showing a residual visual acuity of 0.138±0 cycles/degree. This ability was virtually absent in the rd10 ST age-matched mice (0.063±0.014), t test, p=0.029. No major differences were detected in the morphology of the neurons of the inner retina between the two experimental groups. CONCLUSIONS: The approaches used to test the effects of an EE were consistent in showing significantly better preservation of cones and measurable visual acuity in 1-year-old rd10 EE mice. We therefore confirm and extend previous findings that showed an EE is an effective, minimally invasive tool for promoting long-lasting retinal protection in experimental models of RP.


Assuntos
Atividade Motora/fisiologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/psicologia , Meio Social , Facilitação Social , Animais , Contagem de Células , Sobrevivência Celular , Eletrorretinografia , Feminino , Expressão Gênica , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Opsinas/genética , Opsinas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinite Pigmentosa/genética , Retinite Pigmentosa/metabolismo , Retinite Pigmentosa/patologia , Acuidade Visual
18.
Eur J Neurosci ; 37(11): 1853-62, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23551187

RESUMO

The prevention of cone loss during retinal degeneration is a major goal of most therapeutic strategies in retinal degenerative diseases. An intriguing issue in the current research in this field is to understand why a genetic mutation that affects rods eventually leads to cone death. The main objective of the present study was to investigate to what extent rescuing rods from degeneration affects the survival of cones and prevents functional impairment of the visual performance. To this purpose, we compared rod and cone viabilities by both ex vivo and in vivo determinations in the rd10 mutant mouse, a validated model of human retinitis pigmentosa. The ex vivo experiments included morphological and biochemical tests, whereas in vivo studies compared the rod-mediated scotopic with the cone-mediated photopic electroretinogram. We also determined the overall visual performance by behaviorally testing the visual acuity (VA). The electroretinogram measurements showed that the kinetics of the photopic response in rd10 mice was slowed down with respect to the age-paired wild-type at a very early stage of the disease, when rods were still present and responsive. We then tested cone viability and function under a pharmacological scheme previously shown to prolong rod survival. The treatment consisted of eye drop administration of myriocin, an inhibitor of the biosynthesis of ceramide, a powerful proapoptotic messenger. The results of biochemical, morphological and functional assays converged to show that, in treated rd10 mice cone photoreceptors, the inner retina and overall visual performance were preserved well after rod death.


Assuntos
Células Fotorreceptoras Retinianas Cones/patologia , Retinite Pigmentosa/fisiopatologia , Acuidade Visual , Animais , Apoptose , Sobrevivência Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Monoinsaturados/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/tratamento farmacológico , Retinite Pigmentosa/patologia
19.
PLoS One ; 7(11): e50726, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209820

RESUMO

Slow, progressive rod degeneration followed by cone death leading to blindness is the pathological signature of all forms of human retinitis pigmentosa (RP). Therapeutic schemes based on intraocular delivery of neuroprotective agents prolong the lifetime of photoreceptors and have reached the stage of clinical trial. The success of these approaches depends upon optimization of chronic supply and appropriate combination of factors. Environmental enrichment (EE), a novel neuroprotective strategy based on enhanced motor, sensory and social stimulation, has already been shown to exert beneficial effects in animal models of various disorders of the CNS, including Alzheimer and Huntington disease. Here we report the results of prolonged exposure of rd10 mice, a mutant strain undergoing progressive photoreceptor degeneration mimicking human RP, to such an enriched environment from birth. By means of microscopy of retinal tissue, electrophysiological recordings, visual behaviour assessment and molecular analysis, we show that EE considerably preserves retinal morphology and physiology as well as visual perception over time in rd10 mutant mice. We find that protective effects of EE are accompanied by increased expression of retinal mRNAs for CNTF and mTOR, both factors known as instrumental to photoreceptor survival. Compared to other rescue approaches used in similar animal models, EE is highly effective, minimally invasive and results into a long-lasting retinal protection. These results open novel perspectives of research pointing to environmental strategies as useful tools to extend photoreceptor survival.


Assuntos
Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/fisiologia , Estimulação Física , Retinite Pigmentosa/terapia , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Fator Neurotrófico Ciliar/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/patologia , Retinite Pigmentosa/metabolismo , Serina-Treonina Quinases TOR/genética
20.
Traffic ; 13(8): 1083-9, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22519601

RESUMO

The widely used botulinum neurotoxin A (BoNT/A) blocks neurotransmission via cleavage of the synaptic protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Recent evidence demonstrating long-distance propagation of SNAP-25 proteolysis has challenged the idea that BoNT/A remains localized to the injection site. However, the extent to which distant neuronal networks are impacted by BoNT/A retrograde trafficking remains unknown. Importantly, no studies have addressed whether SNAP-25 cleavage translates into structural and functional changes in distant intoxicated synapses. Here we show that the BoNT/A injections into the adult rat optic tectum result in SNAP-25 cleavage in retinal neurons two synapses away from the injection site, such as rod bipolar cells and photoreceptors. Retinal endings displaying cleaved SNAP-25 were enlarged and contained an abnormally high number of synaptic vesicles, indicating impaired exocytosis. Tectal injection of BoNT/A in rat pups resulted in appearance of truncated-SNAP-25 in cholinergic amacrine cells. Functional imaging with calcium indicators showed a clear reduction in cholinergic-driven wave activity, demonstrating impairments in neurotransmission. These data provide the first evidence for functional effects of the retrograde trafficking of BoNT/A, and open the possibility of using BoNT/A fragments as drug delivery vehicles targeting the central nervous system.


Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/antagonistas & inibidores , Animais , Sinalização do Cálcio , Ratos , Ratos Long-Evans , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/metabolismo , Neurônios Retinianos/ultraestrutura , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , Proteína 25 Associada a Sinaptossoma/metabolismo
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