Antiapoptotic Strategies in Retinal Degeneration: a Biochemical and Functional Approach

The sphingolipid ceramide exerts a pro-apoptotic role in variety of cellular and organ systems and increased of de-novo synthesis of ceramide are associated with initiation of cell death. In Retinitis Pigmentosa (RP) photoreceptor death occurs by apoptosis but the individual pathways of this process are unknown. We employed an animal model of RP, the rd10 mutant mouse, to assess the role of ceramide in inherited photoreceptor degeneration. We used Myriocin, a known inhibitor of serine palmitoyltransferase (SPT, the rate-limiting enzyme of ceramide biosynthesis) which was either injected intravitreally in a single dose or administered daily to rd10 mice as eye drops of Solid Lipid Nanoparti- cles (SLNs). Control mice were given intravitreal injections of vehicle alone or unloaded lipid particles, respectively. We found that retinal ceramide levels in rd10 mice double from P14 to P30, the time interval of maximum photoreceptor death in this strain. Intraocular treatment with Myriocin decreases the number of pycnotic photoreceptors in rd10 mice by approximately 50%. Electroretino- gram (ERG) recordings were obtained from animals of various ages chronically treated with Myriocin-SLNs. ERG a-waves persist after P30 in treated mice while these responses are virtually extinct in control littermates. Retinal sec- tions from ERG recorded animals were examined at a confocal microscope to estimate photoreceptor survival. Morphometric analysis of retinas from rd10 mice aged P24 (peak of rod apoptosis) up to P30 showed prolonged survival of photoreceptors in treated animals. This study demonstrates in a mammalian model of RP that it is possible to decrease the rate of apoptotic death of pho- toreceptors in vivo by lowering retinal ceramide levels through inhibition of the de-novo biosynthesis of this molecule. Non-invasive, chronic administrations of nanoparticles loaded with SPT inhibitors are effective in prolonging survival and light responsiveness of photoreceptors

A preclinical model for the ATLL lymphoma subtype with insights into the role of microenvironment in HTLV-1-mediated lymphomagenesis

Abstract \uef7f View references (83) Adult T cell Leukemia/Lymphoma (ATLL) is a mature T cell malignancy associated with Human T cell Leukemia Virus type 1 (HTLV-1) infection. Among its four main clinical subtypes, the prognosis of acute and lymphoma variants remains poor. The long latency (3-6 decades) and low incidence (3-5%) of ATLL imply the involvement of viral and host factors in full-blown malignancy. Despite multiple preclinical and clinical studies, the contribution of the stromal microenvironment in ATLL development is not yet completely unraveled. The aims of this study were to investigate the role of the host microenvironment, and specifically fibroblasts, in ATLL pathogenesis and to propose a murine model for the lymphoma subtype. Here we present evidence that the oncogenic capacity of HTLV-1-immortalized C91/PL cells is enhanced when they are xenotransplanted together with human foreskin fibroblasts (HFF) in immunocompromised BALB/c Rag2-/-\u3b3c -/-mice. Moreover, cell lines derived from a developed lymphoma and their subsequent in vivo passages acquired the stable property to induce aggressive T cell lymphomas. In particular, one of these cell lines, C91/III cells, consistently induced aggressive lymphomas also in NOD/SCID/IL2R\u3b3c KO (NSG) mice. To dissect the mechanisms linked to this enhanced tumorigenic ability, we quantified 45 soluble factors released by these cell lines and found that 21 of them, mainly pro-inflammatory cytokines and chemokines, were significantly increased in C91/III cells compared to the parental C91/PL cells. Moreover, many of the increased factors were also released by human fibroblasts and belonged to the known secretory pattern of ATLL cells. C91/PL cells co-cultured with HFF showed features reminiscent of those observed in C91/III cells, including a similar secretory pattern and a more aggressive behavior in vivo. On the whole, our data provide evidence that fibroblasts, one of the major stromal components, might enhance tumorigenesis of HTLV-1-infected and immortalized T cells, thus throwing light on the role of microenvironment contribution in ATLL pathogenesis. We also propose that the lymphoma induced in NSG mice by injection with C91/III cells represents a new murine preclinical ATLL model that could be adopted to test novel therapeutic interventions for the aggressive lymphoma subtype

Nutraceutical Approach for the Treatment of Retinal Inflammation after Infections

This chapter described the ability of Zika virus, a hemostat-borne flavivirus, to infect retinal pigment epithelium cells and to generate chronic inflammation capable of generating permanent damage in the host that can progress eventually to the onset of pathology related to retinal degeneration. In addition, given the lack of an effective vaccine against ZIRK, the possibility of using as a therapeutic strategy the reduction of inflammatory processes that are established as a result of viral infection through the use of bioactive phytonutrients was analyzed

Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes

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

The bacterial toxin CNF1 as a tool to induce retinal degeneration reminiscent of retinitis pigmentosa.

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 degeneratio

Expression of calcium-binding proteins and selected neuropeptides in the human, chimpanzee, and crab-eating macaque claustrum

The claustrum is present in all mammalian species examined so far and its morphology, chemoarchitecture, physiology, phylogenesis and ontogenesis are still a matter of debate. Several morphologically distinct types of immunostained cells were described in different mammalian species. To date, a comparative study on the neurochemical organization of the human and non-human primates claustrum has not been fully described yet, partially due to technical reasons linked to the postmortem sampling interval. The present study analyze the localization and morphology of neurons expressing parvalbumin (PV), calretinin (CR), NPY, and somatostatin (SOM) in the claustrum of man (# 5), chimpanzee (# 1) and crab-eating monkey (# 3). Immunoreactivity for the used markers was observed in neuronal cell bodies and processes distributed throughout the anterior-posterior extent of human, chimpanzee and macaque claustrum. Both CR- and PV-immunoreactive (ir) neurons were mostly localized in the central and ventral region of the claustrum of the three species while SOM- and NPY-ir neurons seemed to be equally distributed throughout the ventral-dorsal extent. In the chimpanzee claustrum SOM-ir elements were not observed. No co-localization of PV with CR was found, thus suggesting the existence of two non-overlapping populations of PV and CR-ir interneurons. The expression of most proteins (CR, PV, NPY), was similar in all species. The only exception was the absence of SOM-ir elements in the claustrum of the chimpanzee, likely due to species specific variability. Our data suggest a possible common structural organization shared with the adjacent insular region, a further element that emphasizes a possible common ontogeny of the claustrum and the neocortex

Retinal defects in mice lacking the autism-associated gene Engrailed-2

Defective cortical processing of visual stimuli and altered retinal function have been described in autism spectrum disorder (ASD)patients. In keeping with these findings, anatomical and functional defects have been found in the visual cortex and retina of mice bearing mutations for ASD-associated genes. Here we sought to investigate the anatomy and function of the adult retina of Engrailed 2 knockout (En2 −/− )mice, a model for ASD. Our results showed that En2 is expressed in all three nuclear layers of the adult retina. When compared to age-matched En2 +/+ controls, En2 −/− adult retinas showed a significant decrease in the number of calbindin + horizontal cells, and a significant increase in calbindin + amacrine/ganglion cells. The total number of ganglion cells was not altered in the adult En2 −/− retina, as shown by Brn3a + cell counts. In addition, En2 −/− adult mice showed a significant reduction of photoreceptor (rhodopsin)and bipolar cell (Pcp2, PKCα)markers. Functional defects were also present in the retina of En2 mutants, as indicated by electroretinogram recordings showing a significant reduction in both a-wave and b-wave amplitude in En2 −/− mice as compared to controls. These data show for the first time that anatomical and functional defects are present in the retina of the En2 ASD mouse model

A Nutraceutical Strategy to Slowing Down the Progression of Cone Death in an Animal Model of Retinitis Pigmentosa

Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by progressive degeneration of the visual cells and abnormalities in retinal pigment epithelium, the vision is lost slowly, and the final outcome is total blindness. RP primarily affects rods, but cones can also be affected as a secondary effect. Photoreceptor cell death is usually triggered by apoptosis, however the molecular mechanisms linking the rod degeneration to the secondary cone death are poorly understood. Possible causes of the secondary cone death are oxidative stress and/ or the release of toxic factors from dying rods. The aim of this study is to analyze the effect of nutraceutical molecules with antioxidant properties, on the progression of the disease in an established animal model of RP, and rd10 mice. We show that chronic treatment per os with a flavanone (naringenin) or a flavonol (quercetin) present in citrus fruits, grapes and apples, preserves retinal morphology, and ameliorates functionality. These actions are associated with a significant reduction of stress-oxidative markers, such as the detoxifying enzymes Sod1 and Sod2. In addition, naringenin and quercetin treatment reduces the levels of acrolein staining associated with a reduction of ROS in the cellular environment. The study demonstrates the beneficial effects of naringenin and quercetin, two molecules that possess antioxidant properties, limiting neurodegeneration, and thus preventing cone damage

TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit

Photoreceptors rely upon highly specialized synapses to efficiently transmit signals to multiple postsynaptic targets. Calcium influx in the presynaptic terminal is mediated by voltage-gated calcium channels (VGCC). This event triggers neurotransmitter release, but also gates calcium-activated chloride channels (TMEM), which in turn regulate VGCC activity. In order to investigate the relationship between VGCC and TMEM channels, we analyzed the retina of wild type (WT) and Cacna2d4 mutant mice, in which the VGCC auxiliary a2d4 subunit carries a nonsense mutation, disrupting the normal channel function. Synaptic terminals of mutant photoreceptors are disarranged and synaptic proteins as well as TMEM16A channels lose their characteristic localization. In parallel, calcium-activated chloride currents are impaired in rods, despite unaltered TMEM16A protein levels. Co-immunoprecipitation revealed the interaction between VGCC and TMEM16A channels in the retina. Heterologous expression of these channels in tsA-201 cells showed that TMEM16A associates with the CaV1.4 subunit, and the association persists upon expression of the mutant a2d4 subunit. Collectively, our experiments show association between TMEM16A and the a1 subunit of VGCC. Close proximity of these channels allows optimal function of the photoreceptor synaptic terminal under physiological conditions, but also makes TMEM16A channels susceptible to changes occurring to calcium channels

Attività biologica e studi di docking di derivati bis-ammidici dell'acido 5,6- diidrossiindolo-2-carbossilico come inibitori dell'enzima HIV-1 integrasi

In un precedente studio, nel tentativo di identificare nuovi inibitori dell'HIV-1 integrasi, è stata da noi considerata la modifica strutturale dell'estere feniletilico dell'acido caffeico I (CAPE), il primo prodotto naturale inibitore dell'IN identificato, incorporando il legame vinilico del CAPE all'interno di una struttura rigida costituita dall'anello pirrolico. In questa comunicazione, a completamento dello studio svolto, noi presentiamo i dati relativi all'attività  inibitoria enzimatica per i derivati IIIe-i, lo studio SAR per tutti i derivati ed uno studio di docking nel sito attivo dell'HIV-1 IN eseguito al fine di investigare le possibili interazioni tra i ligandi e gli a.a. del sito catalitico