Ilaria Barone

Long-term retinal function and structure rescue using capsid mutant AAV8 vector in the rd10 mouse, a model of recessive retinitis pigmentosa.

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β subunit of rod cGMP-phosphodiesterase (PDEβ). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEβ was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEβ expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEβ-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEβ-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration.

Botulinum Neurotoxin A Impairs Neurotransmission Following Retrograde Transynaptic Transport

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.

Neural stem cells modified to express BDNF antagonize trimethyltin-induced neurotoxicity through PI3K/Akt and MAP kinase pathways.

In vitro expansion of neural stem cells (NSC) lentivirally transduced with human BDNF may serve as better cellular source for replacing degenerating neurons in disease, trauma and toxic insults. In this study, we evaluate the functional role of forced BDNF expression by means of NSC (M3GFP‐BDNF) obtained from cerebral cortex of 1‐day‐old mice respect to NSC‐control (M3GFP). We find that M3GFP‐BDNF induced to differentiate significantly accumulate BDNF and undergone to high potassium‐mediated depolarization, show rapid BDNF recycle and activation of Trk receptors signaling. Differentiated M3GFP‐BDNF exhibit neurons and oligodendrocytes with extended processes although quantitative analyses of NSC‐derived cell lineages show none statistical significance between both cell populations. Moreover, those cells show a significant induction of neuronal and oligodendroglial markers by RT‐PCR and Western blot respect to M3GFP, such as βIII‐Tubulin, microtubule associated protein 2 (MAP2), neurofilaments heavy (NF‐H), oligodendroglial myelin glycoprotein (OMG) and some molecules involved in glutamatergic synapse maturation, such as receptors tyrosine kinases (TRKs), post‐synaptic density (PSD‐95) and N‐methyl‐D‐aspartate receptors 2 A/B (NMDA2A/B). After treatment with the neurotoxicant trimethyltin (TMT), differentiated M3GFP‐BDNF exhibit an attenuation of cellular damage which correlates with a significant activation of MAPK and PI3K/Akt signaling and delayed activation of death signals, while on M3GFP, TMT induces a significant reduction of cell survival, neuronal differentiation and concomitant earlier activation of cleaved caspase‐3. We demonstrate that overexpression of BDNF firmly regulate cell survival and differentiation of NSC and protects differentiated NSC against TMT‐induced neurotoxicity through the PI3K/Akt and MAPK signaling pathways. J. Cell. Physiol. 224: 710–721, 2010.

Environmental Enrichment Extends Photoreceptor Survival and Visual Function in a Mouse Model of Retinitis Pigmentosa

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.

Identification of a novel mutation in the polymerase delta 1 (POLD1) gene in a lipodystrophic patient affected by mandibular hypoplasia, deafness, progeroid features (MDPL) syndrome ☆

OBJECTIVE Progressive lipodystrophy is one of the major features of the rare MDPL syndrome. Until now, 9 patients affected by this syndrome have been described and a recent study identified in 4 of them an in-frame deletion (Ser605del) of a single codon in the POLD1 gene. Sequence alterations of the POLD1 gene at different sites have been previously reported in human colorectal and endometrial carcinomas. MATERIALS/METHODS A 48-year-old woman was admitted to our department for the assessment of a previously diagnosed lipodystrophy. She did not report a family history of diabetes or other metabolic disorders. Hypertriglyceridemia was diagnosed incidentally when she was 25years old. At that time she was also diagnosed with sensorineural bilateral hearing loss. At physical examination she presented lipoatrophy affecting nearly the entire body, mandibular hypoplasia, bird-like face, beaked nose, progeroid facial features, with crowded teeth, small mouth and uvula. Abdominal ultrasound showed hepatomegaly and hepatosteatosis. Fat mass index measured with DXA was 4.59kg/m(2), indicating a fat deficit; the oral glucose tolerance test showed an impaired glucose tolerance. RESULTS Sequence analysis of the entire coding region of the POLD1 gene, disclosed a novel heterozygous mutation in exon 13 (R507C). CONCLUSION The MDPL patient herein described harbors a novel mutation in the exonuclease domain of POLD1. This new variant provides further evidence for a role of POLD1 in the pathogenesis of MDPL. The mechanisms that link changes at various sites of the protein with different diseases remain to be clarified.

Complexity of retinal cone bipolar cells.

An open issue of retinal organization and function is the comprehension of the different tasks specifically performed by bipolar cells, the neurons that collect information from photoreceptors in the outer retina and convey the signal to the inner plexiform layer. Particularly interesting is to understand the unique contribution to the visual signal brought by cone bipolar cells, neurons typical of the mammalian retina and especially dedicated to receive synaptic input from cones. In all the species studied so far, it has been shown that cone bipolar cells occur in about ten different types, which form distinct clusters identified with a panel of both classical and modern genetic methods. Reviewed here is current literature illustrating the occurrence of morphological, molecular and architectural features that confer to each bipolar cell type exclusive fingerprints, ultimately predicting the emergence of similarly unique, albeit still partially unraveled, functional properties. Thus, differences among cone bipolar cells lay the ground for the genesis in the outer retina of parallel channels, which convey to the inner retina separate information, among others, about contrast, chromatic features and temporal properties of the visual signal.

The Multifaceted Haptoglobin in the Context of Adipose Tissue and Metabolism

Obesity is a low chronic inflammatory state because several inflammatory factors are increased in obese subjects, this having important implications for the onset of obesity-associated complications. The source of most of these inflammatory molecules is white adipose tissue (WAT), which upon excessive weight gain, becomes infiltrated with macrophages and lymphocytes and undergoes important changes in its gene expression. Haptoglobin (Hp), a typical marker of inflammation in clinical practice, main carrier of free hemoglobin, and long known to be part of the hepatic acute phase response, perfectly sits in the intersection between obesity and inflammation: it is expressed by adipocytes and its abundance in WAT and in plasma positively relates to the degree of adiposity. In the present review, we will analyze causes and consequences of Hp expression and regulation in WAT and how these relate to the obesity/inflammation paradigm and comorbidities.

The antidepressant fluoxetine acts on energy balance and leptin sensitivity via BDNF

Leptin and Brain Derived Neurotrophic Factor (BDNF) pathways are critical players in body weight homeostasis. Noninvasive treatments like environmental stimulation are able to increase response to leptin and induce BDNF expression in the brain. Emerging evidences point to the antidepressant selective serotonin reuptake inhibitor Fluoxetine (FLX) as a drug with effects similar to environmental stimulation. FLX is known to impact on body weight, with mechanisms yet to be elucidated. We herein asked whether FLX affects energy balance, the leptin system and BDNF function. Adult lean male mice chronically treated with FLX showed reduced weight gain, higher energy expenditure, increased sensitivity to acute leptin, increased hypothalamic BDNF expression, associated to changes in white adipose tissue expression typical of “brownization”. In the Ntrk2tm1Ddg/J model, carrying a mutation in the BDNF receptor Tyrosine kinase B (TrkB), these effects are partially or totally reversed. Wild type obese mice treated with FLX showed reduced weight gain, increased energy output, and differently from untreated obese mice, a preserved acute response to leptin in terms of activation of the intracellular leptin transducer STAT3. In conclusion, FLX impacts on energy balance and induces leptin sensitivity and an intact TrkB function is required for these effects to take place.

Lipodystrophy and obesity are associated with decreased number of T cells with regulatory function and pro-inflammatory macrophage phenotype

Background/Objectives:In lipodystrophy (LD) adipose tissue function to store lipids is impaired, leading to metabolic syndrome, similar to that found in obesity. Emerging evidence links dysmetabolism with disorders of the immune system. Our aim is to investigate whether T-cell populations with regulatory function and monocyte-derived macrophages (MDMs) are affected by LD and obesity.Subjects/Methods:Blood was collected from 16 LD, 16 obese (OB, BMI>30 kg m−2) and 16 healthy normal-weight women (CNT). Physical parameters, plasma lipid profile, glucose, HbA1c, leptin levels were determined. Flow cytometry was employed to assess the number of circulating CD4+/CD25hi regulatory T cells (Tregs) and invariant natural killer T (iNKT) cells. Characterization of MDMs included: 1. morphological/oil-Red-O staining analyses to define two morphotypes: lipid laden (LL) and spindle-like (sp) MDM; 2. gene expression studies; 3. use of conditioned medium from MDMs (MDMs CM) on human SGBS cells.Results:As compared to CNT, LD and, to a lesser extent, obesity were associated with reduced Tregs and iNKTs (P<0.001 and P<0.01 for LD and OB, respectively), higher number of LL-MDMs (P<0.001 and P<0.01 for LD and OB, respectively), lower number of sp-MDMs (P<0.001 for both LD and OB), which correlated with increased paracrine stimulation of lipid accumulation in cells (P<0.001 and P<0.01 for LD and OB, respectively). LD MDMs showed decreased and increased expression for anti-inflammatory (IL10 and CD163) and pro-inflammatory (CD68 and CCL20) marker genes, respectively. Analysis of correlation indicated that Tregs are directly related with HDL (P<0.01) and inversely related with LL-MDM (P<0.001) and that LL-MDM are directly related with triglycerides (P<0.01) and oxidized LDL (P<0.01).Conclusions:LD and obesity are associated with changes in the immune system: a significant reduction in the number of T cells with regulatory function and a shift of MDM towards lipid accumulation. Lipid profile of the patients correlates with these changes.