Intravitreal air tamponade after AAV2 subretinal injection modifies retinal EGFP distribution.

The subretinal injection protocol for the only approved retinal gene therapy (voretigene neparvovec-rzyl) includes air tamponade at the end of the procedure, but its effects on the subretinal bleb have not been described. In the present study, we evaluated the distribution of enhanced green fluorescent protein (EGFP) after subretinal injection of AAV2 in non-human primates (NHP) without (group A = 3 eyes) or with (group B = 3 eyes) air tamponade. The retinal expression of EGFP was assessed 1 month after subretinal injection with in vivo fundus photographs and fundus autofluorescence. In group A (without air), EGFP expression was limited to the area of the initial subretinal bleb. In group B (with air), EGFP was expressed in a much wider area. These data show that the buoyant force of air on the retina causes a wide subretinal diffusion of vector, away from the injection site. In the present paper, we discuss the beneficial and deleterious clinical effects of this finding. Whereas subretinal injection is likely to become more common with the coming of new gene therapies, the effects of air tamponade should be explored further to improve efficacy, reproducibility, and safety of the protocol.

Fisioterapia extra-hospitalar durante a pandemia: a visão e o posicionamento dos profissionais / Fisioterapia extrahospitalaria durante la pandemia: la visión y el posicionamiento de los profesionales / Out-of-hospital physical therapy during pandemic: the vision and positioning of professionals

RESUMO A pandemia da COVID-19 provocou momentos atípicos para a população. A fim de promover medidas de controle da disseminação viral, decretos emitidos pelos diversos níveis governamentais indicaram serviços essenciais e não essenciais que poderiam permanecer em funcionamento no Brasil durante determinado período. A fisioterapia extra-hospitalar foi considerada não essencial. O artigo tem como objetivo verificar se a fisioterapia no âmbito da atuação extra-hospitalar é considerada um serviço essencial, na visão de fisioterapeutas, durante os períodos de crises humanitárias, a exemplo da pandemia causada pela COVID-19, e identificar os tipos de procedimentos assistenciais executados. Para isso, foi realizada pesquisa transversal, quantitativa e descritiva do tipo survey com análise descritiva. Realizada por meio de um questionário eletrônico publicado nos sites dos Conselhos Regionais de Fisioterapia e Terapia Ocupacional (Crefito) do Paraná, de Santa Catarina, e do Rio Grande do Sul (respectivamente, Crefito 8, 10 e 5). Nos resultados, foi observado que 78% dos voluntários são do sexo feminino, sendo que 44% estão registrados no Crefito 8, 40% são fisioterapeutas do Crefito 5, 16% são registrados no Crefito 10 e 100% da amostra considerou a fisioterapia extra-hospitalar um serviço essencial. Em relação ao grau de formação, 70% dos profissionais que responderam possuem pós-graduação lato-sensu e 54% atuam em estabelecimentos privados. Durante o decreto de serviços essenciais, 56% dos profissionais não atuaram. Com isso, conclui-se que a fisioterapia extra-hospitalar é essencial em crises pandêmicas. Além de dar início e proporcionar continuidade ao tratamento do paciente, evita visitas desnecessárias aos hospitais.

RESUMEN La pandemia del COVID-19 provocó momentos atípicos para la población de todo el mundo. Con el fin de promover medidas para evitar la propagación del virus por Brasil, los decretos emitidos por los gobiernos indicaron servicios esenciales y no esenciales que podrían permanecer en funcionamiento por determinado periodo. La fisioterapia extrahospitalaria se consideró no esencial. Este artículo tiene por objetivo verificar si los profesionales consideran la fisioterapia en el contexto de la acción extrahospitalaria como un servicio esencial durante periodos de crisis humanitaria, como la pandemia provocada por el COVID-19, así como identificar los tipos de procedimientos asistenciales realizados. Para ello, se realizó una investigación transversal, cuantitativa y descriptiva, del tipo encuesta, con análisis descriptivo. Se usó un cuestionario electrónico publicado en los sitios web de los Consejos Regionales de Fisioterapia y Terapia Ocupacional (Crefito) de Paraná, de Santa Catarina y de Rio Grande do Sul (Crefito 8, 10 y 5, respectivamente). Los resultados indican que el 78% de los voluntarios son mujeres, el 44% están registrados en Crefito 8, el 40% son fisioterapeutas en Crefito 5, el 16% están registrados en Crefito 10, y el 100% de la muestra considera la fisioterapeuta extrahospitalaria un servicio esencial. En cuanto al nivel educativo, el 70% de los profesionales que respondieron tiene posgrado lato-sensu y el 54% trabajan en establecimientos privados. Durante el decreto sobre servicios esenciales, el 56% de los profesionales no trabajaban. Esto permite concluir que la fisioterapia extrahospitalaria es fundamental en crisis pandémicas. Además de iniciar y dar continuidad al tratamiento del paciente, evita que este vaya a hospitales sin necesidad.

ABSTRACT The COVID-19 pandemic caused atypical moments for the population. In Brazil, to promote measures to control viral dissemination, Decrees issued by several government levels indicated the essential and non-essential services that could remain in operation during a certain period. Out-of-hospital physical therapy was considered nonessential. This article aims to verify whether physical therapists consider the practice of out-of-hospital physical therapy as an essential service during periods of humanitarian crises, such as the COVID-19 pandemic. Additionally, we aim to identify the types of care procedures performed during this period. We performed a cross-sectional, quantitative, and descriptive survey with descriptive analysis, conducted by an electronic questionnaire published on the websites of the Regional Councils of Physical Therapy and Occupational Therapy (CREFITO's) of Paraná - CREFITO 8, Santa Catarina - CREFITO 10, and Rio Grande do Sul - CREFITO 5. 78% of the volunteers are female, and 44% are registered in CREFITO 8, 40% are physical therapist of CREFITO 5, 16% are registered in CREFITO 10, and 100% of the sample considered out-of-hospital physical therapy an essential service. Regarding the schooling level, 70% have a graduate degree and 54% work in private establishments. During the decree of essential services, 56% of the professionals did not practice. Out-of-hospital physical therapy is essential in pandemic crises. In addition to providing initiation and continuity to patient treatment, it avoids unnecessary visits to hospitals.

Versatile template-directed synthesis of gold nanocages with a predefined number of windows.

Highly symmetrical gold nanocages can be produced with a controllable number of circular windows of either 2, 3, 4, 6 or 12 via an original fabrication route. The synthetic pathway includes three main stages: the synthesis of silica/polystyrene multipod templates, the regioselective seeded growth of a gold shell on the unmasked part of the silica surface and the development of gold nanocages by dissolving/etching the templates. Electron microscopy and tomography provide evidence of the symmetrical features of the as-obtained nanostructures. The optical properties of nanocages with 4 and 12 windows were measured at the single particle level by spatial modulation spectroscopy and correlated with numerical simulations based on finite-element modeling. The new multi-step synthesis approach reported here also allows the synthesis of rattle-like nanostructures through filling of the nanocages with a guest nano-object. With the potential to adjust the chemical composition, size and geometry of both the guest particle and the host cage, it opens new routes towards the fabrication of hollow nanostructures of high interest for a variety of applications including sensing devices, catalytic reactors and biomedicine.

Rapid synthesis of ultra-long silver nanowires for high performance transparent electrodes.

By using 1,2-propanediol instead of the classic polyol solvent, ethylene glycol, ultra-long silver nanowires are obtained in only 1 h. These nanowires lead to transparent electrodes with a sheet resistance of 5 Ohms per sq at a transparency of 94%, one of the highest figures of merit for nanowire electrodes ever reported.

Increasing Silver Nanowire Network Stability through Small Molecule Passivation.

Silver nanowire (AgNW) transparent electrodes show promise as an alternative to indium tin oxide (ITO). However, these nanowire electrodes degrade in air, leading to significant resistance increases. We show that passivating the nanowire surfaces with small organic molecules of 11-mercaptoundecanoic acid (MUA) does not affect electrode transparency contrary to typical passivation films, and is inexpensive and simple to deposit. The sheet resistance of a 32 nm diameter silver nanowire network coated with MUA increases by only 12% over 120 days when exposed to atmospheric conditions but kept in the dark. The increase is larger when exposed to daylight (588%), but is still nearly two orders of magnitude lower than the resistance increase of unpassivated networks. The difference between the experiments performed under daylight versus the dark exemplifies the importance of testing passivation materials under light exposure.

Outer Plexiform Layer Structures Are Not Altered Following AAV-Mediated Gene Transfer in Healthy Rat Retina.

Ocular gene therapy approaches have been developed for a variety of different diseases. In particular, clinical gene therapy trials for RPE65 mutations, X-linked retinoschisis, and choroideremia have been conducted at different centers in recent years, showing that adeno-associated virus (AAV)-mediated gene therapy is safe, but limitations exist as to the therapeutic benefit and long-term duration of the treatment. The technique of vector delivery to retinal cells relies on subretinal injection of the vector solution, causing a transient retinal detachment. Although retinal detachments are known to cause remodeling of retinal neuronal structures as well as significant cell loss, the possible effects of this short-term therapeutic retinal detachment on retinal structure and circuitry have not yet been studied in detail. In this study, retinal morphology and apoptotic status were examined in healthy rat retinas following AAV-mediated gene transfer via subretinal injection with AAV2/5.CMV.d2GFP or sham injection with fluorescein. Outer plexiform layer (OPL) morphology was assessed by immunohistochemical labeling, laser scanning confocal microscopy, and electron microscopy. The number of synaptic contacts in the OPL was quantified after labeling with structural markers. To assess the apoptotic status, inflammatory and pro-apoptotic markers were tested and TUNEL assay for the detection of apoptotic nuclei was performed. Pre- and postsynaptic structures in the OPL, such as synaptic ribbons or horizontal and bipolar cell processes, did not differ in size or shape in injected versus non-injected areas and control retinas. Absolute numbers of synaptic ribbons were not altered. No signs of relevant gliosis were detected. TUNEL labeling of retinal cells did not vary between injected and non-injected areas, and apoptosis-inducing factor was not delocalized to the nucleus in transduced areas. The neuronal circuits in the OPL of healthy rat retinas undergoing AAV-mediated gene transfer were not altered by the temporary retinal detachment caused by subretinal injection, the presence of viral particles, or the expression of green fluorescent protein as a transgene. This observation likely requires further investigations in the dog model for RPE65 deficiency in order to determine the impact of RPE65 transgene expression on diseased retinas in animals and men.

Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates.

Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons.

Human Endogenous Retrovirus and Neuroinflammation in Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND: Human endogenous retroviruses HERV-W encode a pro-inflammatory protein, named MSRV-Env from its original identification in Multiple Sclerosis. Though not detected in various neurological controls, MSRV-Env was found in patients with chronic inflammatory demyelinating polyradiculoneuropathies (CIDPs). This study investigated the expression of MSRV in CIDP and evaluated relevant MSRV-Env pathogenic effects. METHODS: 50 CIDP patients, 19 other neurological controls (ONDs) and 65 healthy blood donors (HBDs) were recruited from two different countries. MSRV-env and -pol transcripts, IL6 and CXCL10 levels were quantified from blood samples. MSRV-Env immunohistology was performed in distal sensory nerves from CIDP and neurological controls biopsies. MSRV-Env pathogenic effects and mode of action were assayed in cultured primary human Schwann cells (HSCs). FINDINGS: In both cohorts, MSRV-env and -pol transcripts, IL6 positivity prevalence and CXCL10 levels were significantly elevated in CIDP patients when compared to HBDs and ONDs (statistically significant in all comparisons). MSRV-Env protein was detected in Schwann cells in 5/7 CIDP biopsies. HSC exposed to or transfected with MSRV-env presented a strong increase of IL6 and CXCL10 transcripts and protein secretion. These pathogenic effects on HSC were inhibited by GNbAC1, a highly specific and neutralizing humanized monoclonal antibody targeting MSRV-Env. INTERPRETATION: The present study showed that MSRV-Env may trigger the release of critical immune mediators proposed as instrumental factors involved in the pathophysiology of CIDP. Significant MSRV-Env expression was detected in a significant proportion of patients with CIDP, in which it may play a role according to its presently observed effects on Schwann cells along with previously known effects on immune cells. Experimental results also suggest that a biomarker-driven therapeutic strategy targeting this protein with a neutralizing antibody such as GNbAC1 may offer new perspectives for treating CIDP patients with positive detection of MSRV-Env expression. FUNDING: Geneuro-Innovation, France.

MSRV envelope protein is a potent, endogenous and pathogenic agonist of human toll-like receptor 4: Relevance of GNbAC1 in multiple sclerosis treatment.

Multiple sclerosis associated retrovirus envelope protein (MSRV-Env) was repeatedly detected in brain lesions and blood of multiple sclerosis (MS) patients. We performed the first pharmacological characterisation of MSRV-Env on recombinant and native human TLR4. MSRV-Env is a full and highly potent TLR4 agonist of endogenous origin. MSRV-Env induces TLR4-dependent pro-inflammatory stimulation of immune cells in vitro and in vivo, and impairs oligodendrocytes precursor cells differentiation to myelinating oligodendrocytes. MSRV-Env may play a role in chronic inflammation and impaired remyelination in MS. GNbAC1, a selective monoclonal antibody, antagonizes MSRV-Env pathogenic effects and represents an innovative therapeutic approach of MS.

AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6ß-deficient Dogs.

We previously reported that subretinal injection of AAV2/5 RK.cpde6ß allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.

Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates.

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.

Immuno-histochemical analysis of rod and cone reaction to RPE65 deficiency in the inferior and superior canine retina.

Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.

Successful gene therapy in the RPGRIP1-deficient dog: a large model of cone-rod dystrophy.

For the development of new therapies, proof-of-concept studies in large animal models that share clinical features with their human counterparts represent a pivotal step. For inherited retinal dystrophies primarily involving photoreceptor cells, the efficacy of gene therapy has been demonstrated in canine models of stationary cone dystrophies and progressive rod-cone dystrophies but not in large models of progressive cone-rod dystrophies, another important cause of blindness. To address the last issue, we evaluated gene therapy in the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)-deficient dog, a model exhibiting a severe cone-rod dystrophy similar to that seen in humans. Subretinal injection of AAV5 (n = 5) or AAV8 (n = 2) encoding the canine Rpgrip1 improved photoreceptor survival in transduced areas of treated retinas. Cone function was significantly and stably rescued in all treated eyes (18-72% of those recorded in normal eyes) up to 24 months postinjection. Rod function was also preserved (22-29% of baseline function) in four of the five treated dogs up to 24 months postinjection. No detectable rod function remained in untreated contralateral eyes. More importantly, treatment preserved bright- and dim-light vision. Efficacy of gene therapy in this large animal model of cone-rod dystrophy provides great promise for human treatment.

The DNA copy number of human endogenous retrovirus-W (MSRV-type) is increased in multiple sclerosis patients and is influenced by gender and disease severity.

BACKGROUND: Multiple sclerosis is an autoimmune disease more prevalent in women than in men. Multiple Sclerosis Associated Retrovirus element (MSRV) is a member of type-W endogenous retrovirus family (HERV-W), known to be associated to MS. Most HERVs are unable to replicate but MSRV expression associated with reverse-transcriptase activity in MS would explain reported DNA copy number increase in MS patients. A potential link between HERV-W copies on chromosome X and gender differential prevalence has been suggested. The present study addresses MSRV-type DNA load in relation with the gender differences and clinical status in MS and healthy controls. RESULTS: 178 MS patients (62.9% women) and 124 controls (56.5% women) were included. MSRV env load (copies/pg of DNA) was analyzed by real time qPCR with specific primers and probe for its env gene, in DNA from peripheral blood mononuclear cells (PBMCs). MSRV load was more elevated in MS patients than in controls (p = 4.15e-7). MS women presented higher MSRV load than control women (p = 0.009) and MS men also had higher load than control men (p = 2.77e-6). Besides, women had higher levels than men, both among patients (p = 0.007) and controls (p = 1.24e-6). Concordantly, EDSS and MSSS scores were higher among female patients with an elevated MSRV load (p = 0.03 and p = 0.04, respectively). CONCLUSIONS: MSRV increases its copy number in PBMC of MS patients and particularly in women with high clinical scores. This may explain causes underlying the higher prevalence of MS in women. The association with the clinical severity calls for further investigations on MSRV load in PBMCs as a biomarker for MS.

Restoration of vision in the pde6ß-deficient dog, a large animal model of rod-cone dystrophy.

Defects in the ß subunit of rod cGMP phosphodiesterase 6 (PDE6ß) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6ß (n = 4) or AAV2/8RK.cpde6ß (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6ß- and AAV2/8RK.cpde6ß-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment.

Tianeptine potentiates AMPA receptors by activating CaMKII and PKA via the p38, p42/44 MAPK and JNK pathways.

Impairments of cellular plasticity appear to underlie the pathophysiology of major depression. Recently, elevated levels of phosphorylated AMPA receptor were implicated in the antidepressant effect of various drugs. Here, we investigated the effects of an antidepressant, Tianeptine, on synaptic function and GluA1 phosphorylation using murine hippocampal slices and in vivo single-unit recordings. Tianeptine, but not imipramine, increased AMPA receptor-mediated neuronal responses both in vitro and in vivo, in a staurosporine-sensitive manner. Paired-pulse ratio was unaltered by Tianeptine, suggesting a postsynaptic site of action. Tianeptine, 10 µM, enhanced the GluA1-dependent initial phase of LTP, whereas 100 µM impaired the latter phases, indicating a critical role of GluA1 subunit phosphorylation in the excitation. Tianeptine rapidly increased the phosphorylation level of Ser(831)-GluA1 and Ser(845)-GluA1. Using H-89 and KN-93, we show that the activation of both PKA and CaMKII is critical in the effect of Tianeptine on AMPA responses. Moreover, the phosphorylation states of Ser(217/221)-MEK and Thr(183)/Tyr(185)-p42MAPK were increased by Tianeptine and specific kinase blockers of the MAPK pathways (PD 98095, SB 203580 and SP600125) prevented the effects of Tianeptine. Overall these data suggest that Tianeptine potentiates several signaling cascades associated with synaptic plasticity and provide further evidence that a major mechanism of action for Tianeptine is to act as an enhancer of glutamate neurotransmission via AMPA receptors.

Identification of protein-protein interactions by surface plasmon resonance followed by mass spectrometry.

Elucidation of the function and meaning of the protein networks can be useful in the understanding of many pathological processes and the identification of new therapeutic targets. This unit describes an approach to discover protein-protein interactions by coupling surface plasmon resonance to mass spectrometry. Briefly, a protein is covalently bound to a sensor chip, which is then exposed to brain extracts injected over the surface via a microfluidic system. This allows the monitoring in real-time of the interactions between the immobilized ligand and the extracts. Interacting proteins from the extracts are then recovered, trypsinized, and identified using mass spectrometry. The data obtained are searched against a sequence database using the Mascot software. To exclude nonspecific interactors, control experiments using blank sensor chips, and/or randomized peptides, are performed. The protocol presented here does not require specific labeling or modification of proteins and can be performed in <4 days.

Neonatal systemic delivery of scAAV9 in rodents and large animals results in gene transfer to RPE cells in the retina.

Systemic delivery of recombinant adeno-associated virus (rAAV) vectors has recently been shown to cross the blood brain barrier in rodents and large animals and to efficiently target cells of the central nervous system. Such approach could be particularly interesting to treat lysosomal storage diseases or neurodegenerative disorders characterized by multiple organs injuries especially neuronal and retinal dysfunctions. However, the ability of rAAV vector to cross the blood retina barrier and to transduce retinal cells after systemic injection has not been precisely determined. In this study, gene transfer was investigated in the retina of neonatal and adult rats after intravenous injection of self-complementary (sc) rAAV serotype 1, 5, 6, 8, and 9 carrying a CMV-driven green fluorescent protein (GFP), by fluorescence fundus photography and histological examination. Neonatal rats injected with scAAV2/9 vector displayed the strongest GFP expression in the retina, within the retinal pigment epithelium (RPE) cells. Retinal tropism of scAAV2/9 vector was further assessed after systemic delivery in large animal models, i.e., dogs and cats. Interestingly, efficient gene transfer was observed in the RPE cells of these two large animal models following neonatal intravenous injection of the vector. The ability of scAAV2/9 to transduce simultaneously neurons in the central nervous system, and RPE cells in the retina, after neonatal systemic delivery, makes this approach potentially interesting for the treatment of infantile neurodegenerative diseases characterized by both neuronal and retinal damages.

Caveolin-1 interacts with alpha-synuclein and mediates toxic actions of cellular alpha-synuclein overexpression.

Caveolin-1 (Cav-1) is a transmembrane protein which clusters proteins and lipids at the cell membrane into a subclass of lipid rafts named caveolae. To increase our understanding about putative functions of Cav-1 in neuronal cells, we used mouse brain extracts and a novel technology coupling surface plasmon resonance to mass spectrometry to find binding partners to Cav-1. An interaction between Cav-1 and alpha-synclein was found and confirmed in reciprocal pulldown experiments. Genetic overexpression of alpha-synclein in mouse neuroblastoma Neuro2A cells (N2A) expectedly decreased cell survival, but also significantly increased the levels of Cav-1. Furthermore, si-RNA-mediated knockdown of Cav-1 counteracted cell death induced by overexpression of alpha-synuclein. We also used an inhibitor of proteasome (MG132) to induce cell death in a Parkinson's disease context. Cav-1 knockdown had no effect on cell death induced by MG132. Conversely, treating the cells with mevastatin, an inhibitor of cholesterol synthesis, inhibits cell death induced by MG132, but not by alpha synuclein overexpression. It can be concluded that Cav-1 may play a functional role in neuronal cells by virtue of its physical interaction with alpha-synuclein and regulate alpha synuclein-mediated actions on cell death, processes known to be involved in synucleinopathies including Parkinson's disease.

PAT1 induces cell death signal and SET mislocalization into the cytoplasm by increasing APP/APLP2 at the cell surface.

The cleavage of amyloid precursor protein (APP) by caspases unmasks a domain extending from membrane to caspase cleavage site. This domain induces apoptosis in vitro and in vivo when overexpressed in neurons through the help of an internalization vector. In this model, we previously showed that SET rapidly binds to the internalized domain and is involved in downstream deleterious effects. Under these conditions SET mislocalizes from the nucleus to the cytoplasm, as in Alzheimer's disease (AD). In this report using the same model, we show that PAT1 attaches to the internalized domain earlier than SET and that this binding causes an increase in the levels of APP and APLP2 at the cell surface. Down regulation experiments of PAT1 and of APP and APLP2 show that the increase of the levels of APP and APLP2 at the cell surface triggers the cell death signal and SET mislocalization into the cytoplasm. In the context of AD these data suggest that mislocalization of SET into the cytoplasm may occur downstream of first cell death signal events involving PAT1 protein.