Randomized trial of bilateral gene therapy injection for m.11778G>A MT-ND4 Leber optic neuropathy.

Leber hereditary optic neuropathy (LHON) is an important example of mitochondrial blindness with the m.11778G>A mutation in the MT-ND4 gene being the most common disease-causing mtDNA variant worldwide. The REFLECT phase 3 pivotal study is a randomized, double-masked, placebo-controlled trial investigating the efficacy and safety of bilateral intravitreal injection of lenadogene nolparvovec in patients with a confirmed m.11778G>A mutation, using a recombinant adeno-associated virus vector 2, serotype 2 (rAAV2/2-ND4). The first-affected eye received gene therapy; the fellow (affected/not-yet-affected) eye was randomly injected with gene therapy or placebo. The primary end point was the difference in change from baseline of best-corrected visual acuity (BCVA) in second-affected/not-yet-affected eyes treated with lenadogene nolparvovec versus placebo at 1.5 years post-treatment, expressed in logarithm of the minimal angle of resolution (LogMAR). Forty-eight patients were treated bilaterally and 50 unilaterally. At 1.5 years, the change from baseline in BCVA was not statistically different between second-affected/not-yet-affected eyes receiving lenadogene nolparvovec and placebo (primary end point). A statistically significant improvement in BCVA was reported from baseline to 1.5 years in lenadogene nolparvovec-treated eyes: -0.23 LogMAR for the first-affected eyes of bilaterally treated patients (P < 0.01); and -0.15 LogMAR for second-affected/not-yet-affected eyes of bilaterally treated patients and the first-affected eyes of unilaterally treated patients (P < 0.05). The mean improvement in BCVA from nadir to 1.5 years was -0.38 (0.052) LogMAR and -0.33 (0.052) LogMAR in first-affected and second-affected/not-yet-affected eyes treated with lenadogene nolparvovec, respectively (bilateral treatment group). A mean improvement of -0.33 (0.051) LogMAR and -0.26 (0.051) LogMAR was observed in first-affected lenadogene nolparvovec-treated eyes and second-affected/not-yet-affected placebo-treated eyes, respectively (unilateral treatment group). The proportion of patients with one or both eyes on-chart at 1.5 years was 85.4% and 72.0% for bilaterally and unilaterally treated patients, respectively. The gene therapy was well tolerated, with no systemic issues. Intraocular inflammation, which was mostly mild and well controlled with topical corticosteroids, occurred in 70.7% of lenadogene nolparvovec-treated eyes versus 10.2% of placebo-treated eyes. Among eyes treated with lenadogene nolparvovec, there was no difference in the incidence of intraocular inflammation between bilaterally and unilaterally treated patients. Overall, the REFLECT trial demonstrated an improvement of BCVA in LHON eyes carrying the m.11778G>A mtDNA mutation treated with lenadogene nolparvovec or placebo to a degree not reported in natural history studies and supports an improved benefit/risk profile for bilateral injections of lenadogene nolparvovec relative to unilateral injections.

SCA7 Mouse Cerebellar Pathology Reveals Preferential Downregulation of Key Purkinje Cell-Identity Genes and Shared Disease Signature with SCA1 and SCA2.

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease mainly characterized by motor incoordination because of progressive cerebellar degeneration. SCA7 is caused by polyglutamine expansion in ATXN7, a subunit of the transcriptional coactivator SAGA, which harbors histone modification activities. Polyglutamine expansions in specific proteins are also responsible for SCA1-SCA3, SCA6, and SCA17; however, the converging and diverging pathomechanisms remain poorly understood. Using a new SCA7 knock-in mouse, SCA7140Q/5Q, we analyzed gene expression in the cerebellum and assigned gene deregulation to specific cell types using published datasets. Gene deregulation affects all cerebellar cell types, although at variable degree, and correlates with alterations of SAGA-dependent epigenetic marks. Purkinje cells (PCs) are by far the most affected neurons and show reduced expression of 83 cell-type identity genes, including these critical for their spontaneous firing activity and synaptic functions. PC gene downregulation precedes morphologic alterations, pacemaker dysfunction, and motor incoordination. Strikingly, most PC genes downregulated in SCA7 have also decreased expression in SCA1 and SCA2 mice, revealing converging pathomechanisms and a common disease signature involving cGMP-PKG and phosphatidylinositol signaling pathways and LTD. Our study thus points out molecular targets for therapeutic development, which may prove beneficial for several SCAs. Furthermore, we show that SCA7140Q/5Q males and females exhibit the major disease features observed in patients, including cerebellar damage, cerebral atrophy, peripheral nerves pathology, and photoreceptor dystrophy, which account for progressive impairment of behavior, motor, and visual functions. SCA7140Q/5Q mice represent an accurate model for the investigation of different aspects of SCA7 pathogenesis.SIGNIFICANCE STATEMENT Spinocerebellar ataxia 7 (SCA7) is one of the several forms of inherited SCAs characterized by cerebellar degeneration because of polyglutamine expansion in specific proteins. The ATXN7 involved in SCA7 is a subunit of SAGA transcriptional coactivator complex. To understand the pathomechanisms of SCA7, we determined the cell type-specific gene deregulation in SCA7 mouse cerebellum. We found that the Purkinje cells are the most affected cerebellar cell type and show downregulation of a large subset of neuronal identity genes, critical for their spontaneous firing and synaptic functions. Strikingly, the same Purkinje cell genes are downregulated in mouse models of two other SCAs. Thus, our work reveals a disease signature shared among several SCAs and uncovers potential molecular targets for their treatment.

Polyglutamine-expanded ATXN7 alters a specific epigenetic signature underlying photoreceptor identity gene expression in SCA7 mouse retinopathy.

BACKGROUND: Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder that primarily affects the cerebellum and retina. SCA7 is caused by a polyglutamine expansion in the ATXN7 protein, a subunit of the transcriptional coactivator SAGA that acetylates histone H3 to deposit narrow H3K9ac mark at DNA regulatory elements of active genes. Defective histone acetylation has been presented as a possible cause for gene deregulation in SCA7 mouse models. However, the topography of acetylation defects at the whole genome level and its relationship to changes in gene expression remain to be determined. METHODS: We performed deep RNA-sequencing and chromatin immunoprecipitation coupled to high-throughput sequencing to examine the genome-wide correlation between gene deregulation and alteration of the active transcription marks, e.g. SAGA-related H3K9ac, CBP-related H3K27ac and RNA polymerase II (RNAPII), in a SCA7 mouse retinopathy model. RESULTS: Our analyses revealed that active transcription marks are reduced at most gene promoters in SCA7 retina, while a limited number of genes show changes in expression. We found that SCA7 retinopathy is caused by preferential downregulation of hundreds of highly expressed genes that define morphological and physiological identities of mature photoreceptors. We further uncovered that these photoreceptor genes harbor unusually broad H3K9ac profiles spanning the entire gene bodies and have a low RNAPII pausing. This broad H3K9ac signature co-occurs with other features that delineate superenhancers, including broad H3K27ac, binding sites for photoreceptor specific transcription factors and expression of enhancer-related non-coding RNAs (eRNAs). In SCA7 retina, downregulated photoreceptor genes show decreased H3K9 and H3K27 acetylation and eRNA expression as well as increased RNAPII pausing, suggesting that superenhancer-related features are altered. CONCLUSIONS: Our study thus provides evidence that distinctive epigenetic configurations underlying high expression of cell-type specific genes are preferentially impaired in SCA7, resulting in a defect in the maintenance of identity features of mature photoreceptors. Our results also suggest that continuous SAGA-driven acetylation plays a role in preserving post-mitotic neuronal identity.

In vivo phenotypic and molecular characterization of retinal degeneration in mouse models of three ciliopathies.

Cilia are highly conserved and ubiquitously expressed organelles. Ciliary defects of genetic origins lead to ciliopathies, in which retinal degeneration (RD) is one cardinal clinical feature. In order to efficiently find and design new therapeutic strategies the underlying mechanism of retinal degeneration of three murine model was compared. The rodent models correspond to three emblematic ciliopathies, namely: Bardet-Biedl Syndrome (BBS), Alström Syndrome (ALMS) and CEP290-mediated Leber Congenital Amaurosis (LCA). Scotopic rodent electroretinography (ERG) was used to test the retinal function of mice, Transmitted Electron microscopy (T.E.M) was performed to assess retinal structural defects and real-time PCR for targeted genes was used to monitor the expression levels of the major apoptotic Caspase-related pathways in retinal extracts to identify pathological pathways driving the RD in order to identify potential therapeutic targets. We found that BBS and CEP290-mediated LCA mouse models exhibit perinatal retinal degeneration associated with rhodopsin mislocalization in the photoreceptor and the induction of an Endoplasmic Reticulum (ER) stress. On the other hand, the tested ALMS mouse model, displayed a slower degeneration phenotype, with no Rhodopsin mislocalization nor ER-stress activity. Our data points out that behind the general phenotype of vision loss associated with these ciliopathies, the mechanisms and kinetics of disease progression are different.

Ordering of Saturated and Unsaturated Lipid Membranes near Their Phase Transitions Induced by an Amphiphilic Cyclodextrin and Cholesterol.

When inserted in membranes of dimyristoyl phosphatidylcholine (DMPC), methylated ß-cyclodextrins with one (TrimßMLC) or two (TrimßDLC) lauryl acyl chains grafted onto the hydrophilic cavity exert a "cholesterol-like ordering effect", by straightening the acyl chains in the fluid phase at temperatures near the chain melting transition. This effect may be related to pretransitional events such as the "anomalous swelling" known to occur with saturated phosphatidylcholine membranes. To investigate this model, order profiles and bilayer thicknesses of DMPC and unsaturated 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) membranes containing amphiphilic cyclodextrins or cholesterol were determined by deuterium NMR. The pure lipid membranes display both a qualitatively similar chain ordering upon cooling in the fluid phase, more important at the chain extremity, which gets more pronounced near their fluid-to-gel transitions. Both membranes show a bilayer thickness increase by ∼0.5 Å just above their transition, as observed previously with saturated phosphatidylcholines of various chain lengths. Membrane-insertion of 5% TrimßMLC or cholesterol induces an important ordering of the DMPC acyl chains just above the transition, which is also more pronounced at the chain extremity. There is an additional increase of the bilayer thickness, most probably due to a deep insertion of these amphiphilic molecules, facilitated by increased bilayer softness in the anomalous swelling regime. These effects are more important with TrimßMLC than with cholesterol. By contrast, no enhanced acyl chain ordering was observed when approaching the transition of TrimßMLC-containing POPC membranes, as a possible consequence of an eventual lack of anomalous swelling in unsaturated lipid membranes. Insertion of higher concentrations of TrimßMLC was found to induce a magnetic orientation of the DMPC membranes in the fluid phase with 10% of this derivative, coupled with the appearance of a broad isotropic component when the concentration is raised to 20%. No membrane orientation or isotropic component was detected with TrimßMLC-containing POPC membranes.

Evidence for functional GABAA but not GABAC receptors in mouse cone photoreceptors.

At the first retinal synapse, horizontal cells (HCs) contact both photoreceptor terminals and bipolar cell dendrites, modulating information transfer between these two cell types to enhance spatial contrast and mediate color opponency. The synaptic mechanisms through which these modulations occur are still debated. The initial hypothesis of a GABAergic feedback from HCs to cones has been challenged by pharmacological inconsistencies. Surround antagonism has been demonstrated to occur via a modulation of cone calcium channels through ephaptic signaling and pH changes in the synaptic cleft. GABAergic transmission between HCs and cones has been reported in some lower vertebrates, like the turtle and tiger salamander. In these reports, it was revealed that GABA is released from HCs through reverse transport and target GABA receptors are located at the cone terminals. In mammalian retinas, there is growing evidence that HCs can release GABA through conventional vesicular transmission, acting both on autaptic GABA receptors and on receptors expressed at the dendritic tips of the bipolar cells. The presence of GABA receptors on mammalian cone terminals remains equivocal. Here, we looked specifically for functional GABA receptors in mouse photoreceptors by recording in the whole-cell or amphotericin/gramicidin-perforated patch clamp configurations. Cones could be differentiated from rods through morphological criteria. Local GABA applications evoked a Cl- current in cones but not in rods. It was blocked by the GABAA receptor antagonist bicuculline methiodide and unaffected by the GABAC receptor antagonist TPMPA [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid]. The voltage dependency of the current amplitude was as expected from a direct action of GABA on cone pedicles but not from an indirect modulation of cone currents following the activation of the GABA receptors of HCs. This supports a direct role of GABA released from HCs in the control of cone activity in the mouse retina.

Efficacy and safety of sublingual tablets of house dust mite allergen extracts: Results of a dose-ranging study in an environmental exposure chamber.

BACKGROUND: In a natural field study, sublingual tablets of house dust mite (HDM) allergen extracts (STG320) were efficacious in treating HDM-associated allergic rhinitis. OBJECTIVES: We sought to assess the efficacy and safety of 3 doses of STG320 in an environmental exposure chamber. METHODS: In this randomized, double-blind study, adults with HDM-associated allergic rhinitis were given a daily sublingual tablet containing placebo or STG320 at a dose of 500IR, 300IR, or 100IR (IR, index of reactivity) for 6 months. Participants recorded their rhinitis symptoms during 4-hour HDM EEC challenges at randomization and months 1, 2, 4, and 6. The primary efficacy end point was the change from baseline to end of treatment in the area under the curve of the rhinitis total symptom score (ChBLAUCRTSS 0-4h). Differences from the placebo group were analyzed by analysis of covariance. Adverse events (AEs) and routine safety parameters were recorded. RESULTS: A total of 355 subjects were randomized to 1 of 4 groups: 500IR (n = 93), 300IR (n = 86), 100IR (n = 89), or placebo (n = 87). The least squares mean differences from placebo in ChBLAUCRTSS 0-4h for the 500IR, 300IR, and 100IR groups indicated a dose-dependent effect, with reductions in symptom scores of 33%, 29%, and 20%, respectively. The most frequent AEs were throat irritation and oral pruritus. There were no reports of anaphylaxis or reports consistent with severe laryngopharyngeal disorders and no use of epinephrine. AEs leading to premature discontinuations were more common in the 500IR group. CONCLUSIONS: A dose-dependent effect of sublingual HDM immunotherapy was demonstrated in this environmental exposure chamber study, supporting further development of this treatment.

Tools to Perform Local Dense 3D Reconstruction of Shallow Water Seabed.

Tasks such as distinguishing or identifying individual objects of interest require the production of dense local clouds at the scale of these individual objects of interest. Due to the physical and dynamic properties of an underwater environment, the usual dense matching algorithms must be rethought in order to be adaptive. These properties also imply that the scene must be observed at close range. Classic robotized acquisition systems are oversized for local studies in shallow water while the systematic acquisition of data is not guaranteed with divers. We address these two major issues through a multidisciplinary approach. To efficiently acquire on-demand stereoscopic pairs using simple logistics in small areas of shallow water, we devised an agile light-weight dedicated system which is easy to reproduce. To densely match two views in a reliable way, we devised a reconstruction algorithm that automatically accounts for the dynamics, variability and light absorption of the underwater environment. Field experiments in the Mediterranean Sea were used to assess the results.

Proteomic survey reveals altered energetic patterns and metabolic failure prior to retinal degeneration.

Inherited mutations that lead to misfolding of the visual pigment rhodopsin (Rho) are a prominent cause of photoreceptor neuron (PN) degeneration and blindness. How Rho proteotoxic stress progressively impairs PN viability remains unknown. To identify the pathways that mediate Rho toxicity in PNs, we performed a comprehensive proteomic profiling of retinas from Drosophila transgenics expressing Rh1(P37H), the equivalent of mammalian Rho(P23H), the most common Rho mutation linked to blindness in humans. Profiling of young Rh1(P37H) retinas revealed a coordinated upregulation of energy-producing pathways and attenuation of energy-consuming pathways involving target of rapamycin (TOR) signaling, which was reversed in older retinas at the onset of PN degeneration. We probed the relevance of these metabolic changes to PN survival by using a combination of pharmacological and genetic approaches. Chronic suppression of TOR signaling, using the inhibitor rapamycin, strongly mitigated PN degeneration, indicating that TOR signaling activation by chronic Rh1(P37H) proteotoxic stress is deleterious for PNs. Genetic inactivation of the endoplasmic reticulum stress-induced JNK/TRAF1 axis as well as the APAF-1/caspase-9 axis, activated by damaged mitochondria, dramatically suppressed Rh1(P37H)-induced PN degeneration, identifying the mitochondria as novel mediators of Rh1(P37H) toxicity. We thus propose that chronic Rh1(P37H) proteotoxic stress distorts the energetic profile of PNs leading to metabolic imbalance, mitochondrial failure, and PN degeneration and therapies normalizing metabolic function might be used to alleviate Rh1(P37H) toxicity in the retina. Our study offers a glimpse into the intricate higher order interactions that underlie PN dysfunction and provides a useful resource for identifying other molecular networks that mediate Rho toxicity in PNs.

Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival.

Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance.

Hyperglycemia and circadian disruption lead to retinal dysfunction in a stabilized colony of the fat sand rat Psammomys obesus.

PURPOSE: The Fat Sand Rat (Psammomys obesus) recapitulates several features of human pre-proliferative diabetic retinopathy, but data are restricted to wild animals, incompatible with stringent biomedical research criteria. To overcome this barrier, we characterized retinal changes in a colony of P. obsesus maintained under strictly controlled housing conditions. METHODS: Animals were maintained on low or high caloric energy diets, and raised under either standard (12 h light/12 h dark) or shortened (5 h light/5 h dark) photoperiods. Visual responses were tested by electroretinography, while structural/molecular changes were assayed by immunochemistry and molecular biology (RNAseq and qPCR). RESULTS: Whereas high calorie diet alone did not induce hyperglycemia, coupled with short photoperiod >80 % animals developed severe hyper-insulinemia by 15 weeks, and 16 % animals further developed hyperglycemia. In these groups, electroretinography showed significant declines in visual responses in both hyper-insulinemic and hyperglycemic animals, especially in photopic (cone) responses. Transcriptomics analysis of hyperglycemic compared to low caloric controls revealed major upregulation in pathways involved in glial activation, extracellular matrix remodeling, inflammation, cytokine production, partial ischemic responses and angiogenesis. Western blotting against rhodopsin and cone opsin also showed decreased levels in both groups, overall decreases being greater for cones than rods in hyperglycemic animals. CONCLUSIONS: P. obesus maintained in rigorously monitored captive conditions, albeit showing attenuated responses to dietary overload compared to wild counterparts, nevertheless do develop some retinal features of diabetic retinopathy-like degeneration. Such a colony with known sanitary status opens their broader use for biomedical research.

Dynamic lipid lateral segregation driven by lauryl cyclodextrin interactions at the membrane surface.

Amphiphilic cyclodextrins, with a cholesterol anchor (ßChol) or an aspartic acid moiety esterified by two lauryl acyl chains (ßDLC), were designed to combine the inclusion ability of the cyclodextrin cavity with the carrier properties of model membranes. Their insertion in phosphatidylcholine bilayers induces a marked lateral phase separation into a pure lipid phase and a cyclodextrin-rich phase (LCD), organized as a 2D cyclodextrin network stabilized by intermolecular hydrogen bonds between the saccharide headgroups at the membrane surface (Roux, M.; Perly, B.; Djedaïni-Pilard, F. Self-Assemblies of Amphiphilic Cyclodextrins. Eur. Biophys. J.2007, 36, 861-867). We have replaced the dilauryl anchor by a single lauryl chain grafted onto a leucine residue, giving monolauryl-ß-cyclodextrin (ßMLC), which readily inserts into bilayers of chain-deuterated DMPC-d27. The removal of one lauryl acyl chain leads to a dynamic membrane insertion of this new cyclodextrin derivative, with significant lipid exchange on the deuterium NMR time scale between a loosely packed cyclodextrin-enriched phase (L'CD) and free lipid regions, yielding broadened two-component NMR spectra. Like the LCD phases, the cyclodextrin-enriched L'CD regions remain (partially) fluid below the DMPC-d27 main fluid-to-gel transition but do not undergo a clear transition toward a gel state, as observed at 14.5 °C in the LCD phase induced by the dilauryl derivative. Partially fluid lipids of the ßMLC-induced L'CD phase coexist with pure lipids in the Pß' gel phase with possible exchange between them until all of the lipids undergo a transition toward an Lß' gel state at around 7 °C. Trimethylated monolauryl-ß-cyclodextrins induce only an ordering of the lipid acyl chains just above the main transition, without any lateral phase separation. Similar chain ordering is also observed within the ßMLC-induced L'CD phase as a consequence of the deep membrane insertion of the monolauryl nonmethylated cyclodextrin derivative.

Inactivation of VCP/ter94 suppresses retinal pathology caused by misfolded rhodopsin in Drosophila.

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (Rh(P23H)). Unlike the wild-type Rh, mutant Rh(P23H) exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded Rh(P23H) and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that Rh(P23H) is a substrate of the ER-associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1(P37H) (the equivalent of mammalian Rh(P23H)) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1(P37H) toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1(P37H) and further activated the Ire1/Xbp1 ER stress pathway in the Rh1(P37H) retina. Despite this, Rh1(P37H) flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1(P37H) retina. Pharmacological treatment of Rh1(P37H) flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration.

Fragmentation of DMPC Membranes by a Wedge-Shaped Amphiphilic Cyclodextrin into Bicellar-like Aggregates.

Small bilayer lipid aggregates such as bicelles provide useful isotropic or anisotropic membrane mimetics for structural studies of biological membranes. We have shown previously by deuterium NMR that a wedge-shaped amphiphilic derivative of trimethyl ßcyclodextrin anchored in deuterated DMPC-d27 bilayers through a lauryl acyl chain (TrimßMLC) is able to induce magnetic orientation and fragmentation of the multilamellar membranes. The fragmentation process fully detailed in the present paper is observed with 20% cyclodextrin derivative below 37 °C, where pure TrimßMLC self-assembles in water into large giant micellar structures. After deconvolution of a broad composite 2H NMR isotropic component, we propose a model where the DMPC membranes are progressively disrupted by TrimßMLC into small and large micellar aggregates depending whether they are extracted from the outer or inner layers of the liposomes. Below the fluid-to-gel transition of pure DMPC-d27 membranes (Tc = 21.5 °C), the micellar aggregates vanish progressively until complete extinction at 13 °C, with a probable release of pure TrimßMLC micelles leaving lipid bilayers in the gel phase doped with only a small amount of the cyclodextrin derivative. Bilayer fragmentation between Tc and 13 °C was also observed with 10% and 5% of TrimßMLC, with NMR spectra suggesting possible interactions of micellar aggregates with fluid-like lipids of the Pß' ripple phase. No membrane orientation and fragmentation was detected with unsaturated POPC membranes, which are able to accommodate the insertion of TrimßMLC without important perturbation. The data are discussed in relation to the formation of possible DMPC bicellar aggregates such as those known to occur after insertion of dihexanoylphosphatidylcholine (DHPC). These bicelles are in particular associated with similar deuterium NMR spectra exhibiting identical composite isotropic components which were never characterized before.

Retinal dystrophins and the retinopathy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by X-linked inherited or de novo DMD gene mutations predominantly affecting males who develop early-onset muscle degeneration, severely affecting their quality of life and leading to reduced life expectancy. DMD patients may also develop proliferative retinopathy, cataract, ERG abnormalities, altered contrast sensitivity, color vision losses, and elevated flash detection thresholds during dark adaptation. Depending on the position of the genetic alteration in the large DMD gene, it is associated with a lack of the full-length dystrophin protein possibly with an additional loss of one or several other dystrophins, which are normally transcribed from internal promoters in retina and crystalline lens. During the last decades, the properties of the dystrophins have been characterized in patients with different genetic alterations and in genetic mouse models of DMD. The complex expression pattern of the dystrophins in photoreceptors, Müller glial cells and astrocytes, likely influences synaptic transmission, ionic balance and vascular integrity of the retina. However, the specific function of each retinal dystrophin remains largely unknown. This review describes the current knowledge on dystrophin expression, the putative molecular, structural, and physiological properties of retinal dystrophins, and the main clinical implications associated with the loss of dystrophins in DMD patients and mouse models. Current data and working hypotheses warrant future research on retinal dystrophins to increase our understanding of dystrophin function in the central nervous system in general and to unveil new retinal mechanisms and therapeutic avenues for retinal diseases.

Safety of Lenadogene Nolparvovec Gene Therapy Over 5 Years in 189 Patients With Leber Hereditary Optic Neuropathy.

PURPOSE: To evaluate the safety profile of lenadogene nolparvovec (Lumevoq) in patients with Leber hereditary optic neuropathy. DESIGN: Pooled analysis of safety data from 5 clinical studies. METHODS: A total of 189 patients received single unilateral or bilateral intravitreal injections of a recombinant adeno-associated virus 2 (rAAV2/2) vector encoding the human wild-type ND4 gene. Adverse events (AEs) were collected throughout the studies, up to 5 years. Intraocular inflammation and increased intraocular pressure (IOP) were ocular AEs of special interest. Other assessments included ocular examinations, vector bio-dissemination, and systemic immune responses against rAAV2/2. RESULTS: Almost all patients (95.2%) received 9 × 1010 viral genomes and 87.8% had at least 2 years of follow-up. Most patients (75.1%) experienced at least one systemic AE, but systemic treatment-related AEs occurred in 3 patients; none were serious. Intraocular inflammation was reported in 75.6% of lenadogene nolparvovec-treated eyes. Almost all intraocular inflammations occurred in the anterior chamber (58.8%) or in the vitreous (40.3%), and were of mild (90.3%) or moderate (8.8%) intensity; most resolved with topical corticosteroids alone. All IOP increases were mild to moderate in intensity. No AE led to study discontinuation. Bio-dissemination of lenadogene nolparvovec and systemic immune response were limited. The safety profile was comparable for patients treated bilaterally and unilaterally. CONCLUSIONS: Lenadogene nolparvovec had a good overall safety profile with excellent systemic tolerability, consistent with limited bio-dissemination. The product was well tolerated, with mostly mild ocular side effects responsive to conventional ophthalmologic treatments.

Indirect Comparison of Lenadogene Nolparvovec Gene Therapy Versus Natural History in Patients with Leber Hereditary Optic Neuropathy Carrying the m.11778G>A MT-ND4 Mutation.

INTRODUCTION: Lenadogene nolparvovec is a promising novel gene therapy for patients with Leber hereditary optic neuropathy (LHON) carrying the m.11778G>A ND4 mutation (MT-ND4). A previous pooled analysis of phase 3 studies showed an improvement in visual acuity of patients injected with lenadogene nolparvovec compared to natural history. Here, we report updated results by incorporating data from the latest phase 3 trial REFLECT in the pool, increasing the number of treated patients from 76 to 174. METHODS: The visual acuity of 174 MT-ND4-carrying patients with LHON injected in one or both eyes with lenadogene nolparvovec from four pooled phase 3 studies (REVERSE, RESCUE and their long-term extension trial RESTORE; and REFLECT trial) was compared to the spontaneous evolution of an external control group of 208 matched patients from 11 natural history studies. RESULTS: Treated patients showed a clinically relevant and sustained improvement in their visual acuity when compared to natural history. Mean improvement versus natural history was - 0.30 logMAR (+ 15 ETDRS letters equivalent) at last observation (P < 0.01) with a maximal follow-up of 3.9 years after injection. Most treated eyes were on-chart as compared to less than half of natural history eyes at 48 months after vision loss (89.6% versus 48.1%; P < 0.01) and at last observation (76.1% versus 44.4%; P < 0.01). When we adjusted for covariates of interest (gender, age of onset, ethnicity, and duration of follow-up), the estimated mean gain was - 0.43 logMAR (+ 21.5 ETDRS letters equivalent) versus natural history at last observation (P < 0.0001). Treatment effect was consistent across all phase 3 clinical trials. Analyses from REFLECT suggest a larger treatment effect in patients receiving bilateral injection compared to unilateral injection. CONCLUSION: The efficacy of lenadogene nolparvovec in improving visual acuity in MT-ND4 LHON was confirmed in a large cohort of patients, compared to the spontaneous natural history decline. Bilateral injection of gene therapy may offer added benefits over unilateral injection. TRIAL REGISTRATION NUMBERS: NCT02652780 (REVERSE); NCT02652767 (RESCUE); NCT03406104 (RESTORE); NCT03293524 (REFLECT); NCT03295071 (REALITY).

Mutations in Lama1 disrupt retinal vascular development and inner limiting membrane formation.

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.

Mammalian retinal horizontal cells are unconventional GABAergic neurons.

Lateral interactions at the first retinal synapse have been initially proposed to involve GABA by transporter-mediated release from horizontal cells, onto GABA(A) receptors expressed on cone photoreceptor terminals and/or bipolar cell dendrites. However, in the mammalian retina, horizontal cells do not seem to contain GABA systematically or to express membrane GABA transporters. We here report that mouse retinal horizontal cells express GAD65 and/or GAD67 mRNA, and were weakly but consistently immunostained for GAD65/67. While GABA was readily detected after intracardiac perfusion, it was lost during classical preparation for histology or electrophysiology. It could not be restored by incubation in a GABA-containing medium, confirming the absence of membrane GABA transporters in these cells. However, GABA was synthesized de novo from glutamate or glutamine, upon addition of pyridoxal 5'-phosphate, a cofactor of GAD65/67. Mouse horizontal cells are thus atypical GABAergic neurons, with no functional GABA uptake, but a glutamate and/or glutamine transport system allowing GABA synthesis, probably depending physiologically from glutamate released by photoreceptors. Our results suggest that the role of GABA in lateral inhibition may have been underestimated, at least in mammals, and that tissue pre-incubation with glutamine and pyridoxal 5'-phosphate should yield a more precise estimate of outer retinal processing.

Early Miocene stalked crinoids (Echinodermata) from the southern Rhodanian basin (southeastern France). Paleoenvironments and taxonomy.

Using recent samplings and specimens from ancient collections, 14 sites and five species of stalked crinoids have been listed in the Miocene of the southern Rhodanian basin (southeastern France). Three species and two genera are new for science: Papacrinus avignonensis n. gen., n sp. (Balanocrininae), Paraconocrinus rhodanicus n. sp. (Rhizocrinidae) and Gastecrinus vinealis n. gen., n. sp. (Incertae sedis). The identification among the Mediterranean Miocene fauna of the genus Metacrinus, now confined to the Indo-Pacific province, was confirmed by the discovery of brachial ossicles attributed to Metacrinus berthei. The richest and most diversified site was exposed during temporal excavations at the Place du Palais des Papes in Avignon. Four out of the five stalked crinoid species were found in this fossil assemblage in which M. berthei predominates. ?Endoxocrinus gastaldii is associated with M. berthei in several sites. Using dissociated ossicles, differences in quantitative and qualitative characters between these two species are deeply analyzed with their taphonomical, taxonomical and paleoecological consequences. Paleoreliefs and valleys, which had been incised during the Burdigalian, channeled currents. They favored stalked crinoid settlement on various substrates during the late BurdigalianLower Langhian transgression. Comparison with the extant fauna allows us to estimate the depth range of the biotopes with stalked crinoids from 100 to 250 m. These estimates are in agreement with those deduced from other paleontological studies.