AAV-Mediated Restoration of Dystrophin-Dp71 in the Brain of Dp71-Null Mice: Molecular, Cellular and Behavioral Outcomes.

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood-brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients.

ACOX1-mediated peroxisomal fatty acid oxidation contributes to metabolic reprogramming and survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.

Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy.

We report the formulation, characterization, colloidal stability, and in vitro efficiency of Fisetin nanocrystals stabilized by poloxamer P407. Such nanocrystals present a nanometer scale (148.6 ± 1.1 nm) and a high homogeneity (polydispersity index of 0.17 ± 0.01), with a production yield of 97.0 ± 2.5%. The engineered formulations of nanocrystals suspension (pH of 7.4 ± 0.1), stabilized via steric repulsion, are stable for several days in aqueous environment (Milli Q water, NaCl 10 mM or mannitol 5% w/v), for few days in HEPES buffered saline (HBS) (20 / 150 mM) under sink conditions, and in culture medium. After freeze drying in 5% w/v mannitol, the nanocrystal formulations can be stored at -80 °C for at least 120 days. Drug release experiments displayed a 98.7 ± 5.1% cumulative release over 3 days in HBS. Compared to the free drug, the nanocrystal formulations showed an improved cytotoxicity highlighted by the decrease of the half maximal inhibitory concentration for both murine Lewis lung carcinoma (3LL) and human endothelial (EA.hy926) cell lines. In addition, after incubation with Fisetin nanosuspensions, significant changes in the cell morphology for both cell lines were observed, showing an improved anti-angiogenic effect of nanocrystals formulation compared to the free drug. Overall, Fisetin formulated as nanocrystals showed enhanced biopharmaceutical properties and in vitro activity, offering a wide range of indications for challenging applications in the clinic.

Luminogenic HiBiT Peptide-Based NanoBRET Ligand Binding Assays for Melatonin Receptors.

The two human melatonin receptors MT1 and MT2, which belong to the G protein-coupled receptor (GPCR) family, are important drug targets with approved indications for circadian rhythm- and sleep-related disorders and major depression. Currently, most of the pharmacological studies were performed using [3H]melatonin and 2-[125I]iodomelatonin (2-[125I]-MLT) radioligands. Recently, NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a sensitive, nonradioactive alternative for quantifying GPCR ligand engagement on the surface of living cells in equilibrium and real time. However, developing such assays for the two melatonin receptors depends on the availability of fluorescent tracers, which has been challenging predominantly owing to their narrow ligand entry channel and small ligand binding pocket. Here, we generated a set of melatonergic fluorescent tracers and used NanoBRET to evaluate their engagement with MT1 and MT2 receptors that are genetically fused to an N-terminal luminogenic HiBiT-peptide. We identified several nonselective and subtype-selective tracers. Among the selective tracers, PBI-8238 exhibited high nanomolar affinity to MT1, and PBI-8192 exhibited low nanomolar affinity to MT2. The pharmacological profiles of both tracers were in good agreement with those obtained with the current standard 2-[125I]-MLT radioligand. Molecular docking and mutagenesis studies suggested the binding mode of PBI-8192 in MT2 and its selectivity over MT1. In conclusion, we describe the development of the first nonradioactive, real-time binding assays for melatonin receptors expressed at the cell surface of living cells that are likely to accelerate drug discovery for melatonin receptors.

Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels.

COVID-19 is a complex disease with short- and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. Invasion of the brain by SARS-CoV-2 has been observed in humans and is postulated to be involved in post-COVID state. Brain infection is particularly pronounced in the K18-hACE2 mouse model of COVID-19. Prevention of brain infection in the acute phase of the disease might thus be of therapeutic relevance to prevent long-lasting symptoms of COVID-19. We previously showed that melatonin or two prescribed structural analogs, agomelatine and ramelteon delay the onset of severe clinical symptoms and improve survival of SARS-CoV-2-infected K18-hACE2 mice. Here, we show that treatment of K18-hACE2 mice with melatonin and two melatonin-derived marketed drugs, agomelatine and ramelteon, prevents SARS-CoV-2 entry in the brain, thereby reducing virus-induced damage of small cerebral vessels, immune cell infiltration and brain inflammation. Molecular modeling analyses complemented by experimental studies in cells showed that SARS-CoV-2 entry in endothelial cells is prevented by melatonin binding to an allosteric-binding site on human angiotensin-converting enzyme 2 (ACE2), thus interfering with ACE2 function as an entry receptor for SARS-CoV-2. Our findings open new perspectives for the repurposing of melatonergic drugs and its clinically used analogs in the prevention of brain infection by SARS-CoV-2 and COVID-19-related long-term neurological symptoms.

Therapeutic potential of melatonin and melatonergic drugs on K18-hACE2 mice infected with SARS-CoV-2.

As the COVID-19 pandemic grows, several therapeutic candidates are being tested or undergoing clinical trials. Although prophylactic vaccination against SARS-CoV-2 infection has been shown to be effective, no definitive treatment exists to date in the event of infection. The rapid spread of infection by SARS-CoV-2 and its variants fully warrants the continued evaluation of drug treatments for COVID-19, especially in the context of repurposing of already available and safe drugs. Here, we explored the therapeutic potential of melatonin and melatonergic compounds in attenuating COVID-19 pathogenesis in mice expressing human ACE2 receptor (K18-hACE2), strongly susceptible to SARS-CoV-2 infection. Daily administration of melatonin, agomelatine, or ramelteon delays the occurrence of severe clinical outcome with improvement of survival, especially with high melatonin dose. Although no changes in most lung inflammatory cytokines are observed, treatment with melatonergic compounds limits the exacerbated local lung production of type I and type III interferons, which is likely associated with the observed improved symptoms in treated mice. The promising results from this preclinical study should encourage studies examining the benefits of repurposing melatonergic drugs to treat COVID-19 and related diseases in humans.

Emotional behavior and brain anatomy of the mdx52 mouse model of Duchenne muscular dystrophy.

The exon-52-deleted mdx52 mouse is a critical model of Duchenne muscular dystrophy (DMD), as it features a deletion in a hotspot region of the DMD gene, frequently mutated in patients. Deletion of exon 52 impedes expression of several brain dystrophins (Dp427, Dp260 and Dp140), thus providing a key model for studying the cognitive impairment associated with DMD and testing rescuing strategies. Here, using in vivo magnetic resonance imaging and neurohistology, we found no gross brain abnormalities in mdx52 mice, suggesting that the neural dysfunctions in this model are likely at the level of brain cellular functionalities. Then, we investigated emotional behavior and fear learning performance of mdx52 mice compared to mdx mice that only lack Dp427 to focus on behavioral phenotypes that could be used in future comparative preclinical studies. mdx52 mice displayed enhanced anxiety and a severe impairment in learning an amygdala-dependent Pavlovian association. These replicable behavioral outcome measures are reminiscent of the internalizing problems reported in a quarter of DMD patients, and will be useful for preclinical estimation of the efficacy of treatments targeting brain dysfunctions in DMD.

Dp71 contribution to the molecular scaffold anchoring aquaporine-4 channels in brain macroglial cells.

Intellectual disability in Duchenne muscular dystrophy has been associated with the loss of dystrophin-protein 71, Dp71, the main dystrophin-gene product in the adult brain. Dp71 shows major expression in perivascular macroglial endfeet, suggesting that dysfunctional glial mechanisms contribute to cognitive impairments. In the present study, we investigated the molecular alterations induced by a selective loss of Dp71 in mice, using semi-quantitative immunogold analyses in electron microscopy and immunofluorescence confocal analyses in brain sections and purified gliovascular units. In macroglial pericapillary endfeet of the cerebellum and hippocampus, we found a drastic reduction (70%) of the polarized distribution of aquaporin-4 (AQP4) channels, a 50% reduction of ß-dystroglycan, and a complete loss of α1-syntrophin. Interestingly, in the hippocampus and cortex, these effects were not homogeneous: AQP4 and AQP4ex isoforms were mostly lost around capillaries but preserved in large vessels corresponding to pial arteries, penetrating cortical arterioles, and arterioles of the hippocampal fissure, indicating the presence of Dp71-independent pools of AQP4 in these vascular structures. In conclusion, the depletion of Dp71 strongly alters the distribution of AQP4 selectively in macroglial perivascular endfeet surrounding capillaries. This effect likely affects water homeostasis and blood-brain barrier functions and may thus contribute to the synaptic and cognitive defects associated with Dp71 deficiency.

Combined Treatment with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer-PPMO and AAV-U7 Rescues the Severe DMD Phenotype in Mice.

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by an absence of the dystrophin protein, which is essential for muscle fiber integrity. Among the developed therapeutic strategies for DMD, the exon-skipping approach corrects the frameshift and partially restores dystrophin expression. It could be achieved through the use of antisense sequences, such as peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or the small nuclear RNA-U7 carried by an adeno-associated virus (AAV) vector. AAV-based gene therapy approaches have potential for use in DMD treatment but are subject to a major limitation: loss of the AAV genome, necessitating readministration of the vector, which is not currently possible, due to the immunogenicity of the capsid. The PPMO approach requires repeated administrations and results in only weak cardiac dystrophin expression. Here, we evaluated a combination of PPMO- and AAV-based therapy in a mouse model of severe DMD. Striking benefits of this combined therapy were observed in striated muscles, with marked improvements in heart and diaphragm structure and function, with unrivalled extent of survival, opening novel therapeutic perspectives for patients.

Increased creative thinking in narcolepsy.

Some studies suggest a link between creativity and rapid eye movement sleep. Narcolepsy is characterized by falling asleep directly into rapid eye movement sleep, states of dissociated wakefulness and rapid eye movement sleep (cataplexy, hypnagogic hallucinations, sleep paralysis, rapid eye movement sleep behaviour disorder and lucid dreaming) and a high dream recall frequency. Lucid dreaming (the awareness of dreaming while dreaming) has been correlated with creativity. Given their life-long privileged access to rapid eye movement sleep and dreams, we hypothesized that subjects with narcolepsy may have developed high creative abilities. To test this assumption, 185 subjects with narcolepsy and 126 healthy controls were evaluated for their level of creativity with two questionnaires, the Test of Creative Profile and the Creativity Achievement Questionnaire. Creativity was also objectively tested in 30 controls and 30 subjects with narcolepsy using the Evaluation of Potential Creativity test battery, which measures divergent and convergent modes of creative thinking in the graphic and verbal domains, using concrete and abstract problems. Subjects with narcolepsy obtained higher scores than controls on the Test of Creative Profile (mean ± standard deviation: 58.9 ± 9.6 versus 55.1 ± 10, P = 0.001), in the three creative profiles (Innovative, Imaginative and Researcher) and on the Creative Achievement Questionnaire (10.4 ± 25.7 versus 6.4 ± 7.6, P = 0.047). They also performed better than controls on the objective test of creative performance (4.3 ± 1.5 versus 3.7 ± 1.4; P = 0.009). Most symptoms of narcolepsy (including sleepiness, hypnagogic hallucinations, sleep paralysis, lucid dreaming, and rapid eye movement sleep behaviour disorder, but not cataplexy) were associated with higher scores on the Test of Creative Profile. These results highlight a higher creative potential in subjects with narcolepsy and further support a role of rapid eye movement sleep in creativity.

Cerebellar synapse properties and cerebellum-dependent motor and non-motor performance in Dp71-null mice.

Recent emphasis has been placed on the role that cerebellar dysfunctions could have in the genesis of cognitive deficits in Duchenne muscular dystrophy (DMD). However, relevant genotype-phenotype analyses are missing to define whether cerebellar defects underlie the severe cases of intellectual deficiency that have been associated with genetic loss of the smallest product of the dmd gene, the Dp71 dystrophin. To determine for the first time whether Dp71 loss could affect cerebellar physiology and functions, we have used patch-clamp electrophysiological recordings in acute cerebellar slices and a cerebellum-dependent behavioral test battery addressing cerebellum-dependent motor and non-motor functions in Dp71-null transgenic mice. We found that Dp71 deficiency selectively enhances excitatory transmission at glutamatergic synapses formed by climbing fibers (CFs) on Purkinje neurons, but not at those formed by parallel fibers. Altered basal neurotransmission at CFs was associated with impairments in synaptic plasticity and clustering of the scaffolding postsynaptic density protein PSD-95. At the behavioral level, Dp71-null mice showed some improvements in motor coordination and were unimpaired for muscle force, static and dynamic equilibrium, motivation in high-motor demand and synchronization learning. Dp71-null mice displayed altered strategies in goal-oriented navigation tasks, however, suggesting a deficit in the cerebellum-dependent processing of the procedural components of spatial learning, which could contribute to the visuospatial deficits identified in this model. In all, the observed deficits suggest that Dp71 loss alters cerebellar synapse function and cerebellum-dependent navigation strategies without being detrimental for motor functions.