Lipid nanoparticle delivery of TALEN mRNA targeting LPA causes gene disruption and plasma lipoprotein(a) reduction in transgenic mice.

Lipoprotein(a), or Lp(a), is encoded by the LPA gene and is a causal genetic risk factor for cardiovascular disease. Individuals with high Lp(a) are at risk for cardiovascular morbidity and are refractory to standard lipid-lowering agents. Lp(a)-lowering therapies currently in clinical development require repetitive dosing, while a gene editing approach presents an opportunity for a single-dose treatment.

Liver lipopolysaccharide binding protein prevents hepatic inflammation in physiological and pathological non-obesogenic conditions.

Lipopolysaccharide binding protein (LBP) knockout mice models are protected against the deleterious effects of major acute inflammation but its possible physiological role has been less well studied. We aimed to evaluate the impact of liver LBP downregulation (using nanoparticles containing siRNA- Lbp) on liver steatosis, inflammation and fibrosis during a standard chow diet (STD), and in pathological non-obesogenic conditions, under a methionine and choline deficient diet (MCD, 5 weeks). Under STD, liver Lbp gene knockdown led to a significant increase in gene expression markers of liver inflammation (Itgax, Tlr4, Ccr2, Ccl2 and Tnf), liver injury (Krt18 and Crp), fibrosis (Col4a1, Col1a2 and Tgfb1), endoplasmic reticulum (ER) stress (Atf6, Hspa5 and Eif2ak3) and protein carbonyl levels.

Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation.

Circulating lipopolysaccharide-binding protein (LBP) is increased in individuals with liver steatosis. We aimed to evaluate the possible impact of liver LBP downregulation using lipid nanoparticle-containing chemically modified LBP small interfering RNA (siRNA) (LNP- UNA-siRNA) on the development of fatty liver. Weekly LNP- UNA-siRNA was administered to mice fed a standard chow diet, a high-fat and high-sucrose diet, and a methionine- and choline-deficient diet (MCD).

Present and future of lipid nanoparticle-mRNA technology in phenylketonuria disease treatment.

Phenylketonuria (PKU) is a metabolic rare disease characterized by a failure of the body to clear out the high levels of Phenylalanine (Phe), leading to devastating neurological defects and growth retardation. PKU was discovered in 1934 by AsbjrØrn FØlling, and even though there have been continuous efforts from the scientific community to find therapeutic approaches to modulate the high levels of phenylalanine found in the body of the PKU patients, an efficient therapy still needs to be developed. Current standard of care includes low phenylalanine diets, but the strict restrictions for patients and families makes it very difficult to adequately being implemented.

Downregulation of peripheral lipopolysaccharide binding protein impacts on perigonadal adipose tissue only in female mice.

Background And Aims: The sexual dimorphism in fat-mass distribution and circulating leptin and insulin levels is well known, influencing the progression of obesity-associated metabolic disease. Here, we aimed to investigate the possible role of lipopolysaccharide-binding protein (LBP) in this sexual dimorphism.

Methods: The relationship between plasma LBP and fat mass was evaluated in 145 subjects.

Lipid nanoparticle delivers phenylalanine ammonia lyase mRNA to the liver leading to catabolism and clearance of phenylalanine in a phenylketonuria mouse model.

Phenylketonuria (PKU) is a genetic disorder affecting around 1 in 12,000 live births (1), caused by a mutation in the phenylalanine hydroxylase (PAH) gene in the liver which facilitates the catabolism of phenylalanine (Phe). Without a functional copy of PAH, levels of Phe in the blood and tissues rise, resulting in potentially life-threatening damage to the central nervous system. (2) Treatment options for PKU are limited, and center around adherence to a strict PKU diet that suffers from poor patient compliance.

Development of an mRNA replacement therapy for phenylketonuria.

Phenylketonuria (PKU) is an inborn error caused by deficiencies in phenylalanine (Phe) metabolism. Mutations in the phenylalanine hydroxylase (PAH) gene are the main cause of the disease whose signature hallmarks of toxically elevated levels of Phe accumulation in plasma and organs such as the brain, result in irreversible intellectual disability. Here, we present a unique approach to treating PKU deficiency by using an mRNA replacement therapy.

Notch-Mediated Epigenetic Regulation of Voltage-Gated Potassium Currents.

Rationale: Ventricular arrhythmias often arise from the Purkinje-myocyte junction and are a leading cause of sudden cardiac death. Notch activation reprograms cardiac myocytes to an induced Purkinje-like state characterized by prolonged action potential duration and expression of Purkinje-enriched genes.

Objective: To understand the mechanism by which canonical Notch signaling causes action potential prolongation.

Notch-independent RBPJ controls angiogenesis in the adult heart.

Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes.

Developmental origin of age-related coronary artery disease.

Aim: Age and injury cause structural and functional changes in coronary artery smooth muscle cells (caSMCs) that influence the pathogenesis of coronary artery disease. Although paracrine signalling is widely believed to drive phenotypic changes in caSMCs, here we show that developmental origin within the fetal epicardium can have a profound effect as well.

Methods And Results: Fluorescent dye and transgene pulse-labelling techniques in mice revealed that the majority of caSMCs are derived from Wt1(+), Gata5-Cre(+) cells that migrate before E12.

Notch activates cell cycle reentry and progression in quiescent cardiomyocytes.

The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell-derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest.

Coronary development is regulated by ATP-dependent SWI/SNF chromatin remodeling component BAF180.

Dissecting the molecular mechanisms that guide the proper development of epicardial cell lineages is critical for understanding the etiology of both congenital and adult forms of human cardiovascular disease. In this study, we describe the function of BAF180, a polybromo protein in ATP-dependent SWI/SNF chromatin remodeling complexes, in coronary development. Ablation of BAF180 leads to impaired epithelial-to-mesenchymal-transition (EMT) and arrested maturation of epicardium around E11.

Nefopam inhibits calcium influx, cGMP formation, and NMDA receptor-dependent neurotoxicity following activation of voltage sensitive calcium channels.

Nefopam hydrochloride is a potent non sedative benzoxazocine analgesic that possesses a profile distinct from that of anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanism remains unclear. We have investigated the actions of nefopam on voltage sensitive calcium channels and calcium-mediated pathways.

Antihistamine terfenadine inhibits calcium influx, cGMP formation, and NMDA receptor-dependent neurotoxicity following activation of L-type voltage sensitive calcium channels.

We have investigated the actions of the H1 receptor antagonist terfenadine on voltage sensitive calcium channels and calcium-mediated pathways. We found that terfenadine prevented N-methyl-D-aspartate (NMDA)-mediated excitotoxicity following stimulation of L-type voltage sensitive calcium channels by the specific agonist BayK8644. The neuroprotective effect of terfenadine was concentration-dependent, 10 and 100 nM terfenadine providing 50 and 100% neuroprotection, respectively.

RNA synthesis-dependent potentiation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated toxicity by antihistamine terfenadine in cultured rat cerebellar neurons.

We have studied the effects of terfenadine on neurotoxicity and elevation of free cytoplasmic Ca2+ levels upon stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in cultured cerebellar neurons. Pre-exposure to terfenadine (5 microM, 5 h) significantly increased neuronal death following specific stimulation of receptors by 100 microM AMPA or by subtoxic concentrations of domoate (8 microM), stimuli that are non-toxic when applied to terfenadine-untreated sister cultures. Terfenadine potentiation was prevented by the transcription inhibitor actinomycin D and was significantly ameliorated by histamine (1 mM).

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity.

Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 microM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate.

Ontogeny of kainate receptor gene expression in the developing rat midbrain and striatum.

Kainate (KA) receptors are a family of ionotropic glutamate receptors, which mediate the excitatory synaptic transmission in various areas of the mammalian CNS. We have studied the expression pattern of the genes encoding for KA receptor subunits (Glur5-1, Glur5-2, Glur6, Glur7, KA1 and KA2) in rat prenatal (E), postnatal and adult ventral mesencephalon (MES) and striatum (STR) and in fetal midbrain primary cultures. Each receptor subunit shows a unique area- and temporal-expression pattern.

Novel effect of nefopam preventing cGMP increase, oxygen radical formation and neuronal death induced by veratridine.

Nefopam hydrochloride is a potent analgesic compound that possesses a profile distinct from that of opiods or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. Here we have used cultured cerebellar neurons to test the hypothesis that nefopam may modulate voltage sensitive sodium channel (VSSC) activity.

Antihistamine terfenadine potentiates NMDA receptor-mediated calcium influx, oxygen radical formation, and neuronal death.

We previously reported that the histamine H1 receptor antagonist terfenadine enhances the excitotoxic response to N-methyl-D-aspartate (NMDA) receptor agonists in cerebellar neurons. Here we investigated whether this unexpected action of terfenadine relates to its antihistamine activity, and which specific events in the signal cascade coupled to NMDA receptors are affected by terfenadine. Low concentrations of NMDA (100 microM) or glutamate (15 microM) that were only slightly (<20%) toxic when added alone, caused extensive cell death in cultures pre-exposed to terfenadine (5 microM) for 5 h.

NMDA receptor dependent and independent components of veratridine toxicity in cultured cerebellar neurons are prevented by nanomolar concentrations of terfenadine.

Exposure of cultured neurons to nanomolar concentrations of terfenadine prevented the NMDA receptor-mediated early appearance (30min.) of toxicity signs induced by the voltage sensitive sodium channel activator veratridine. Terfenadine also provided an histamine-insensitive protection against delayed neurotoxicity by veratridine (24h), occurring independently of NMDA receptor activation, while not protecting from excitotoxicity following direct exposure of neurons to glutamate.

Terfenadine prevents NMDA receptor-dependent and -independent toxicity following sodium channel activation.

Exposure of cultured cerebellar neurons to terfenadine prevented the N-methyl-D-aspartate (NMDA) receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. Delayed neurotoxicity by veratridine (24 h) occurring independently from NMDA receptor activation was also prevented by terfenadine. Terfenadine did not protect from excitotoxicity following direct exposure of neurons to glutamate.

Terfenadine induces toxicity in cultured cerebellar neurons: a role for glutamate receptors.

Exposure of cultured cerebellar neurons to the histamine H1 receptor antagonist terfenadine resulted in neuronal degeneration and death. Terfenadine neurotoxicity was dependent upon concentration and time of exposure. After 2 h exposure, 20 microM terfenadine reduced the number of surviving neurons by 75%, and as low as 10 nM terfenadine induced significant neurotoxicity after 5 days of exposure.

Inhibition of protein phosphatases induces IGF-1-blocked neurotrophin-insensitive neuronal apoptosis.

We have previously described the marine toxin okadaic acid (OKA) to be a potent neurotoxin for cultured rat cerebellar neurons. Here we show that OKA-induced neurodegeneration involves the DNA fragmentation characteristic of apoptosis and is protein synthesis-dependent. DNA fragmentation and neurotoxicity correlated with inhibition of protein phosphatase (PP) 2A rather than PP1 activity.

Epidermal growth factor receptor in adult human dorsal root ganglia.

Transforming growth factor-alpha (TGFalpha) enhances neuronal survival and neurite outgrowth in cultured dorsal root ganglia (DRG) sensory neurons. It binds a membrane protein, denominated epidermal growth factor receptor (EGFr). EGFr has been localized in developing and adult human DRG.

Beta-amyloid precursor protein in human digital skin.

The occurrence and distribution of beta-amyloid precursor protein (beta APP) and of beta-amyloid peptide (beta/A4) was investigated using immunoblotting and immunohistochemical techniques in the digital skin of healthy adult subjects. beta APP-like proteic bands with apparent molecular masses between 55-60 kDa, 100-125 kDa (corresponding to the full-length beta APP isoforms), 145-150 kDa, and 200 kDa were found in pellets and supernatants of whole skin and dermis. The same proteins, except that of approximately 200 kDa, were also found in pellets from the epidermis, whereas epidermic supernatants were unreactive.