Protease-activated receptor-2 : a novel pathogenic pathway in a murine model of osteoarthritis

Osteoarthritis (OA) is a global clinical challenge for which no effective disease modifying agents currently exist. Herein we identify protease-activated receptor-2 (PAR-2) as a novel pathogenic mechanism and potential therapeutic target in OA. Experimental OA was induced in wild-type and PAR-2 deficient mice by sectioning the medial menisco-tibial ligament (MMTL), leading to development of a mild arthropathy. Cartilage degradation and increased subchondral bone formation were assessed as indicators of OA pathology. Four weeks following MMTL section, cartilage erosion and increased subchondral bone formation was evident in wild type mice but substantially reduced in PAR-2 deficient mice. Crucially, therapeutic inhibition of PAR-2 in wild type mice,using either a PAR-2 antagonist or a monoclonal antibody targeting the protease cleavage site of PAR-2, was also equally effective at reducing OA progression in vivo. PAR-2 wasupregulated in chondrocytes of wild-type but not sham-operated mice. Wild type mice showed further joint degradation eight weeks following induction of OA, but PAR-2 deficient mice were still protected. The substantial protection from pathology afforded by PAR-2 deficiency following induction of OA provides proof of concept that PAR-2 has a key role in OA and suggests this receptor as a potential therapeutic target. Osteoarthritis (OA) is a chronic disabling condition currently affecting millions globally 1 with radiological evidence of OA in approximately 80% of the population aged over 65. 2OA is characterised by cartilage degradation and increased subchondral bone formation (osteosclerosis). Despite extensive pathophysiologic investigations, clinical management has not altered significantly and comprises administration of analgesics and non-steroidal anti-inflammatory agents and recourse upon joint failure to arthroplasty. No unifying pathogenetic model exists - suggested hypotheses encompass primary cartilage metabolic dysregulation, enthesial disease together with biomechanical dysregulation. Thus far, no critical checkpoint pathway has been identified that is essential for disease progression and which might by corollary represent a valid, disease-modifying OA therapeutic target. Protease-activated receptor-2 (PAR-2) is a G-protein coupled receptor whose 'tethered' ligand is activated by serine proteases. 3 PAR-2 is present in chondrocytes in cartilage from OA patients 4, and following its activation, matrix metalloproteases (MMPs) are generated. 5 However, these previous observations are associative and do not establish the role of PAR-2 in the pathogenesis of OA. We here sought direct evidence of a causal relationship between PAR-2 expression and cartilage and bone pathology in a murine model of OA

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

We have assessed the impact of a-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of a-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction.Instituto de Investigaciones Bioquímicas de La Plat

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

We have assessed the impact of a-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of a-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction.Instituto de Investigaciones Bioquímicas de La Plat

Expanding the clinical and genetic spectrum of ALPK3 variants: Phenotypes identified in pediatric cardiomyopathy patients and adults with heterozygous variants

Introduction: Biallelic damaging variants in ALPK3, encoding alpha-protein kinase 3, cause pediatric-onset cardiomyopathy with manifestations that are incompletely defined. Methods and Results: We analyzed clinical manifestations of damaging biallelic ALPK3 variants in 19 pediatric patients, including nine previously published cases. Among these, 11 loss-of-function (LoF) variants, seven compound LoF and deleterious missense variants, and one homozygous deleterious missense variant were identified. Among 18 live-born patients, 8 exhibited neonatal dilated cardiomyopathy (44.4%; 95% CI: 21.5%-69.2%) that subsequently transitioned into ventricular hypertrophy. The majority of patients had extracardiac phenotypes, including contractures, scoliosis, cleft palate, and facial dysmorphisms. We observed no association between variant type or location, disease severity, and/or extracardiac manifestations. Myocardial histopathology showed focal cardiomyocyte hypertrophy, subendocardial fibroelastosis in patients under 4 years of age, and myofibrillar disarray in adults. Rare heterozygous ALPK3 variants were also assessed in adult-onset cardiomyopathy patients. Among 1548 Dutch patients referred for initial genetic analyses, we identified 39 individuals with rare heterozygous ALPK3 variants (2.5%; 95% CI: 1.8%-3.4%), including 26 missense and 10 LoF variants. Among 149 U.S. patients without pathogenic variants in 83 cardiomyopathy-related genes, we identified six missense and nine LoF ALPK3 variants (10.1%; 95% CI: 5.7%-16.1%). LoF ALPK3 variants were increased in comparison to matched controls (Dutch cohort, P = 1.6×10−5; U.S. cohort, P = 2.2×10−13). Conclusion: Biallelic damaging ALPK3 variants cause pediatric cardiomyopathy manifested by DCM transitioning to hypertrophy, often with poor contractile function. Additional extracardiac features occur in most patients, including musculoskeletal abnormalities and cleft palate. Heterozygous LoF ALPK3 variants are enriched in adults with cardiomyopathy and may contribute to their cardiomyopathy. Adults with ALPK3 LoF variants therefore warrant evaluations for cardiomyopathy

Expanding the clinical and genetic spectrum of ALPK3 variants: phenotypes identified in pediatric cardiomyopathy patients and adults with heterozygous variants

Introduction Biallelic damaging variants in ALPK3, encoding alpha-protein kinase 3, cause pediatric-onset cardiomyopathy with manifestations that are incompletely defined.Methods and Results We analyzed clinical manifestations of damaging biallelic ALPK3 variants in 19 pediatric patients, including nine previously published cases. Among these, 11 loss-of-function (LoF) variants, seven compound LoF and deleterious missense variants, and one homozygous deleterious missense variant were identified. Among 18 live-born patients, 8 exhibited neonatal dilated cardiomyopathy (44.4%; 95% CI: 21.5%-69.2%) that subsequently transitioned into ventricular hypertrophy. The majority of patients had extracardiac phenotypes, including contractures, scoliosis, cleft palate, and facial dysmorphisms. We observed no association between variant type or location, disease severity, and/or extracardiac manifestations. Myocardial histopathology showed focal cardiomyocyte hypertrophy, subendocardial fibroelastosis in patients under 4 years of age, and myofibrillar disarray in adults.Rare heterozygous ALPK3 variants were also assessed in adult-onset cardiomyopathy patients. Among 1548 Dutch patients referred for initial genetic analyses, we identified 39 individuals with rare heterozygous ALPK3 variants (2.5%; 95% CI: 1.8%3.4%), including 26 missense and 10 LoF variants. Among 149 U.S. patients without pathogenic variants in 83 cardiomyopathy-related genes, we identified six missense and nine LoF ALPK3 variants (10.1%; 95% CI: 5.7%-1 6.1%). LoF ALPK3 variants were increased in comparison to matched controls (Dutch cohort, P = 1.6x10(-5); U.S. cohort, P = 2.2x10(-13)).Conclusion Biallelic damaging ALPK3 variants cause pediatric cardiomyopathy manifested by DCM transitioning to hypertrophy, often with poor contractile function. Additional extracardiac features occur in most patients, including musculoskeletal abnormalities and cleft palate. Heterozygous LoF ALPK3 variants are enriched in adults with cardiomyopathy and may contribute to their cardiomyopathy. Adults with ALPK3 LoF variants therefore warrant evaluations for cardiomyopathy.Genetics of disease, diagnosis and treatmen

Protease-activated receptor-2 (PAR-2): a potential new target in arthritis

Protease-activated receptors (PARs) are a novel family of seven-transmembrane G-protein-coupled receptors. The unique feature of this family is that activation is initiated by cleavage of the N-terminus by serine or other proteases, thereby unmasking a tethered ligand that then interacts with the receptor, leading to activation. PARs have been described in the context of inflammation, and recent evidence indicates a particular role for the second member of this family, PAR-2, in arthritis. Synovial expression of this receptor is greatly upregulated in murine models of arthritis, and both acute and chronic experimental monoarthritis are substantially attenuated in Par2 knockout mice, suggesting a key role for PAR-2 in inflammatory joint disease. These findings translate to inflammatory disease in humans, since PAR-2 expression is upregulated in synovial tissues from patients with rheumatoid arthritis (RA), and appears to be an upstream regulator of proinflammatory cytokine generation, including tumor necrosis factor alpha (TNF-alpha). These findings identify PAR-2 as a new therapeutic target in the management of RA, and the challenge is now to develop potent and selective agents to prevent activation of this receptor