Human HPSE2 gene transfer ameliorates bladder pathophysiology in a mutant mouse model of urofacial syndrome.

Rare early-onset lower urinary tract disorders include defects of functional maturation of the bladder. Current treatments do not target the primary pathobiology of these diseases. Some have a monogenic basis, such as urofacial, or Ochoa, syndrome (UFS). Here, the bladder does not empty fully because of incomplete relaxation of its outflow tract, and subsequent urosepsis can cause kidney failure. UFS is associated with biallelic variants of HPSE2, encoding heparanase-2. This protein is detected in pelvic ganglia, autonomic relay stations that innervate the bladder and control voiding. Bladder outflow tracts of Hpse2 mutant mice display impaired neurogenic relaxation. We hypothesized that HPSE2 gene transfer soon after birth would ameliorate this defect and explored an adeno-associated viral (AAV) vector-based approach. AAV9/HPSE2, carrying human HPSE2 driven by CAG, was administered intravenously into neonatal mice. In the third postnatal week, transgene transduction and expression were sought, and ex vivo myography was undertaken to measure bladder function. In mice administered AAV9/HPSE2, the viral genome was detected in pelvic ganglia. Human HPSE2 was expressed and heparanase-2 became detectable in pelvic ganglia of treated mutant mice. On autopsy, wild-type mice had empty bladders, whereas bladders were uniformly distended in mutant mice, a defect ameliorated by AAV9/HPSE2 treatment. Therapeutically, AAV9/HPSE2 significantly ameliorated impaired neurogenic relaxation of Hpse2 mutant bladder outflow tracts. Impaired neurogenic contractility of mutant detrusor smooth muscle was also significantly improved. These results constitute first steps towards curing UFS, a clinically devastating genetic disease featuring a bladder autonomic neuropathy.

Experimental long-term diabetes mellitus alters the transcriptome and biomechanical properties of the rat urinary bladder.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease and diabetic nephropathy is widely studied. In contrast, the pathobiology of diabetic urinary bladder disease is less understood despite dysfunctional voiding being common in DM. We hypothesised that diabetic cystopathy has a characteristic molecular signature. We therefore studied bladders of hyperglycaemic and polyuric rats with streptozotocin (STZ)-induced DM. Sixteen weeks after induction of DM, as assessed by RNA arrays, wide-ranging changes of gene expression occurred in DM bladders over and above those induced in bladders of non-hyperglycaemic rats with sucrose-induced polyuria. The altered transcripts included those coding for extracellular matrix regulators and neural molecules. Changes in key genes deregulated in DM rat bladders were also detected in db/db mouse bladders. In DM rat bladders there was reduced birefringent collagen between detrusor muscle bundles, and atomic force microscopy showed a significant reduction in tissue stiffness; neither change was found in bladders of sucrose-treated rats. Thus, altered extracellular matrix with reduced tissue rigidity may contribute to voiding dysfunction in people with long-term DM. These results serve as an informative stepping stone towards understanding the complex pathobiology of diabetic cystopathy.

Envisioning treating genetically-defined urinary tract malformations with viral vector-mediated gene therapy.

Human urinary tract malformations can cause dysfunctional voiding, urosepsis and kidney failure. Other affected individuals, with severe phenotypes on fetal ultrasound screening, undergo elective termination. Currently, there exist no specific treatments that target the primary biological disease mechanisms that generate these urinary tract malformations. Historically, the pathogenesis of human urinary tract malformations has been obscure. It is now established that some such individuals have defined monogenic causes for their disease. In health, the implicated genes are expressed in either differentiating urinary tract smooth muscle cells, urothelial cells or peripheral nerve cells supplying the bladder. The phenotypes arising from mutations of these genes include megabladder, congenital functional bladder outflow obstruction, and vesicoureteric reflux. We contend that these genetic and molecular insights can now inform the design of novel therapies involving viral vector-mediated gene transfer. Indeed, this technology is being used to treat individuals with early onset monogenic disease outside the urinary tract, such as spinal muscular atrophy. Moreover, it has been contended that human fetal gene therapy, which may be necessary to ameliorate developmental defects, could become a reality in the coming decades. We suggest that viral vector-mediated gene therapies should first be tested in existing mouse models with similar monogenic and anatomical aberrations as found in people with urinary tract malformations. Indeed, gene transfer protocols have been successfully pioneered in newborn and fetal mice to treat non-urinary tract diseases. If similar strategies were successful in animals with urinary tract malformations, this would pave the way for personalized and potentially curative treatments for people with urinary tract malformations.

ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder.

ACTB encodes ß-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, ß-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic ß-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, ß-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.

Heimler Syndrome Is Caused by Hypomorphic Mutations in the Peroxisome-Biogenesis Genes PEX1 and PEX6.

Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. We ascertained eight families affected by HS and, by using a whole-exome sequencing approach, identified biallelic mutations in PEX1 or PEX6 in six of them. Loss-of-function mutations in both genes are known causes of a spectrum of autosomal-recessive peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS-affected family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define HS as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6.

LRIG2 mutations cause urofacial syndrome.

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract.

Regulation of protein kinase D activity in adult myocardium: novel counter-regulatory roles for protein kinase Cepsilon and protein kinase A.

Protein kinase D (PKD) is activated downstream of protein kinase C (PKC) in many cell types, although little is known about the mechanisms that regulate PKD in adult myocardium. Exposure of cultured adult rat ventricular myocytes (ARVM) to phorbol 12-myristate 13-acetate (PMA; 100 nM for 5 min) activated PKD, as evidenced by significantly increased phosphorylation at Ser744/8 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). PKD activation occurred concomitantly with translocation of the enzyme from the cytosolic to the particulate fraction. The role of PKC was confirmed by pretreatment (15 min) of ARVM with the PKC inhibitors GF109203X (1 microM) and Ro31-8220 (1 microM), both of which prevented PKD phosphorylation on subsequent exposure to PMA. Exposure of ARVM to endothelin-1 (ET1; 100 nM for 10 min) also activated PKD by a PKC-dependent mechanism. To determine the PKC isoform(s) involved in the ET1-induced PKD activation, ARVM were infected with adenoviral vectors encoding dominant-negative (DN) mutants of PKCalpha, PKCdelta and PKCepsilon. Expression of DN-PKCalpha and DN-PKCdelta had little effect on ET1-induced PKD activation, whilst this was significantly attenuated by expression of DN-PKCepsilon, indicating that PKCepsilon plays a predominant role in the pertinent ET1 signaling pathway. Intriguingly, prior exposure to the adenylyl cyclase activator forskolin (1 microM for 5 min) or the beta-adrenergic agonist isoprenaline (100 nM for 5 min) markedly attenuated ET1-induced PKD activation, but not PMA-induced PKD activation. The ET1-induced response was rescued when protein kinase A (PKA) was inhibited (H89, 10 microM) before exposure to isoprenaline. These results show that ET1-induced PKD activation in ARVM is mediated by PKC, primarily the PKCepsilon isoform, and is suppressed by PKA activation.

Effects of bisindolylmaleimide PKC inhibitors on p90RSK activity in vitro and in adult ventricular myocytes.

1 Bisindolylmaleimide inhibitors of protein kinase C (PKC), such as GF109203X and Ro31-8220, have been used to investigate the roles of PKC isoforms in many cellular processes in cardiac myocytes, but these agents may also inhibit p90RSK activity. 2 In in vitro kinase assays utilising 50 microM [ATP], GF109203X and Ro31-8220 inhibited p90RSK isoforms (IC50 values for inhibition of RSK1, RSK2 and RSK3, respectively, were 610, 310 and 120 nM for GF109203X, and 200, 36 and 5 nM for Ro31-8220) as well as classical and novel PKC isoforms (IC50 values for inhibition of PKCalpha and PKCepsilon, respectively, were 8 and 12 nM for GF109203X, and 4 and 8 nM for Ro31-8220). 3 At physiological [ATP] (5 mM), both GF109203X and Ro31-8220 exhibited reduced potency as inhibitors of RSK2, PKCalpha and PKCepsilon (IC50 values of 7400, 310 and 170 nM, respectively, for GF109203X, and 930, 150 and 140 nM, respectively, for Ro31-8220), with the latter agent retaining its relatively greater potency. 4 To determine the effects of GF109203X and Ro31-8220 on p90RSK activity in cultured adult rat ventricular myocytes (ARVM), phosphorylation of the eukaryotic elongation factor 2 kinase (eEF2K) at Ser366, a known p90RSK target, was used as the index of such activity. Adenoviral expression of a constitutively active form of mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) was used to induce PKC-independent p90RSK activation and downstream phosphorylation of eEF2K. 5 eEF2K phosphorylation was abolished by U0126 (1 microM), a selective inhibitor of MEK1, and was significantly reduced by GF109203X at > or =3 microM and by Ro31-8220 at > or =1 microM. At 1 microM, both agents inhibited PMA-induced PKC activity in ARVM. 6 These data show that GF109203X and Ro31-8220 inhibit various isoforms of PKC and p90RSK in vitro and in intact ARVM, with the former agent exhibiting relatively greater selectivity for PKC.