Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from loss of acid sphingomyelinase (ASM) activity. We have used a knockout mouse model of NPD (ASMKO mice) to evaluate the effects of direct intracerebral transplantation of bone marrow–derived mesenchymal stem cells (MSCs) on the progression of neurological disease in this disorder. MSCs were transduced with a retroviral vector to overexpress ASM and were injected into the hippocampus and cerebellum of 3-week-old ASMKO pups. Transplanted cells migrated away from the injection sites and survived at least 6 months after transplantation. Seven of 8 treated mice, but none of the untreated controls, survived for ≥ 7 months after transplant. Survival times were greater in sex-matched than in sex-mismatched transplants. Transplantation significantly delayed the Purkinje cell loss that is characteristic of NPD, although the protective effect declined with distance from the injection site. Overall ASM activity in brain homogenates was low, but surviving Purkinje cells contained the retrovirally expressed human enzyme, and transplanted animals showed a reduction in cerebral sphingomyelin. These results reveal the potential of treating neurodegenerative lysosomal storage disorders by intracerebral transplantation of bone marrow–derived MSCs.
Hee Kyung Jin, Janet E. Carter, George W. Huntley, Edward H. Schuchman
Gaucher disease, the most common lysosomal storage disease, is caused by a deficiency of glucocerebrosidase resulting in the impairment of glucosylceramide degradation. The hallmark of the disease is the presence of the Gaucher cell, a macrophage containing much of the stored glucosylceramide found in tissues, which is believed to cause many of the clinical manifestations of the disease. We have developed adult mice carrying the Gaucher disease L444P point mutation in the glucocerebrosidase (Gba) gene and exhibiting a partial enzyme deficiency. The mutant mice demonstrate multisystem inflammation, including evidence of B cell hyperproliferation, an aspect of the disease found in some patients. However, the mutant mice do not accumulate large amounts of glucosylceramide or exhibit classic Gaucher cells in tissues.
Hiroki Mizukami, Yide Mi, Ryuichi Wada, Mari Kono, Tadashi Yamashita, Yujing Liu, Norbert Werth, Roger Sandhoff, Konrad Sandhoff, Richard L. Proia
The treatment of chronic inflammatory diseases is complicated by their unpredictable, relapsing clinical course. Here, we describe a new strategy in which an inflammation-regulated therapeutic transgene is introduced into the joints to prevent recurrence of arthritis. To this end, we designed a recombinant adenoviral vector containing a two-component, inflammation-inducible promoter controlling the expression of human IL-10 (hIL-10) cDNA. When tested in vitro, this system had a low-level basal activity and was activated four to five orders of magnitude by various inflammatory stimuli, including TNF-α, IL-1β, IL-6, and LPS. When introduced in joints of rats with recurrent streptococcal cell wall–induced arthritis, the IL-10 transgene was induced in parallel with disease recurrence and effectively prevented the influx of inflammatory cells and the associated swelling of the joints. Levels of inflammation-inducible hIL-10 protein within the joints correlated closely with the severity of recurrence. An endogenously regulated therapeutic transgene can thus establish negative feedback and restore homeostasis in vivo while minimizing host exposure to the recombinant drug.
A.V. Miagkov, A.W. Varley, R.S. Munford, S.S. Makarov
Dominant mutations in sarcomere protein genes cause hypertrophic cardiomyopathy, an inherited human disorder with increased ventricular wall thickness, myocyte hypertrophy, and disarray. To understand the early consequences of mutant sarcomere proteins, we have studied mice (designated αMHC403/+) bearing an Arg403Gln missense mutation in the α cardiac myosin heavy chain. We demonstrate that Ca2+ is reduced in the sarcoplasmic reticulum of αMHC403/+ mice, and levels of the sarcoplasmic reticulum Ca2+-binding protein calsequestrin are diminished in advance of changes in cardiac histology or morphology. Further evidence for dysregulation of sarcoplasmic reticulum Ca2+ in these animals is seen in their decreased expression of the ryanodine receptor Ca2+-release channel and its associated membrane proteins and in an increase in ryanodine receptor phosphorylation. Early administration of the L-type Ca2+ channel inhibitor diltiazem restores normal levels of these sarcoplasmic reticular proteins and prevents the development of pathology in αMHC403/+ mice. We conclude that disruption of sarcoplasmic reticulum Ca2+ homeostasis is an important early event in the pathogenesis of this disorder and suggest that the use of Ca2+ channel blockers in advance of established clinical disease could prevent hypertrophic cardiomyopathy caused by sarcomere protein gene mutations.
Christopher Semsarian, Imran Ahmad, Michael Giewat, Dimitrios Georgakopoulos, Joachim P. Schmitt, Bradley K. McConnell, Steven Reiken, Ulrike Mende, Andrew R. Marks, David A. Kass, Christine E. Seidman, J.G. Seidman
LMX1B encodes a LIM-homeodomain transcription factor. Mutations in LMX1B cause nail-patella syndrome (NPS), an autosomal dominant disease with skeletal abnormalities, nail hypoplasia, and nephropathy. Expression of glomerular basement membrane (GBM) collagens is reduced in Lmx1b–/– mice, suggesting one basis for NPS nephropathy. Here, we show that Lmx1b–/– podocytes have reduced numbers of foot processes, are dysplastic, and lack typical slit diaphragms, indicating an arrest in development. Using antibodies to podocyte proteins important for podocyte function, we found that Lmx1b–/– podocytes express near-normal levels of nephrin, synaptopodin, ZO-1, α3 integrin, and GBM laminins. However, mRNA and protein levels for CD2AP and podocin were greatly reduced, suggesting a cooperative role for these molecules in foot process and slit diaphragm formation. We identified several LMX1B binding sites in the putative regulatory regions of both CD2AP and NPHS2 (podocin) and demonstrated that LMX1B binds to these sequences in vitro and can activate transcription through them in cotransfection assays. Thus, LMX1B regulates the expression of multiple podocyte genes critical for podocyte differentiation and function. Our results indicate that reduced levels of proteins associated with foot processes and the glomerular slit diaphragm likely contribute, along with reduced levels of GBM collagens, to the nephropathy associated with NPS.
Jeffrey H. Miner, Roy Morello, Kaya L. Andrews, Cong Li, Corinne Antignac, Andrey S. Shaw, Brendan Lee
Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the α4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV α4).
Claudia Rohr, Jürgen Prestel, Laurence Heidet, Hiltraud Hosser, Wilhelm Kriz, Randy L. Johnson, Corinne Antignac, Ralph Witzgall
Regulatory subunit KCNE3 (E3) interacts with KCNQ1 (Q1) in epithelia, regulating its activation kinetics and augmenting current density. Since E3 is expressed weakly in the heart, we hypothesized that ectopic expression of E3 in cardiac myocytes might abbreviate action potential duration (APD) by interacting with Q1 and augmenting the delayed rectifier current (IK). Thus, we transiently coexpressed E3 with Q1 and KCNE1 (E1) in Chinese hamster ovary cells and found that E3 coexpression increased outward current at potentials by ≥ –80 mV and accelerated activation. We then examined the changes in cardiac electrophysiology following injection of adenovirus-expressed E3 into the left ventricular cavity of guinea pigs. After 72 hours, the corrected QT interval of the electrocardiogram was reduced by ∼10%. APD was reduced by >3-fold in E3-transduced cells relative to controls, while E-4031–insensitive IK and activation kinetics were significantly augmented. Based on quantitative modeling of a transmural cardiac segment, we demonstrate that the degree of QT interval abbreviation observed results from electrotonic interactions in the face of limited transduction efficiency and that heterogeneous transduction of E3 may actually potentiate arrhythmias. Provided that fairly homogeneous ectopic ventricular expression of regulatory subunits can be achieved, this approach may be useful in enhancing repolarization and in treating long QT syndrome.
Reza Mazhari, H. Bradley Nuss, Antonis A. Armoundas, Raimond L. Winslow, Eduardo Marbán
Neuropeptide Y (NPY) is a downstream modulator of leptin action, possibly at the level of the arcuate nucleus where NPY neurons are known to express both leptin receptors and Y2 receptors. In addition to the well-described role of NPY and leptin in energy balance and obesity, intracerebroventricular administration of NPY or leptin also causes bone loss. Here we show that Y2 receptor–deficient mice have a twofold increase in trabecular bone volume as well as greater trabecular number and thickness compared with control mice. We also demonstrate that central Y2 receptors are crucial for this process, since selective deletion of hypothalamic Y2 receptors in mature conditional Y2 knockout mice results in an identical increase in trabecular bone volume within 5 weeks. This hypothalamus-specific Y2 receptor deletion stimulates osteoblast activity and increases the rate of bone mineralization and formation, with no effect on osteoblast or osteoclast surface measurements. The lack of any changes in plasma total calcium, leptinemia, or hypothalamo-pituitary-corticotropic, -thyrotropic, -somatotropic, or -gonadotropic output suggests that Y2 receptors do not modulate bone formation by humoral mechanisms, and that alteration of autonomic function through hypothalamic Y2 receptors may play a key role in a major central regulatory circuit of bone formation.
Paul A. Baldock, Amanda Sainsbury, Michelle Couzens, Ronaldo F. Enriquez, Gethin P. Thomas, Edith M. Gardiner, Herbert Herzog
Based on studies by our group and others, we hypothesized that IL-7 may possess antifibrotic activities in an IFN-γ–dependent and independent manner. Here, we have evaluated the antifibrotic therapeutic potential of IL-7 in both in vitro and in vivo pulmonary fibrosis models. IL-7 inhibited both TGF-β production and signaling in fibroblasts and required an intact JAK1/STAT1 signal transduction pathway. IL-7–mediated inhibition of TGF-β signaling was found to be associated with an increase in Smad7, a major inhibitory regulator in the SMAD family. In the presence of IL-7, Smad7 dominant negative fibroblasts restored TGF-β–induced collagen synthesis, indicating that an IL-7–mediated increase in Smad7 suppressed TGF-β signaling. Consistent with these in vitro findings, recombinant IL-7 decreased bleomycin-induced pulmonary fibrosis in vivo, independent of IFN-γ. The antifibrotic activities of IL-7 merit further basic and clinical investigation for the treatment of pulmonary fibrosis.
Min Huang, Sherven Sharma, Li X. Zhu, Michael P. Keane, Jie Luo, Ling Zhang, Marie D. Burdick, Ying Q. Lin, Mariam Dohadwala, Brian Gardner, Raj K. Batra, Robert M. Strieter, Steven M. Dubinett
Deficiency of the Golgi enzyme UDP-Gal:N-acetylglucosamine β-1,4-galactosyltransferase I (β4GalT I) (E.C.184.108.40.206) causes a new congenital disorder of glycosylation (CDG), designated type IId (CDG-IId), a severe neurologic disease characterized by a hydrocephalus, myopathy, and blood-clotting defects. Analysis of oligosaccharides from serum transferrin by HPLC, mass spectrometry, and lectin binding revealed the loss of sialic acid and galactose residues. In skin fibroblasts and leukocytes, galactosyltransferase activity was reduced to 5% that of controls. In fibroblasts, a truncated polypeptide was detected that was about 12 kDa smaller in size than wild-type β4GalT I and that failed to localize to the Golgi apparatus. Sequencing of the β4GalT I cDNA and gene revealed an insertion of a single nucleotide (1031-1032insC) leading to premature translation stop and loss of the C-terminal 50 amino acids of the enzyme. The patient was homozygous and his parents heterozygous for this mutation. Expression of a corresponding mutant cDNA in COS-7 cells led to the synthesis of a truncated, inactive polypeptide, which localized to the endoplasmic reticulum.
Bengt Hanßke, Christian Thiel, Torben Lübke, Martin Hasilik, Stefan Höning, Verena Peters, Peter H. Heidemann, Georg F. Hoffmann, Eric G. Berger, Kurt von Figura, Christian Körner