Search

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose‐1‐phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno‐associated virus (AAV)‐mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV‐mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFβRII protein levels and that these effectors of cellular homeostasis could be restored following AAV‐mediated expression of GALT. Finally, we show initial in vivo proof‐of‐concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV‐GALT. [ABSTRACT FROM AUTHOR]

Background: Gangliosides are highly enriched in the brain and are critical for its normal development and function. However, in some rare neurometabolic diseases, a deficiency in lysosomal ganglioside hydrolysis is pathogenic and leads to early-onset neurodegeneration, neuroinflammation, demyelination, and dementia. Increasing evidence also suggests that more subtle ganglioside accumulation contributes to the pathogenesis of more common neurological disorders including Alzheimer's disease (AD). Notably, ganglioside GM3 levels are elevated in the brains of AD patients and in several mouse models of AD, and plasma GM3 levels positively correlate with disease severity in AD patients. Methods: Tg2576 AD model mice were fed chow formulated with a small molecule inhibitor of glucosylceramide synthase (GCSi) to determine whether reducing glycosphingolipid synthesis affected aberrant GM3 accumulation, amyloid burden, and disease manifestations in cognitive impairment. GM3 was measured with LC-MS, amyloid burden with ELISA and amyloid red staining, and memory was assessed using the contextual fear chamber test. Results: GCSi mitigated soluble Aβ42 accumulation in the brains of AD model mice when treatment was started prophylactically. Remarkably, GCSi treatment also reduced soluble Aβ42 levels and amyloid plaque burden in aged (i.e., 70 weeks old) AD mice with preexisting neuropathology. Our analysis of contextual memory in Tg2576 mice showed that impairments in remote (cortical-dependent) memory consolidation preceded deficits in short-term (hippocampal-dependent) contextual memory, which was consistent with soluble Aβ42 accumulation occurring more rapidly in the cortex of AD mice compared to the hippocampus. Notably, GCSi treatment significantly stabilized remote memory consolidation in AD mice—especially in mice with enhanced cognitive training. This finding was consistent with GCSi treatment lowering aberrant GM3 accumulation in the cortex of AD mice. Conclusions: Collectively, our results indicate that glycosphingolipids regulated by GCS are important modulators of Aβ neuropathology and that glycosphingolipid homeostasis plays a critical role in the consolidation of remote memories. [ABSTRACT FROM AUTHOR]

Most lysosomal storage diseases (LSDs) have a significant neurological component, including types 2 and 3 Gaucher disease (neuronal forms of Gaucher disease; nGD). No therapies are currently available for nGD since the recombinant enzymes used in the systemic form of Gaucher disease do not cross the blood–brain barrier (BBB). However, a number of promising approaches are currently being tested, including substrate reduction therapy (SRT), in which partial inhibition of the synthesis of the glycosphingolipids (GSLs) that accumulate in nGD lowers their accumulation. We now induce nGD in mice by injection with conduritol B‐epoxide (CBE), an irreversible inhibitor of acid beta‐glucosidase (GCase), the enzyme defective in nGD, with or without co‐injection with Genz‐667161, a prototype for SRT which crosses the BBB. Significant neuropathology, and a reduction in lifespan, was observed upon CBE injection, and this was largely reversed by co‐injection with Genz‐667161, along with a reduction in glucosylceramide and glucosylsphingosine levels. Analysis of gene expression by RNAseq revealed that Genz‐667161 largely reversed the changes in genes and pathways that were differentially expressed upon CBE injection, specifically pathways of GSL metabolism, lipoproteins and other lipid metabolic pathways, lipid droplets, astrocyte activation, neuronal function, and to some extent, neuroinflammation. Together, this demonstrates the efficacy of SRT to reverse the effects of substrate accumulation on pathological components and pathways in nGD brain. [ABSTRACT FROM AUTHOR]

Citral is a mixture of neral and geranial, which are of great interest to the fragrance industry due to its lemon-scented aroma. A newly characterized nerol dehydrogenase of Persicaria minor (PmNeDH) from our recent findings has shown a capacity to convert citral from nerol. Differential gene expression analysis revealed that the expression level of PmNeDH was highly upregulated during early treatment of several stress-related phytohormones i.e. methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA). SA and ABA were shown to have a prolonged effect on PmNeDH expression level until second day of treatment. The findings were in agreement with the cis-regulatory elements predicted from the gene promoter. The phylogenetic relationship of PmNeDH with its homologs from the medium-chain dehydrogenases/reductases (MDR) superfamily was also mentioned. In this study, we proposed a possible biological function of PmNeDH gene in P. minor, which might play significant roles in plant defence mechanism. [ABSTRACT FROM AUTHOR]

Safe and effective gene delivery: Niemann-Pick type A disease (NPD-A) is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM). Patients present mental retardation with prominent loss of early motor skills and cognitive decline and early death. Gene replacement therapy has been shown to be effective in other monogenic neurological disorders. Now, Samaranch et al. evaluated the safety and efficacy of adeno-associated viral vector serotype 9 (AAV9)–based gene therapy. AAV9-mediated delivery of human ASM in the cerebellomedullary cistern allowed widespread gene expression in the brain and spinal cord of nonhuman primates without signs of toxicity. The treatment prevented motor and memory impairment and increased survival in a mouse model of NPD-A. Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders. [ABSTRACT FROM AUTHOR]

Agarwood is a resinous part of the non-timber Aquilaria tree, which is a highly valuable product for medicine and fragrance purposes. To protect the endangered Aquilaria species, mass plantation of Aquilaria trees has become a sustainable way in Asian countries to obtain the highly valuable agarwood. As only physiologically triggered Aquilaria tree can produce agarwood, effective induction methods are long sought in the agarwood industry. In this paper, we attempt to provide an overview for the past efforts toward the understanding of agarwood formation, the evolvement of induction methods and their further development prospects by integrating it with high-throughput omics approaches. [ABSTRACT FROM AUTHOR]

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α- galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy. [ABSTRACT FROM AUTHOR]

Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson's disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, GbaD409V/D409V and a A53T–α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T–SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of GbaD409V/D409V mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T–SNCA mice with GZ667161 reduced membrane- associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a diseasemodifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease. [ABSTRACT FROM AUTHOR]

TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%( v/ v) ethylene glycol and 5%( v/ v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å. [ABSTRACT FROM AUTHOR]

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent intracellular accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb). Vascular endothelial cells may play important roles in disease pathogenesis, and are one of the main target cell types in therapeutic interventions. In this study, we generated immortalized aortic endothelial cell lines from a mouse model of Fabry disease. These cells retained endothelial cell-specific markers and functions. Gb expression level in one of these clones (referred to as FMEC2) was highly susceptible to culture media, and appeared to be regulated by glucosylceramide synthase. Results also showed that Gb could be upregulated by hydrocortisone. FMEC2 express the mannose 6-phosphate receptor and sortilin but not the mannose receptor. Uptake studies suggested that sortilin plays a role in the binding and internalization of mammalian cell-produced α-galactosidase A. Moss-aGal (a plant-made enzyme) was endocytosed by FMEC2 via a receptor other than the aforementioned receptors. In conclusion, this study suggests that glucosylceramide synthase and hydrocortisone may play important roles in modulating Gb levels in Fabry mouse aortic endothelial cells, and that endocytosis of recombinant α-galactosidase A involves a combination of multiple receptors depending on the properties of the enzyme. [ABSTRACT FROM AUTHOR]

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age-related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin-1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin-1 (Fbn1Prx1-/- mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1Prx1-/- mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1Prx1-/- mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1-silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARg levels. Consonant with fibrillin-1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1Prx1-/- limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin-1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches. [ABSTRACT FROM AUTHOR]

Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2. [ABSTRACT FROM AUTHOR]

Metabolic dysfunction is an important modulator of disease course in amyotrophic lateral sclerosis (ALS). We report here that a familial mouse model (transgenic mice over-expressing the G93A mutation of the Cu/Zn superoxide dismutase 1 gene) of ALS enters a progressive state of acidosis that is associated with several metabolic (hormonal) alternations that favor lipolysis. Extensive investigation of the major determinants of H+ concentration (i.e., the strong ion difference and the strong ion gap) suggests that acidosis is also due in part to the presence of an unknown anion. Consistent with a compensatory response to avert pathological acidosis, ALS mice harbor increased accumulation of glycogen in CNS and visceral tissues. The altered glycogen is associated with fluctuations in lysosomal and neutral α-glucosidase activities. Disease-related changes in glycogen, glucose, and α-glucosidase activity are also found in spinal cord tissue samples of autopsied patients with ALS. Collectively, these data provide insights into the pathogenesis of ALS as well as potential targets for drug development. [ABSTRACT FROM AUTHOR]

Background: Huntington's disease (HD) is a neurological disorder caused by mutations in the huntingtin (HTT) gene, the product of which leads to selective and progressive neuronal cell death in the striatum and cortex. Transcriptional dysregulation has emerged as a core pathologic feature in the CNS of human and animal models of HD. It is still unclear whether perturbations in gene expression are a consequence of the disease or importantly, contribute to the pathogenesis of HD. Objective: To examine if transcriptional dysregulation can be ameliorated with antisense oligonucleotides that reduce levels of mutant Htt and provide therapeutic benefit in the YAC128 mouse model of HD. Methods: Quantitative real-time PCR analysis was used to evaluate dysregulation of a subset of striatal genes in the YAC128 mouse model. Transcripts were then evaluated following ICV delivery of antisense oligonucleotides (ASO). Rota rod and Porsolt swim tests were used to evaluate phenotypic deficits in these mice following ASO treatment. Results: Transcriptional dysregulation was detected in the YAC128 mouse model and appears to progress with age. ICV delivery of ASOs directed against mutant Htt resulted in reduction in mutant Htt levels and amelioration in behavioral deficits in the YAC128 mouse model. These improvements were correlated with improvements in the levels of several dysregulated striatal transcripts. Conclusions: The role of transcriptional dysregulation in the pathogenesis of Huntington's disease is not well understood, however, a wealth of evidence now strongly suggests that changes in transcriptional signatures are a prominent feature in the brains of both HD patients and animal models of the disease. Our study is the first to show that a therapeutic agent capable of improving an HD disease phenotype is concomitantly correlated with normalization of a subset of dysregulated striatal transcripts. Our data suggests that correction of these disease-altered transcripts may underlie, at least in part, the therapeutic efficacy shown associated with ASO-mediated correction of HD phenotypes and may provide a novel set of early biomarkers for evaluating future therapeutic concepts for HD. [ABSTRACT FROM AUTHOR]

Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3+ regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9–12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D). However, a longer course of treatment (10 weeks) of 4–13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing β-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D. [ABSTRACT FROM AUTHOR]

Mutations of GBA1, the gene encoding glucocerebrosidase, represent a common genetic risk factor for developing the synucleinopathies Parkinson disease (PD) and dementia with Lewy bodies. PD patients with or without GBA1 mutations also exhibit lower enzymatic levels of glucocerebrosidase in the central nervous system (CNS), suggesting a possible link between the enzyme and the development of the disease. Previously, we have shown that early treatment with glucocerebrosidase can modulate a-synuclein aggregation in a presymptomatic mouse model of Gaucher-related synucleinopathy (Gba1D409V/D409V) and ameliorate the associated cognitive deficit. To probe this link further, we have now evaluated the efficacy of augmenting glucocerebrosidase activity in the CNS of symptomatic Gba1D409V/D409V mice and in a transgenic mouse model overexpressing A53T α-synuclein. Adeno-associated virus-mediated expression of glucocerebrosidase in the CNS of symptomatic Gba1D409V/D409V mice completely corrected the aberrant accumulation of the toxic lipid glucosylsphingosine and reduced the levels of ubiquitin, tau, and proteinase K-resistant α-synuclein aggregates. Importantly, hippocampal expression of glucocerebrosidase in Gba1D409V/D409V mice (starting at 4 or 12 mo of age) also reversed their cognitive impairment when examined using a novel object recognition test. Correspondingly, overexpression of glucocerebrosidase in the CNS of A53T α-synuclein mice reduced the levels of soluble α-synuclein, suggesting that increasing the glycosidase activity can modulate a-synuclein processing and may modulate the progression of α-synucleinopathies. Hence, increasing glucocerebrosidase activity in the CNS represents a potential therapeutic strategy for GBA1-related and non-GBA1-associated synucleinopathies, including PD. [ABSTRACT FROM AUTHOR]

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA−/− mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA−/− mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA−/− mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA−/− mice, phosphorylase activity is suppressed in the GAA−/− mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity. [ABSTRACT FROM AUTHOR]

Transcriptional control of transgene expression is crucial to successful gene therapy, yet few promoter/enhancer combinations have been tested in clinical trials. We created a simple, desktop computer database and populated it with promoter sequences from publicly available sources. From this database, we rapidly identified novel CpG-free promoter sequences suitable for use in non-inflammatory, non-viral in vivo gene transfer. In a simple model of lung gene transfer, five of the six promoter elements selected, chosen without prior knowledge of their transcriptional activities, directed significant transgene expression. Each of the five novel promoters directed transgene expression for at least 14 days post-delivery, greatly exceeding the duration achieved with the commonly used CpG-rich viral enhancer/promoters. Novel promoter activity was also evaluated in a more clinically relevant model of aerosol-mediated lung gene transfer and in the liver following delivery via high-pressure tail vein injection. In each case, the novel CpG-free promoters exhibited higher and/or more sustained transgene expression than commonly used CpG-rich enhancer/promoter sequences. This study demonstrates that novel CpG-free promoters can be readily identified and that they can direct significant levels of transgene expression. Furthermore, the database search criteria can be quickly adjusted to identify other novel promoter elements for a variety of transgene expression applications. [ABSTRACT FROM AUTHOR]

Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type ofthe disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUGexp) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUGexp RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence. [ABSTRACT FROM AUTHOR]

Background The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. Methods We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug). Results TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls. Conclusions Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Heterozygous mutations in the glucocerebrosidase gene (GBA1) are associated with increased risk for α-synuclein aggregation disorders ('synucleinopathies'), which include Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Homozygous GBA1 mutations lead to reduced GBA1 lysosomal activity underlying three variants of Gaucher disease (GD). Despite the wealth of clinical and genetic evidence supporting the association between mutant genotypes and synucleinopathy risk, the precise mechanisms by which GBA1 mutations lead to PD and DLB remain unclear. Here, we summarize recent findings that highlight the complexity of this pathogenetic link. In neural cells, both gain and loss of function mechanisms, as conferred by mutant GBA1 expression and activity loss, respectively, seem to promote aberrant α-synuclein processing. In addition, we draw attention to recent insights gleaned from GD animal models regarding axonal pathology, brain inflammation and memory dysfunction. From a translational perspective, we discuss the concepts of neural enzyme replacement therapy and pharmacological agents as potential treatment strategies for GBA1-associated synucleinopathies. Finally, we touch on the issue whether aberrant α-synuclein species may coregulate GBA1 activity in the vertebrate brain, thereby providing a reverse link, i.e., between an important synucleinopathy risk factor and the enzyme's lysosomal function. In summary, several leads connecting GBA1 mutations with α-synuclein misprocessing have emerged as potential targets for the treatment of GBA1-related synucleinopathies, and possibly, for non-GBA1-associated neurodegenerative diseases. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron cell loss, muscular atrophy, and a shortened life span. Survival is highly variable, as some patients die within months, while others live for many years. Exposure to stress or the development of a nonoptimal stress response to disease might account for some of this variability. We show in the SODIG93A mouse model of ALS that recurrent exposure to restraint stress led to an earlier onset of astrogliosis and microglial activation within the spinal cord, accelerated muscular weakness, and a significant decrease in median survival (105 vs. 122 d) when compared to non-stressed animals. Moreover, during normal disease course, ALS mice display a cacostatic stress response by developing an aberrant serum corticosterone circadian rhythm. Interestingly, we also found that higher corticosterone levels were significantly correlated with both an earlier onset of paralysis (males: r²=0.746; females: r²=0.707) and shorter survival times (males: r²=0.680; females: r²=0.552) in ALS mice. These results suggest that stress is capable of accelerating disease progression and that strategies that modulate glucocorticoid metabolism might be a viable treatment approach for ALS. [ABSTRACT FROM AUTHOR]

Treating the central nervous system manifestations of subjects with neuropathic lysosomal storage diseases remains a major technical challenge. This is because of the low efficiency by which lysosomal enzymes in systemic circulation are able to traverse the blood brain barrier into the central nervous system. Intracranial transplantation of neural stems cells genetically modified to overexpress the respective deficient enzymes represents a potential approach to addressing this group of diseases. The unique properties of neural stem cells and progenitor cells, such as their ability to migrate to distal sites, differentiate into various cell types and integrate within the host brain without disrupting normal function, making them particularly attractive therapeutic agents. In addition, neural stem cells are amenable to ex vivo propagation and modification by gene transfer vectors. In this regard, transplanted cells can serve not only as a source of lysosomal enzymes but also as a means to potentially repair the injured brain by replenishing the organ with healthy cells and effecting the release of neuroprotective factors. This review discusses some of the well-characterized neural stem cell types and their possible use in treating neuropathic lysosomal storage diseases such as the Niemann Pick A disease. [ABSTRACT FROM AUTHOR]

Several diseases of the nervous system are characterized by neurodegeneration and death in childhood. Conventional medicine is ineffective, but fetal or neonatal gene therapy may provide an alternative route to treatment. We evaluated the ability of single-stranded and self-complementary adeno-associated virus pseudotype 2/9 (AAV2/9) to transduce the nervous system and target gene expression to specific neural cell types following intravenous injection into fetal and neonatal mice, using control uninjected age-matched mice. Fetal and neonatal administration produced global delivery to the central (brain, spinal cord, and all layers of the retina) and peripheral (myenteric plexus and innervating nerves) nervous system but with different expression profiles within the brain; fetal and neonatal administration resulted in expression in neurons and protoplasmic astrocytes, respectively. Neither single-stranded nor self-complementary AAV2/9 triggered a microglia-mediated immune response following either administration. In summary, intravenous AAV2/9 targets gene expression to specific neural cell types dependent on developmental stage. This represents a powerful tool for studying nervous system development and disease. Furthermore, it may provide a therapeutic strategy for treatment of early lethal genetic diseases, such as Gaucher disease, and for disabling neuropathies, such as preterm brain injury. [ABSTRACT FROM AUTHOR]

Emerging genetic and clinical evidence suggests a link between Gaucher disease and the synucleinopathies Parkinson disease and dementia with Lewy bodies. Here, we provide evidence that a mouse model of Gaucher disease (Gba1D409V/D409V) exhibits characteristics of synucleinopathies, including progressive accumulation of proteinase K-resistant α-synuclein/ubiquitin aggregates in hippocampal neurons and a coincident memory deficit. Analysis of homozygous (Gba1D409V/D409V) and heterozygous (Gba1D409V/+ and Gba1+/-) Gaucher mice indicated that these pathologies are a result of the combination of a loss of glucocerebrosidase activity and a toxic gain-of-function resulting from expression of the mutant enzyme. Importantly, adeno-associated virus-mediated expression of exogenous glucocerebrosidase injected into the hippocampus of Gba1D409V/D409V mice ameliorated both the histopathological and memory aberrations. The data support the contention that mutations in GBA1 can cause Parkinson disease-like α-synuclein pathology, and that rescuing brain glucocerebrosidase activity might represent a therapeutic strategy for GBA1-associated synucleinopathies. [ABSTRACT FROM AUTHOR]

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects. [ABSTRACT FROM AUTHOR]

Background The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis. Methods Apolipoprotein E deficient (ApoE [ABSTRACT FROM AUTHOR]

Niemann-Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we reported that biochemical and functional abnormalities observed in ASM knockout (ASMKO) mice could be partially alleviated by intracerebroventricular (ICV) infusion of hASM. We now show that this route of delivery also results in widespread enzyme distribution throughout the rat brain and spinal cord. However, enzyme diffusion into CNS parenchyma did not occur in a linear dose-dependent fashion. Moreover, although the levels of hASM detected in the rat CNS were determined to be within the range shown to be therapeutic in ASMKO mice, the absolute amounts represented less than 1% of the total dose administered. Finally, our results also showed that similar levels of enzyme distribution are achieved across rodent species when the dose is normalized to CNS weight as opposed to whole body weight. Collectively, these data suggest that the efficacy observed following ICV delivery of hASM in ASMKO mice could be scaled to CNS of the rat. [ABSTRACT FROM AUTHOR]

Grass bridges (caoqiao) were floating bridges made of grass, twigs, and ropes that enabled river crossing at different water levels -- due to seasonal change -- in North China. Prior to modern architectural technology, these temporary grass bridges were popular. This article discusses the building of grass bridges over the Zhang River during the Qing dynasty. The Zhang River grass bridges were built in the fall, when water levels were low, and removed in spring, when water levels rose. The locations of these bridges changed each year. Because these bridges often crossed county borders, conflicts sometimes arose over which jurisdiction was financially responsible for construction. The ecological impact and location selection of the bridges are also analyzed.

Fabry disease is an X-linked glycosphingolipid storage disorder caused by a deficiency in the activity of the lysosomal hydrolase α-galactosidase A (α-gal). This deficiency results in accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in lysosomes. Endothelial cell storage of GL-3 frequently leads to kidney dysfunction, cardiac and cerebrovascular disease. The current treatment for Fabry disease is through infusions of recombinant α-gal (enzyme-replacement therapy; ERT). Although ERT can markedly reduce the lysosomal burden of GL-3 in endothelial cells, variability is seen in the clearance from several other cell types. This suggests that alternative and adjuvant therapies may be desirable. Use of glucosylceramide synthase inhibitors to abate the biosynthesis of glycosphingolipids (substrate reduction therapy, SRT) has been shown to be effective at reducing substrate levels in the related glycosphingolipidosis, Gaucher disease. Here, we show that such an inhibitor (eliglustat tartrate, Genz-112638) was effective at lowering GL-3 accumulation in a mouse model of Fabry disease. Relative efficacy of SRT and ERT at reducing GL-3 levels in Fabry mouse tissues differed with SRT being more effective in the kidney, and ERT more efficacious in the heart and liver. Combination therapy with ERT and SRT provided the most complete clearance of GL-3 from all the tissues. Furthermore, treatment normalized urine volume and uromodulin levels and significantly delayed the loss of a nociceptive response. The differential efficacies of SRT and ERT in the different tissues indicate that the combination approach is both additive and complementary suggesting the possibility of an improved therapeutic paradigm in the management of Fabry disease. [ABSTRACT FROM AUTHOR]

Studies examining the effects of hypoxia upon osteoclast biology have consistently revealed a stimulatory effect; both osteoclast differentiation and resorption activity have been shown to be enhanced in the presence of hypoxia. In the present study we examined the effects of the hypoxia mimetics dimethyloxallyl glycine (DMOG) and desferrioxamine (DFO) upon osteoclastogenesis. In contrast to hypoxia, our studies revealed a dose-dependent inhibition of osteoclast formation from macrophages treated with DMOG and DFO. Moreover, expression of a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha) did not enhance osteoclastogenesis and actually attenuated the differentiation process. DMOG did not affect cell viability or receptor activator of nuclear factor kappaB ligand (RANKL)-dependent phosphorylation of mitogen-activated protein (MAP) kinases. However, RANKL-dependent transcription of tartrate-resistant acid phosphatase (TRAP) was reduced in the presence of DMOG. Additionally, DMOG promoted transcription of the pro-apoptotic mediator B-Nip3. These studies suggest that a hypoxia-responsive factor other than HIF-1alpha is necessary for enhancing the formation of osteoclasts in hypoxic settings. [ABSTRACT FROM AUTHOR]

Lipid rafts reportedly play an important role in modulating the activation of mast cells and granulocytes, the primary effector cells of airway hyperresponsiveness and asthma. Activation is mediated through resident signaling molecules whose activity, in part, may be modulated by the composition of glycosphingolipids (GSLs) in membrane rafts. In this study, we evaluated the impact of inhibiting GSL biosynthesis in mast cells and in the ovalbumin (OVA)-induced mouse model of asthma using either a small molecule inhibitor or anti-sense oligonucleotides (ASOs) directed against specific enzymes in the GSL pathway. Lowering GSL levels in mast cells through inhibition of glucosylceramide synthase (GCS) reduced phosphorylation of Syk tyrosine kinase and phospholipase C gamma 2 (PLC-γ2) as well as cytoplasmic Ca2+ levels. Modulating these intracellular signaling events also resulted in a significant decrease in mast cell degranulation. Primary mast cells isolated from a GM3 synthase (GM3S) knockout mouse exhibited suppressed activation-induced degranulation activity further supporting a role of GSLs in this process. In previously OVA-sensitized mice, intra-nasal administration of ASOs to GCS, GM3S or lactosylceramide synthase (LCS) significantly suppressed metacholine-induced airway hyperresponsiveness and pulmonary inflammation to a subsequent local challenge with OVA. However, administration of the ASOs into mice that had been sensitized and locally challenged with the allergen did not abate the consequent pulmonary inflammatory sequelae. These results suggest that GSLs contribute to the initiation phase of the pathogenesis of airway hyperreactivity and asthma and lowering GSL levels may offer a novel strategy to modulate these manifestations. [ABSTRACT FROM AUTHOR]

Background: Obesity is characterized by the accumulation of fat in the liver and other tissues, leading to insulin resistance. We have previously shown that a specific inhibitor of glucosylceramide synthase, which inhibits the initial step in the synthesis of glycosphingolipids (GSLs), improved glucose metabolism and decreased hepatic steatosis in both ob/ob and diet-induced obese (DIO) mice. Here we have determined in the DIO mouse model the efficacy of a related small molecule compound, Genz-112638, which is currently being evaluated clinically for the treatment of Gaucher disease, a lysosomal storage disorder. Methodology/Principal Findings: DIO mice were treated with the Genz-112638 for 12 to 16 weeks by daily oral gavage. Genz-112638 lowered HbA1c levels and increased glucose tolerance. Whole body adiposity was not affected in normal mice, but decreased in drug-treated obese mice. Drug treatment also significantly lowered liver triglyceride levels and reduced the development of hepatic steatosis. We performed hyperinsulinemic-euglycemic clamps on the DIO mice treated with Genz-112638 and showed that insulin-mediated suppression of hepatic glucose production increased significantly compared to the placebo treated mice, indicating a marked improvement in hepatic insulin sensitivity. Conclusions/Significance: These results indicate that GSL inhibition in obese mice primarily results in an increase in insulin action in the liver, and suggests that GSLs may have an important role in hepatic insulin resistance in conditions of obesity. [ABSTRACT FROM AUTHOR]

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid β-glucosidase), with consequent cellular accumulation of glucosylceramide (GL-1). The disease is managed by intravenous administrations of recombinant glucocerebrosidase (imiglucerase), although symptomatic patients with mild to moderate type 1 Gaucher disease for whom enzyme replacement therapy (ERT) is not an option may also be treated by substrate reduction therapy (SRT) with miglustat. To determine whether the sequential use of both ERT and SRT may provide additional benefits, we compared the relative pharmacodynamic efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V/null). As expected, ERT with recombinant glucocerebrosidase was effective in reducing the burden of GL-1 storage in the liver, spleen, and lung of 3-month-old Gaucher mice. SRT using a novel inhibitor of glucosylceramide synthase (Genz-112638) was also effective, albeit to a lesser degree than ERT. Animals administered recombinant glucocerebrosidase and then Genz-112638 showed the lowest levels of GL-1 in all the visceral organs and a reduced number of Gaucher cells in the liver. This was likely because the additional deployment of SRT following enzyme therapy slowed the rate of reaccumulation of GL-1 in the affected organs. Hence, in patients whose disease has been stabilized by intravenously administered recombinant glucocerebrosidase, orally administered SRT with Genz-112638 could potentially be used as a convenient maintenance therapy. In patients naïve to treatment, ERT followed by SRT could potentially accelerate clearance of the offending substrate. [ABSTRACT FROM AUTHOR]

Background When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs). Methods We have compared laser microdissection, pronase digestion and nasal brushing for: (i) the ability to enrich RECs from the wild-type mouse nose and (ii) the length of time to perform the procedure. Using TaqMan, we subsequently assessed gene transfer in enriched RECs after nasal perfusion of GL67A/pCF1-CFTR complexes in a CF mouse model. Results Laser microdissection successfully isolated RECs; however, time-consuming sample preparation made this technique unsuitable for high-throughput studies. Pronase digestion was sufficiently rapid but only yielded 19% (range = 13%) RECs ( n = 6). The nasal brushing method was superior, yielding 92% (range = 15%) RECs ( n = 8) and was equally effective in CF knockout mice (91%, range = 14%, n = 10). Importantly, gene transfer was detectable in brushed RECs from 70% of perfused mice and the number of vector-specific transcripts was comparable to 3.5% of endogenous wild-type Cftr levels. Conclusions Isolation of RECs by brushing allows accurate assessment of GTA transfection efficiency in an experimental system that is relevant for CF gene therapy. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]