Prediction of drug-drug interaction risk of P-glycoprotein substrate in drug discovery.

We aimed at predicting the drug-drug interaction (DDI) risk of P-glycoprotein (P-gp) substrates by using P-gp expressing LLC-PK1 cells and its knockout mice (KO). The area under the curve (AUC) of 16 marketed drugs and plasma concentration (Cplasma) of 207 screening compounds, with corrected efflux ratio (CER) ≥ 2, were compared between P-gp KO mice and wild type mice (WT). At permeability (Papp) ≥ 10 × 10-6 cm/s in parent LLC-PK1 cells, AUC ratios (KO/WT) and Cplasma ratios (KO/WT) of these compounds were within 3-fold. AUC ratios (KO/WT) of clinical P-gp substrates, with human AUC ratios with and without P-gp inhibitor administration ≥2, were higher than 8.7. These observations led us to establish a work-flow of P-gp substrate assessment with the threshold AUC ratio (KO/WT) ≥ 9 leading to a DDI risk of AUC ratio (human) ≥ 2. A screening compound showing high CER (=57.6) was found, but its AUC ratio (KO/WT) was 3.7, had been presumed to be a weak risk and its AUC ratio (human) was 1.2 in a later clinical DDI study. Our proposed workflow should be useful for predicting the DDI risk of P-gp substrates in drug discovery.

Near full-automation of COPMAN using a LabDroid enables high-throughput and sensitive detection of SARS-CoV-2 RNA in wastewater as a leading indicator.

Wastewater-based epidemiology (WBE) is a promising tool to efficiently monitor COVID-19 prevalence in a community. For WBE community surveillance, automation of the viral RNA detection process is ideal. In the present study, we achieved near full-automation of a previously established method, COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater), which was then applied to detect SARS-CoV-2 in wastewater for half a year. The automation line employed the Maholo LabDroid and an automated-pipetting device to achieve a high-throughput sample-processing capability of 576 samples per week. SARS-CoV-2 RNA was quantified with the automated COPMAN using samples collected from two wastewater treatment plants in the Sagami River basin in Japan between 1 November 2021 and 24 May 2022, when the numbers of daily reported COVID-19 cases ranged from 0 to 130.3 per 100,000 inhabitants. The automated COPMAN detected SARS-CoV-2 RNA from 81 out of 132 samples at concentrations of up to 2.8 × 105 copies/L. These concentrations showed direct correlations with subsequently reported clinical cases (5-13 days later), as determined by Pearson's and Spearman's cross-correlation analyses. To compare the results, we also conducted testing with the EPISENS-S (Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids, Ando et al., 2022), a previously reported detection method. SARS-CoV-2 RNA detected with EPISENS-S correlated with clinical cases only when using Spearman's method. Our automated COPMAN was shown to be an efficient method for timely and large-scale monitoring of viral RNA, making WBE more feasible for community surveillance.

A neuronal cell-based reporter system for monitoring the activity of HDAC2.

Given that histone acetylation via histone acetyltransferases (HATs) and histone deacetylases (HDACs) is significant in memory formation, HDAC2 has been thoroughly investigated as a potential therapeutic target for the treatment of cognitive dysfunction. Although HDAC inhibitors have been discovered through in vitro enzyme assay, off-target effects on other HDACs are common due to their conserved catalytic domains. Each HDAC could be regulated by specific intracellular molecular mechanisms, raising the possibility that a cell-based assay could identify selective inhibitors targeting specific HDACs through their regulatory mechanisms. Here, we propose a versatile, cell-based reporter system for screening HDAC2 inhibitors. Through RNA-sequencing from human cultured neuronal cells, we determined that expression of a transcriptional repressor, inhibitor of DNA binding 1 (ID1), is increased by knockdown of HDAC2. We also established the knock-in neuronal cell lines of a bioluminescence reporter gene to ID1. The knock-in cell lines showed significant reporter activity by known HDAC inhibitors and by HDAC2-knockdown but not by HDAC1-knockdown. Thus, our neuronal cell-based reporter system is a promising method for screening the specific inhibitors of HDAC2 but not HDAC1, by potentially targeting not only HDAC2, but also the regulatory mechanisms of HDAC2 in neurons.

Species difference in brain penetration of P-gp and BCRP substrates among monkey, dog and mouse.

The brain penetration of 19 drugs, including P-glycoprotein (P-gp) and/or breast cancer resistance protein (BCRP) substrates, was compared among mice, cynomolgus monkeys and beagle dogs. The brain-to-plasma concentration ratios (Kp,brain) of the tested compounds in monkey and dog showed good correlation, whereas species differences were observed between non-rodents (monkey/dog) and rodents (mouse). In particular, the Kp,brain values of 7 compounds out of 12 P-gp substrates (Kp,brain ratio in P-gp knockout mice versus wild-type mice ≥3) in monkey and dog were more than three-fold higher than those in mice and a similar trend was observed in the brain-to-plasma unbound concentration ratios (Kp,uu,brain). The cerebral spinal fluid (CSF) drug concentrations (CCSF), a surrogate for unbound brain concentration (Cu,brain), were also compared between dog and monkey, and the CSF-to-plasma unbound concentration ratios (Kp,uu,CSF) of BCRP substrates in dog were notably higher than those in monkey, although non-bcrp substrates showed good correlation. Also, the Kp,uu,CSF values of BCRP substrates in dog were clearly higher than the Kp,uu,brain values, indicating that the dog CCSF of BCRP substrates was not suitable as a surrogate of Cu,brain. These observations should be useful when selecting the appropriate animal models for CNS drug discovery.

Acsl1 is essential for skin barrier function through the activation of linoleic acid and biosynthesis of ω-O-acylceramide in mice.

The long-chain acyl-CoA synthase1 (Acsl1) is a major enzyme that converts long-chain fatty acids to acyl-CoAs. The role of Acsl1 in energy metabolism has been elucidated in the adipose tissue, heart, and skeletal muscle. Here, we demonstrate that systemic deficiency of Acsl1 caused severe skin barrier defects, leading to embryonic lethality. Acsl1 mRNA and protein are expressed in the Acsl1+/+ epidermis, which are absent in Acsl1-/- mice. In Acsl1-/- mice, epidermal ceramide [EOS] (Cer[EOS]) containing ω-O-esterified linoleic acid, a lipid essential for the skin barrier, was significantly reduced. Conversely, ω-hydroxy ceramide (Cer[OS]), a precursor of Cer[EOS], was increased. Moreover, the levels of triglyceride (TG) species containing linoleic acids were lower in Acsl1-/- mice, whereas those not containing linoleic acid were comparable to Acsl1+/+ mice. As TG is considered to work as a reservoir of linoleic acid for the biosynthesis of Cer[EOS] from Cer[OS], our results suggest that Acsl1 plays an essential role in ω-O-acylceramide synthesis by providing linoleic acid for ω-O-esterification. Therefore, our findings identified a new biological role of Acsl1 as a regulator of the skin barrier.

Visualization of specific collagen-producing cells by Col1-GFP transgenic mice revealed novel type I collagen-producing cells other than fibroblasts in systemic organs/tissues.

Type I collagen is one of the most abundant proteins in mammals and plays important roles in maintaining the integrity of many tissues. Although fibroblasts are the main source of type I collagen, other cells also produce it; however, these cells are not well-defined owing to the lack of a specific marker. A transgenic (Tg) mouse line has been generated in which type I collagen-producing cells are labeled with enhanced green fluorescent protein (EGFP), which enables the monitoring of these cells without requiring an additional cell marker. This Tg mouse line has since been widely used to study type I collagen-producing cells and fibrosis; one study revealed that podocytes, which were not previously considered to produce type I collagen, expressed EGFP. This raises a question regarding the specificity of EGFP expression in this Tg mouse line. To exclude the possibility of non-specific EGFP expression in the existing Tg mouse line and specifically monitor type I collagen-producing cells, we generated a new Tg mouse line and histologically confirmed the specificity of EGFP expression throughout the body. Moreover, we explored type I collagen-producing cells other than fibroblasts and revealed for the first time that Leydig cells have the ability to produce type I collagen. Because of its highly specific and physiologically accurate expression, our new Tg mouse line will help to accurately elucidate not only type I collagen-producing cells in normal tissues but also the potential cells in fibrotic tissues, providing new insights into the pathology of fibrosis.

The role of acylated-ghrelin in the regulation of sucrose intake.

The octanoyl modification of ghrelin by ghrelin O-acyltransferase (GOAT) is essential for exerting its physiologic actions. Since exogenous acylated-ghrelin has shown to stimulate food intake in humans and rodents, GOAT has been regarded as a promising target for modulating appetite, thereby treating obesity and diabetes. However, GOAT-knockout (KO) mice have been reported to show no meaningful body weight reduction, when fed a high-fat diet. In this study, we sought to determine whether GOAT has a role in the regulation of body weight and food intake when fed a dietary sucrose. We found that GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, when fed a high-fat + high-sucrose diet. In addition, GOAT KO mice fed a medium-chain triglyceride (MCT) + high-sucrose diet showed a marked resistance to obesity and reduced feed efficiency. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity caused by overconsumption of palatable food.

Potential role of IL-17-producing CD4/CD8 double negative αß T cells in psoriatic skin inflammation in a TPA-induced STAT3C transgenic mouse model.

BACKGROUND: Psoriasis is one of the most common immune-mediated chronic inflammatory skin disorders and is accompanied by erythematous scaly plaques. There is growing evidence that the IL-23/Th17 axis plays a critical role in development of the disease. It was recently shown that in addition to CD4+ Th17 cells, various IL-17-producing cell subsets such as CD8+ Tc17 cells, dermal γδ T cells, and innate lymphoid cells are also involved in the development of psoriatic inflammation in humans. OBJECTIVE: To investigate which subsets of IL-17-producing cells are involved in psoriasis-like skin inflammation in a TPA (tumor promoter 12-O-tetradecanoylphorbol-13-acetate)-induced K14.Stat3C mouse model. METHOD: Skin-infiltrating cells were isolated from inflamed lesions of TPA-treated K14.Stat3C transgenic mice, and analyzed for IL-17 producing cell subsets by flow cytometry. RESULTS: We observed significantly increased numbers of IL-17-producing CD4+ T cells, CD8+ T cells and dermal γδ T cells in TPA-induced skin lesions of K14.Stat3C mice. Additionally, we found that another IL-17-producing T cell subset, αß-TCR+ CD4CD8 double negative T cells (DN αß T cells), was also increased in lesional skin. These IL-17-producing DN αß T cells are NK1.1 negative, suggesting they are not natural killer T cells or mucosal associated invariant T cells. As well as other IL-17-producing cells, DN αß T cells in the inflamed skin can also respond to IL-23 stimulation to produce IL-17. It is also suggested that DN αß T cells may express retinoic acid-related orphan receptor gamma t and CC chemokine receptor 6. CONCLUSION: In TPA-induced lesional skin of K14.Stat3C mice, IL-17-producing CD4+ Th17 cells, CD8+ Tc17 cells, dermal γδ T cells and TCR- cells probably containing ILCs all participated in skin inflammation, which is similar to human clinical psoriatic features. Furthermore, we showed for the first time the possibility that an IL-17-producing DN αß T cell subset is also involved in psoriatic inflammation.

Reduced intake of carbohydrate prevents the development of obesity and impaired glucose metabolism in ghrelin O-acyltransferase knockout mice.

A close relationship between acylated-ghrelin and sucrose intake has been reported. However, little has been examined about the physiological action of ghrelin on preference for different types of carbohydrate such as glucose, fructose, and starch. The current study was aimed to investigate the role of acylated-ghrelin in the determinants of the choice of carbohydrates, and pathogenesis of chronic disorders, including obesity and insulin resistance. In a two-bottle-drinking test, ghrelin O-acyltransferase (GOAT) knockout (KO) mice consumed a less amount of glucose and maltodextrin, and almost the same amount of fructose and saccharin solution compared to WT littermates. The increased consumption of glucose and maltodextrin was observed when acylated-ghrelin, but not unacylated-ghrelin, was exogeneously administered in normal C57BL/6J mice, suggesting an association of acylated-ghrelin with glucose-containing carbohydrate intake. When fed a diet rich in maltodextrin, starch and fat for 12 weeks, GOAT KO mice showed less food intake and weight gain, as well as improved glucose tolerance and insulin sensitivity than WT mice. Our data suggests that blockade of GOAT activity may offer a therapeutic option for treatment of obesity and its associated metabolic syndrome by preventing from overconsumption of carbohydrate-rich food.

Characterization of the role of sphingomyelin synthase 2 in glucose metabolism in whole-body and peripheral tissues in mice.

Sphingomyelin synthase 2 (SMS2) is a proposed potential therapeutic target for obesity and insulin resistance. However, the contributions of SMS2 to glucose metabolism in tissues and its possible therapeutic mechanisms remain unclear. Thus, to determine whole-body glucose utilization and the contributions of each insulin-targeted tissue to glucose uptake, we performed a glucose kinetics study, using the radiolabeled glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG), in wild-type (WT) and SMS2 knockout (KO) mice. Insulin signaling was enhanced in the liver, white adipose tissue and skeletal muscle of SMS2 KO mice compared with those of WT mice. In addition, compared with in WT mice, blood clearance of (18)F-FDG was accelerated in SMS2 KO mice when they were fed either a normal or a high fat diet. (18)F-FDG uptake was also increased in insulin-targeted tissues such as skeletal muscle in the SMS2 KO mice. Whereas skeletal muscle sphingolipid content was not clearly affected, plasma levels of very long-chain fatty acid (VLCFA)-containing ceramides were markedly increased in SMS2 KO mice, compared with in WT mice. We also generated liver-conditional SMS2 KO mice and performed glucose and insulin tolerance tests on mice with a high fat diet. However, no significant effect was observed. Thus, our study provided evidence that genetic inhibition of SMS2 elevated glucose clearance through activation of glucose uptake into insulin-targeted tissues such as skeletal muscle by a mechanism independent of hepatic SMS2. Our findings further indicate that this occurs, at least in part, via indirect mechanisms such as elevation of VLCFA-containing ceramides.

Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice.

Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18-C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18-C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.

Ghrelin O-acyltransferase knockout mice show resistance to obesity when fed high-sucrose diet.

Ghrelin is an appetite-stimulating hormone secreted from stomach. Since the discovery that acylation of the serine-3 residue by ghrelin O-acyltransferase (GOAT) is essential for exerting its functions, GOAT has been regarded as an therapeutic target for attenuating appetite, and thus for the treatment of obesity and diabetes. However, contrary to the expectations, GOAT-knockout (KO) mice have not shown meaningful body weight reduction, under high-fat diet. Here, in this study, we sought to determine whether GOAT has a role in body weight regulation and glucose metabolism with a focus on dietary sucrose, because macronutrient composition of diet is important for appetite regulation. We found that peripherally administered acylated-ghrelin, but not unacylated one, stimulated sucrose consumption in a two-bottle-drinking test. The role of acylated-ghrelin in sucrose preference was further supported by the finding that GOAT KO mice consumed less sucrose solution compared with WT littermates. Then, we investigated the effect of dietary composition of sucrose on food intake and body weight in GOAT KO and WT mice. As a result, when fed on high-fat diet, food intake and body weight were similar between GOAT KO and WT mice. However, when fed on high-fat, high-sucrose diet, GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, leading to amelioration of glucose metabolism. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity and metabolic disorders caused by overeating of palatable food.

Ndrg1 is a T-cell clonal anergy factor negatively regulated by CD28 costimulation and interleukin-2.

Induction of T-cell clonal anergy involves serial activation of transcription factors, including NFAT and Egr2/3. However, downstream effector mechanisms of these transcription factors are not fully understood yet. Here we identify Ndrg1 as an anergy factor induced by Egr2. Ndrg1 is upregulated by anergic signalling and maintained at high levels in resting anergic T cells. Overexpression of Ndrg1 mimics the anergic state and knockout of the gene prevents anergy induction. Interestingly, Ndrg1 is phosphorylated and degraded by CD28 signalling in a proteasome-dependent manner, explaining the costimulation dependence of anergy prevention. Similarly, IL-2 treatment of anergic T cells, under conditions that lead to the reversal of anergy, also induces Ndrg1 phosphorylation and degradation. Finally, older Ndrg1-deficient mice show T-cell hyperresponsiveness and Ndrg1-deficient T cells aggravate inducible autoimmune inflammation. Thus, Ndrg1 contributes to the maintenance of clonal anergy and inhibition of T-cell-mediated inflammation.

An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing. Only about 20,000 patterns in total were needed for SIRAS phasing while single wavelength anomalous dispersion (SAD) phasing was unsuccessful with more than 80,000 patterns of derivative crystals. We employed high energy X-rays from SACLA (12.6 keV) to take advantage of the large anomalous enhancement near the LIII absorption edge of Hg, which is one of the most widely used heavy atoms for phasing in conventional protein crystallography. Hard XFEL is of benefit for de novo phasing in the use of routinely used heavy atoms and high resolution data collection.

Exploring new gene integration sites for gene knock-in by gene-trapping strategy.

The knock-in mouse is a powerful tool for biological research, but the stability of expression of an integrated gene strongly depends on where it is integrated in the mouse genome. At present, there are an insufficient number of loci suitable for gene knock-in, such as the Rosa26 locus. Therefore, in this study, we developed an efficient strategy for identifying genome loci suitable for gene knock-in and characterized the properties of such loci for gene integration. For efficient discovery and characterization, we constructed a new gene-trapping vector that enables monitoring of the expression of both trapped and integrated genes using fluorescence. We successfully obtained fluorescent-positive mouse embryonic stem cell (mESC) clones with the vector. Thorough analysis of the expression of fluorescent proteins in chimera embryos generated with the obtained mESC clones, some of the gene-trapped chimera embryos showed stable and ubiquitous expression of the integrated gene. Furthermore, adult mice derived from one of the gene-trapped mESC clones showed ubiquitous expression of the integrated gene in various tissues without any unusual phenotype. This indicated that the identified locus possesses high potential for foreign gene integration. Our strategy allows for efficient discovery and characterization of mouse genome loci for gene integration.

Twisted gastrulation, a BMP antagonist, exacerbates podocyte injury.

Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7) in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1), a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs) and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.

Derlin-1 deficiency is embryonic lethal, Derlin-3 deficiency appears normal, and Herp deficiency is intolerant to glucose load and ischemia in mice.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes a cellular condition called ER stress. To overcome ER stress, unfolded proteins are eliminated by an ER-associated degradation (ERAD) system. To explore the physiological requirements for ERAD-related membrane proteins in mammals, we generated Derlin-1-, Derlin-3-, and Herp-deficient mice by gene targeting. Complete loss of Derlin-1 caused embryonic lethality at around E7-E8 (early somite stages). In contrast, Derlin-3- and Herp-deficient mice were born alive with the expected Mendelian frequency, and were superficially indistinguishable from wild-type mice. However, in the Derlin-3- and Herp-deficient mouse organs, the expression levels of ERAD-related proteins were affected under both normal and ER stress conditions; specific effects differed among the organs. Degradation of ERAD substrates was reduced in the Herp-deficient liver, and Herp-deficient mice exhibited impaired glucose tolerance and vulnerability to brain ischemic injury, both of which are known to be implicated in ER stress. Our findings indicate that ERAD or uncharacterized functions involving Derlin-1 are essential in early embryonic development. Derlin-3- and Herp-deficient mice may become useful model animals for investigations of the physiological contribution of ERAD under stressful or pathological conditions.

NDRG4 protein-deficient mice exhibit spatial learning deficits and vulnerabilities to cerebral ischemia.

The N-myc downstream-regulated gene (NDRG) family consists of four related proteins, NDRG1-NDRG4, in mammals. We previously generated NDRG1-deficient mice that were unable to maintain myelin sheaths in peripheral nerves. This condition was consistent with human hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D, caused by a nonsense mutation of NDRG1. In contrast, the effects of genetic defects of the other NDRG members remain unknown. In this study, we focused on NDRG4, which is specifically expressed in the brain and heart. In situ mRNA hybridization on the brain revealed that NDRG4 was expressed in neurons of various areas. We generated NDRG4-deficient mice that were born normally with the expected Mendelian frequency. Immunochemical analysis demonstrated that the cortex of the NDRG4-deficient mice contained decreased levels of brain-derived neurotrophic factor (BDNF) and normal levels of glial cell line-derived neurotrophic factor, NGF, neurotrophin-3, and TGF-ß1. Consistent with BDNF reduction, NDRG4-deficient mice had impaired spatial learning and memory but normal motor function in the Morris water maze test. When temporary focal ischemia of the brain was induced, the sizes of the infarct lesions were larger, and the neurological deficits were more severe in NDRG4-deficient mice compared with the control mice. These findings indicate that NDRG4 contributes to the maintenance of intracerebral BDNF levels within the normal range, which is necessary for the preservation of spatial learning and the resistance to neuronal cell death caused by ischemic stress.

The effect of Ndrg2 expression on astroglial activation.

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the central nervous system (CNS). To study the expression and possible role of Ndrg2 in quiescent and activated astrocytes, mice were administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP), a Parkinson disease (PD)-related neurotoxin which causes both neurodegeneration and glial activation. Immunohistological analysis revealed that Ndrg2 was highly expressed in both types of astrocytes, but less so in astrocytes during the early process of activation. Ndrg2 was also expressed in astrocyte-like cells, but not in neurons, in human brains from PD and Cortico-basal degeneration (CBD) patients. In cultured astrocytes, gene silencing of Ndrg2 significantly enhanced the numbers of 5-bromo-2'-deoxy-uridine (BrdU)-incorporated and proliferating cell nuclear antigen (PCNA)-positive cells, and reduced the length of cell processes and the amount of F-actin. In contrast, adenovirus-mediated overexpression of Ndrg2 significantly reduced the numbers of BrdU-incorporated and PCNA-positive cells, and enhanced the amount of F-actin. Fractionation and immunocytochemical analysis further revealed that Ndrg2 was located in different cellular fractions including the cytosol and cell surface membranes. These results suggest that Ndrg2 may regulate astroglial activation through the suppression of cell proliferation and stabilization of cell morphology.

Ndrg1 in development and maintenance of the myelin sheath.

CMT4D disease is a severe autosomal recessive demyelinating neuropathy with extensive axonal loss leading to early disability, caused by mutations in the N-myc downstream regulated gene 1 (NDRG1). NDRG1 is expressed at particularly high levels in the Schwann cell (SC), but its physiological function(s) are unknown. To help with their understanding, we characterise the phenotype of a new mouse model, stretcher (str), with total Ndrg1 deficiency, in comparison with the hypomorphic Ndrg1 knock-out (KO) mouse. While both models display normal initial myelination and a transition to overt pathology between weeks 3 and 5, the markedly more severe str phenotype suggests that even low Ndrg1 expression results in significant phenotype rescue. Neither model replicates fully the features of CMT4D: although axon damage is present, regenerative capacity is unimpaired and the mice do not display the early severe axonal loss typical of the human disease. The widespread large fibre demyelination coincides precisely with the period of rapid growth of the animals and the dramatic (160-500-fold) increase in myelin volume and length in large fibres. This is followed by stabilisation after week 10, while small fibres remain unaffected. Gene expression profiling of str peripheral nerve reveals non-specific secondary changes at weeks 5 and 10 and preliminary data point to normal proteasomal function. Our findings do not support the proposed roles of NDRG1 in growth arrest, terminal differentiation, gene expression regulation and proteasomal degradation. Impaired SC trafficking failing to meet the considerable demands of nerve growth, emerges as the likely pathogenetic mechanism in NDRG1 deficiency.