New rabies viral resources for multi-scale neural circuit mapping.

Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer's disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.

Molecular Mechanism of SR Protein Kinase 1 Inhibition by the Herpes Virus Protein ICP27.

Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins. Mutational analysis previously showed that the RGG box motif of ICP27 is required for interaction with SRPK1; however, the mechanism for the inhibition and the exact role of the RGG box was unknown. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to demonstrate that the isolated peptide comprising the RGG box of ICP27 binds to SRPK1 with high affinity, competing with a native substrate, the SR repeat region of SR protein SRSF1. We determined the crystal structure of the complex between SRPK1 and an RGG box peptide, which revealed that the viral peptide binds to the substrate docking groove, mimicking the interactions of SR repeats. Site-directed mutagenesis within the RGG box further confirmed the importance of selected arginine residues for interaction, relocalization, and inhibition of SRPK1 in vivo Together these data reveal the molecular mechanism of the competitive inhibition of cellular SRPK1 by viral ICP27, which modulates SRPK1 activity.IMPORTANCE Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.

Sensitivity of lipid metabolism and insulin signaling to genetic alterations in hepatic peroxisome proliferator-activated receptor-gamma coactivator-1alpha expression.

OBJECTIVE: The peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1 family of transcriptional coactivators controls hepatic function by modulating the expression of key metabolic enzymes. Hepatic gain of function and complete genetic ablation of PGC-1alpha show that this coactivator is important for activating the programs of gluconeogenesis, fatty acid oxidation, oxidative phosphorylation, and lipid secretion during times of nutrient deprivation. However, how moderate changes in PGC-1alpha activity affect metabolism and energy homeostasis has yet to be determined. RESEARCH DESIGN AND METHODS: To identify key metabolic pathways that may be physiologically relevant in the context of reduced hepatic PGC-1alpha levels, we used the Cre/Lox system to create mice heterozygous for PGC-1alpha specifically within the liver (LH mice). RESULTS: These mice showed fasting hepatic steatosis and diminished ketogenesis associated with decreased expression of genes involved in mitochondrial beta-oxidation. LH mice also exhibited high circulating levels of triglyceride that correlated with increased expression of genes involved in triglyceride-rich lipoprotein assembly. Concomitant with defects in lipid metabolism, hepatic insulin resistance was observed both in LH mice fed a high-fat diet as well as in primary hepatocytes. CONCLUSIONS: These data highlight both the dose-dependent and long-term effects of reducing hepatic PGC-1alpha levels, underlining the importance of tightly regulated PGC-1alpha expression in the maintenance of lipid homeostasis and glucose metabolism.

Gender-dependent HLA-DR-restricted epitopes identified from herpes simplex virus type 1 glycoprotein D.

In recent clinical trials, a herpes simplex virus (HSV) recombinant glycoprotein D (gD) vaccine was more efficacious in woman than in men. Here we report six HLA-DR-restricted T-cell gD epitope peptides that bind to multiple HLA-DR (DR1, DR4, DR7, DR13, DR15, and DRB5) molecules that represent a large proportion of the human population. Four of these peptides recalled naturally primed CD4(+) T cells in up to 45% of the 46 HSV-seropositive, asymptomatic individuals studied. For the gD(49-82), gD(77-104), and gD(121-152) peptides, the CD4(+) T-cell responses detected in HSV-seropositive, asymptomatic women were higher and more frequent than the responses detected in men. Immunization of susceptible DRB1*0101 transgenic mice with a mixture of three newly identified, gender-dependent, immunodominant epitope peptides (gD(49-82), gD(77-104), and gD(121-152)) induced a gender- and CD4(+) T-cell-dependent immunity against ocular HSV type 1 challenge. These results revealed a gender-dependent T-cell response to a discrete set of gD epitopes and suggest that while a T-cell epitope-based HSV vaccine that targets a large percentage of the human population may be feasible with a limited number of immunodominant promiscuous HLA-DR-restricted epitopes, gender should be taken into account during evaluations of such vaccines.

Regulation of energy substrate utilization and hepatic insulin sensitivity by phosphatidylcholine transfer protein/StarD2.

Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is a highly specific intracellular lipid binding protein with accentuated expression in oxidative tissues. Here we show that decreased plasma concentrations of glucose and free fatty acids in fasting PC-TP-deficient (Pctp(-/-)) mice are attributable to increased hepatic insulin sensitivity. In hyperinsulinemic-euglycemic clamp studies, Pctp(-/-) mice exhibited profound reductions in hepatic glucose production, gluconeogenesis, glycogenolysis, and glucose cycling. These changes were explained in part by the lack of PC-TP expression in liver per se and in part by marked alterations in body fat composition. Reduced respiratory quotients in Pctp(-/-) mice were indicative of preferential fatty acid utilization for energy production in oxidative tissues. In the setting of decreased hepatic fatty acid synthesis, increased clearance rates of dietary triglycerides and increased hepatic triglyceride production rates reflected higher turnover in Pctp(-/-) mice. Collectively, these data support a key biological role for PC-TP in the regulation of energy substrate utilization.

Loss of the PlagL2 transcription factor affects lacteal uptake of chylomicrons.

Enterocytes assemble dietary lipids into chylomicron particles that are taken up by intestinal lacteal vessels and peripheral tissues. Although chylomicrons are known to assemble in part within membrane secretory pathways, the modifications required for efficient vascular uptake are unknown. Here we report that the transcription factor pleomorphic adenoma gene-like 2 (PlagL2) is essential for this aspect of dietary lipid metabolism. PlagL2(-/-) mice die from postnatal wasting owing to failure of fat absorption. Lipids modified in the absence of PlagL2 exit from enterocytes but fail to enter interstitial lacteal vessels. Dysregulation of enterocyte genes closely linked to intracellular membrane transport identified candidate regulators of critical steps in chylomicron assembly. PlagL2 thus regulates important aspects of dietary lipid absorption, and the PlagL2(-/-) animal model has implications for the amelioration of obesity and the metabolic syndrome.

Interacting proteins dictate function of the minimal START domain phosphatidylcholine transfer protein/StarD2.

The Star (steroidogenic acute regulatory protein)-related transfer (START) domain superfamily is characterized by a distinctive lipid-binding motif. START domains typically reside in multidomain proteins, suggesting their function as lipid sensors that trigger biological activities. Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is an example of a START domain minimal protein that consists only of the lipid-binding motif. PC-TP, which binds phosphatidylcholine exclusively, is expressed during embryonic development and in several tissues of the adult mouse, including liver. Although it catalyzes the intermembrane exchange of phosphatidylcholines in vitro, this activity does not appear to explain the various metabolic alterations observed in mice lacking PC-TP. Here we demonstrate that PC-TP function may be mediated via interacting proteins. Yeast two-hybrid screening using libraries prepared from mouse liver and embryo identified Them2 (thioesterase superfamily member 2) and the homeodomain transcription factor Pax3 (paired box gene 3), respectively, as PC-TP-interacting proteins. These were notable because the START domain superfamily contains multidomain proteins in which the START domain coexists with thioesterase domains in mammals and with homeodomain transcription factors in plants. Interactions were verified in pulldown assays, and colocalization with PC-TP was confirmed within tissues and intracellularly. The acyl-CoA thioesterase activity of purified recombinant Them2 was markedly enhanced by recombinant PC-TP. In tissue culture, PC-TP coactivated the transcriptional activity of Pax3. These findings suggest that PC-TP functions as a phosphatidylcholine-sensing molecule that engages in diverse regulatory activities that depend upon the cellular expression of distinct interacting proteins.

Structure and function of phosphatidylcholine transfer protein (PC-TP)/StarD2.

Phosphatidylcholine transfer protein (PC-TP) is a highly specific soluble lipid binding protein that transfers phosphatidylcholine between membranes in vitro. PC-TP is a member of the steroidogenic acute regulatory protein-related transfer (START) domain superfamily. Although its biochemical properties and structure are well characterized, the functions of PC-TP in vivo remain incompletely understood. Studies of mice with homozygous disruption of the Pctp gene have largely refuted the hypothesis that this protein participates in the hepatocellular selection and transport of biliary phospholipids, in the production of lung surfactant, in leukotriene biosynthesis and in cellular phosphatidylcholine metabolism. Nevertheless, Pctp(-/-) mice exhibit interesting defects in lipid homeostasis, the understanding of which should elucidate the biological functions of PC-TP.

A polymorphism in New Zealand inbred mouse strains that inactivates phosphatidylcholine transfer protein.

New Zealand obese (NZO/HlLt) male mice develop polygenic diabetes and altered phosphatidylcholine metabolism. The gene encoding phosphatidylcholine transfer protein (PC-TP) is sited within the support interval for Nidd3, a recessive NZO-derived locus on Chromosome 11 identified by prior segregation analysis between NZO/HlLt and NON/Lt. Sequence analysis revealed that the NZO-derived PC-TP contained a non-synonymous point mutation that resulted in an Arg120His substitution, which was shared by the related NZB/BlNJ and NZW/LacJ mouse strains. Consistent with the structure-based predictions, functional studies demonstrated that Arg120His PC-TP was inactive, suggesting that this mutation contributes to the deficiencies in phosphatidylcholine metabolism observed in NZO mice.

Phosphatidylcholine transfer protein regulates size and hepatic uptake of high-density lipoproteins.

Phosphatidylcholine transfer protein (PC-TP) is a steroidogenic acute regulatory-related transfer domain protein that is enriched in liver cytosol and binds phosphatidylcholines with high specificity. In tissue culture systems, PC-TP promotes ATP-binding cassette protein A1-mediated efflux of cholesterol and phosphatidylcholine molecules as nascent pre-beta-high-density lipoprotein (HDL) particles. Here, we explored a role for PC-TP in HDL metabolism in vivo utilizing 8-wk-old male Pctp(-/-) and wild-type littermate C57BL/6J mice that were fed for 7 days with either chow or a high-fat/high-cholesterol diet. In chow-fed mice, neither plasma cholesterol concentrations nor the concentrations and compositions of plasma phospholipids were influenced by PC-TP expression. However, in Pctp(-/-) mice, there was an accumulation of small alpha-migrating HDL particles. This occurred without changes in hepatic expression of ATP-binding cassette protein A1 or in proteins that regulate the intravascular metabolism and clearance of HDL particles. In Pctp(-/-) mice fed the high-fat/high-cholesterol diet, HDL particle sizes were normalized, whereas plasma cholesterol and phospholipid concentrations were increased compared with wild-type mice. In the absence of upregulation of hepatic ATP-binding cassette protein A1, reduced HDL uptake from plasma into livers of Pctp(-/-) mice contributed to higher plasma lipid concentrations. These data indicate that PC-TP is not essential for the enrichment of HDL with phosphatidylcholines but that it does modulate particle size and rates of hepatic clearance.

Altered hepatic cholesterol metabolism compensates for disruption of phosphatidylcholine transfer protein in mice.

Phosphatidylcholine transfer protein (PC-TP) is a member of the steroidogenic acute regulatory transfer protein-related domain superfamily and is enriched in liver. To explore a role for PC-TP in hepatic cholesterol metabolism, Pctp-/- and wild-type C57BL/6J mice were fed a standard chow diet or a high-fat, high-cholesterol lithogenic diet. In chow-fed Pctp-/- mice, acyl CoA:cholesterol acyltransferase (Acat) activity was markedly increased, 3-hydroxy-3-methylglutaryl-CoA reductase activity was unchanged, and cholesterol 7alpha-hydroxylase activity was reduced. Consistent with increased Acat activity, esterified cholesterol concentrations in livers of Pctp-/- mice were increased, whereas unesterified cholesterol concentrations were reduced. Hepatic phospholipid concentrations were also decreased in the absence of PC-TP and consequently, unesterified cholesterol-to-phospholipid ratios in liver remained unchanged. The lithogenic diet downregulated 3-hydroxy-3-methylglutaryl-CoA reductase in wild-type and Pctp-/- mice, whereas Acat was increased only in wild-type mice. In response to the lithogenic diet, a greater reduction in cholesterol 7alpha-hydroxylase activity in Pctp-/- mice could be attributed to increased size and hydrophobicity of the bile salt pool. Despite higher hepatic phospholipid concentrations, the unesterified cholesterol-to-phospholipid ratio increased. The lack of Acat upregulation suggests that, in the setting of the dietary challenge, the capacity for esterification to defend against hepatic accumulation of unesterified cholesterol was exceeded in the absence of PC-TP expression. We speculate that regulation of cholesterol homeostasis is a physiological function of PC-TP in liver, which can be overcome with a cholesterol-rich lithogenic diet.

A biphasic response of hepatobiliary cholesterol metabolism to dietary fat at the onset of obesity in the mouse.

Human obesity is associated with abnormal hepatic cholesterol homeostasis and resistance to leptin action. Because leptin administration to rodents promotes the biliary elimination of plasma cholesterol, this study was designed to elucidate a pathophysiological role for leptin during the development of obesity. We fed mice diets containing high or low saturated fat contents. Before and after the onset of obesity, we measured downstream targets of leptin action and evaluated plasma, hepatic, and biliary cholesterol metabolism. Although not obese at 28 days, mice fed a high fat diet became hyperleptinemic. Sensitivity to leptin was evidenced by downregulation of both hepatic stearoyl CoA desaturase-1 and fatty acid synthase. Due principally to upregulation of adenosine triphosphate-binding cassette proteins A1 and G5, plasma high density lipoprotein (HDL) cholesterol concentrations increased, as did relative secretion rates of biliary cholesterol. A smaller, more hydrophilic bile salt pool decreased intestinal cholesterol absorption. In this setting, hepatic cholesterol synthesis was downregulated, indicative of increased uptake of plasma cholesterol. After 56 days of high fat feeding, obesity was associated with leptin resistance, as evidenced by marked hyperleptinemia without downregulation of stearoyl CoA desaturase-1 or fatty acid synthase and by upregulation of hepatic cholesterol and bile salt synthesis. Hypercholesterolemia was attributable to overproduction and decreased clearance of large HDL(1) particles. In conclusion, before the onset of obesity, preserved leptin sensitivity promotes biliary elimination of endogenous cholesterol in response to dietary fat. Leptin resistance due to obesity leads to a maladaptive response whereby newly synthesized cholesterol in the liver is eliminated via bile.

Impaired response of biliary lipid secretion to a lithogenic diet in phosphatidylcholine transfer protein-deficient mice.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein that is highly expressed in liver and catalyzes intermembrane transfer of phosphatidylcholines in vitro. To explore a role for PC-TP in the hepatocellular trafficking of biliary phosphatidylcholines, we characterized biliary lipid secretion using Pctp(-/-) and wild-type littermate control mice with C57BL/6J and FVB/NJ genetic backgrounds, which express PC-TP at relatively high and low levels in liver, respectively. Eight-week-old male Pctp(-/-) and wild-type mice were fed a chow diet or a lithogenic diet, which served to upregulate biliary lipid secretion. In chow-fed mice, the absence of PC-TP did not reduce biliary phospholipid secretion or alter the phospholipid composition of biles. However, the responses in secretion of biliary phospholipids, cholesterol, and bile salts to the lithogenic diet were impaired in Pctp(-/-) mice from both genetic backgrounds. Alterations in biliary lipid secretion could not be attributed to transcriptional regulation of the expression of canalicular membrane lipid transporters, but possibly to a defect in their trafficking to the canalicular membrane. These findings support a role for PC-TP in the response of biliary lipid secretion to a lithogenic diet, but not specifically in the hepatocellular transport and secretion of phosphatidylcholines.