Adeno-Associated Virus-Mediated Dorsal Root Ganglion Toxicity in the New Zealand White Rabbit.

Recombinant adeno-associated virus (AAV)-mediated degeneration of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) has been observed in non-human primates (NHPs) following intravenous (IV) and intrathecal (IT) delivery. Administration of recombinant AAV encoding a human protein transgene via a single intra-cisterna magna (ICM) injection in New Zealand white rabbits resulted in histopathology changes very similar to NHPs: mononuclear cell infiltration, degeneration/necrosis of sensory neurons, and nerve fiber degeneration of sensory tracts in the spinal cord and of multiple nerves. AAV-associated clinical signs and incidence/severity of histologic findings indicated that rabbits were equally or more sensitive than NHPs to sensory neuron damage. Another study using human and rabbit transgene constructs of the same protein demonstrated comparable changes suggesting that the effects are not an immune response to the non-self protein transgene. Rabbit has not been characterized as a species for general toxicity testing of AAV gene therapies, but these studies suggest that it may be an alternative model to investigate mechanisms of AAV-mediated neurotoxicity and test novel AAV designs mitigating these adverse effects.

RNAi screening in primary human hepatocytes of genes implicated in genome-wide association studies for roles in type 2 diabetes identifies roles for CAMK1D and CDKAL1, among others, in hepatic glucose regulation.

Genome-wide association (GWA) studies have described a large number of new candidate genes that contribute to of Type 2 Diabetes (T2D). In some cases, small clusters of genes are implicated, rather than a single gene, and in all cases, the genetic contribution is not defined through the effects on a specific organ, such as the pancreas or liver. There is a significant need to develop and use human cell-based models to examine the effects these genes may have on glucose regulation. We describe the development of a primary human hepatocyte model that adjusts glucose disposition according to hormonal signals. This model was used to determine whether candidate genes identified in GWA studies regulate hepatic glucose disposition through siRNAs corresponding to the list of identified genes. We find that several genes affect the storage of glucose as glycogen (glycolytic response) and/or affect the utilization of pyruvate, the critical step in gluconeogenesis. Of the genes that affect both of these processes, CAMK1D, TSPAN8 and KIF11 affect the localization of a mediator of both gluconeogenesis and glycolysis regulation, CRTC2, to the nucleus in response to glucagon. In addition, the gene CDKAL1 was observed to affect glycogen storage, and molecular experiments using mutant forms of CDK5, a putative target of CDKAL1, in HepG2 cells show that this is mediated by coordinate regulation of CDK5 and PKA on MEK, which ultimately regulates the phosphorylation of ribosomal protein S6, a critical step in the insulin signaling pathway.

Identification of the CREB-binding protein/p300-interacting protein CITED2 as a peroxisome proliferator-activated receptor alpha coregulator.

Like other nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) use a wide variety of protein-protein interactions to properly regulate transcription of target genes. In an attempt to identify novel PPAR-interacting proteins, a cDNA expression library was screened with bacterially expressed PPARalpha. One of the genes identified as a PPARalpha-associated protein by interaction cloning was the CREB-binding protein/p300-interacting transactivator with ED-rich tail 2 (CITED2, also called p35srj/mrg1/msg1). This coactivator interacted directly with PPARalpha in the presence or absence of ligand predominantly via the ligand binding domain of the nuclear receptor. In transient transfection reporter assays, CITED2 acted as a dose-dependent coactivator of PPARalpha-dependent transcriptional regulation in the presence of several exogenous ligands. CITED2 also increased PPARgamma-dependent regulation of reporter genes but had no effect on PPARbeta activity. To determine whether CITED2 affects endogenous gene expression, this protein was stably overexpressed (CITED2+) or repressed by small inhibitor RNA (CITED2-) in immortalized mouse hepatocytes. Relative to the control stably transfected or CITED2-cells, CITED2+ cells had an increased rate of cell proliferation. Microarray analysis and real time PCR showed that several genes are differentially affected by PPARalpha ligands in CITED2+ versus CITED2-cells. Genes that were affected by PPARalpha ligands in a CITED2-modulatory manner include angiopoietin-like protein 4, forkhead C2, hypoxia-inducible factor-1alpha, and MAPK phosphatase 1. Interestingly these genes share common functions in that they are known to promote vascularization and angiogenesis in response to hypoxia. The results described here suggest that CIT-ED2 is a coactivator of PPARalpha and that both proteins may participate in signaling cascades of hypoxic response and angiogenesis.

Comprehensive gene expression analysis of peroxisome proliferator-treated immortalized hepatocytes: identification of peroxisome proliferator-activated receptor alpha-dependent growth regulatory genes.

Chemicals known as peroxisome proliferators (PPs) are the subject of intense study because of their ability to cause hepatocellular carcinoma in laboratory rodents. These chemicals act through a family of proteins termed the peroxisome proliferator-activated receptors (PPARs), in particular PPARalpha. It has become increasingly apparent that the role of the PPs in the development of cancer encompasses many different aspects of cell growth regulation. Immortalized hepatocytes from wild-type (PPARalpha(+/+)) and PPARalpha(-/-) mice were generated using a temperature-sensitive SV40 virus. Characterization of the murine SV40 hepatocytes (MuSH) generated from both genotypes (MuSHalpha(+/+), MuSHalpha(-/-)) show markers of differentiation such as albumin expression, but is devoid of Kupffer cell contamination. Hallmark PPARalpha-mediated responses such as induction of acyl-CoA oxidase mRNA by PPs are present in the MuSHalpha(+/+) but are absent in MuSHalpha(-/-) cells. In contrast to most cell culture systems, the wild-type MuSH hepatocytes retain the mitogenic activity of PPs, whereas the MuSHalpha(-/-) does not respond in this manner, thus making this cell culture system an ideal tool to examine growth regulatory gene expression affected by PPs. Microarray experiments performed on both cell types identified many genes in which regulation is dependent on the presence of PPARalpha, and these changes were verified with reverse transcriptase-PCR. Genes involved in carcinogenesis and control of the cell cycle that are regulated by PPs in a PPARalpha-dependent manner include ubiquitin COOH-terminal hydrolase 37 (also known as UCT-L5) and cyclin T1. These results show that MuSH cells reflect the biological properties of both the wild-type and PPARalpha-null animals and can be used to identify novel PPARalpha-regulated genes that could be involved in regulation of the cell cycle and carcinogenesis.

Heat shock protein-90 (Hsp90) acts as a repressor of peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARbeta activity.

The nuclear receptor (NR) peroxisome proliferator-activated receptor-alpha (PPARalpha) mediates the effects of several hypolipidemic drugs, endogenous fatty acids, and peroxisome proliferators. Despite belonging to a class of NR not known to interact with cytosolic chaperone complexes, we have recently shown that PPARalpha interacts with heat shock protein 90 (Hsp90), although the biological consequence of this association was unknown. In the present study, PPARalpha directly associated with Hsp90 in vitro to a much greater extent than either PPARbeta or PPARgamma. This interaction is similar to other NR-Hsp90 complexes with association occurring between the middle of Hsp90 and the hinge (D) and ligand binding domain (EF) of PPARalpha. Using several different approaches to disrupt Hsp90 complexes within the cell, we demonstrate that Hsp90 is a repressor of both PPARalpha and PPARbeta activity. Treatment with geldanamycin (GA) increased the activity of PPARalpha and in the presence of ligand in transient transfection assays. PPARalpha-response element (PPRE)-reporter assays in a stable cell line treated with GA resulted in enhanced expression of a known target gene, acyl-CoA oxidase. Similarly, overexpression of the tetratricopeptide repeat (TPR) of protein phosphatase 5 (PP5) increased PPARalpha or PPARbeta activity in a PPRE-reporter assay and decreased the interaction between PPARalpha or PPARbeta and Hsp90 in a mammalian two-hybrid assay. Finally, cotransfection with the C-terminal hsp-interacting protein (CHIP) construct, a TPR-containing ubiquitin ligase that interacts with hsp90, increased PPARalpha's and decreased PPARbeta's ability to regulate PPRE-reporter activity upon ligand activation. All three methods to disrupt Hsp90 function (GA, PP5-TPR, CHIP) resulted in an alteration in PPARalpha or PPARbeta activity to a much greater extent than PPARgamma. While FKBP52 had no effect on PPARalpha activity, p23 greatly enhanced constitutive and Wy14 643 induced PPRE-reporter activity. Thus, we describe the chaperone complex as being a regulator of PPARalpha and PPARbeta activity and have identified a novel, subtype-specific, inhibitory role for Hsp90.

Evidence that peroxisome proliferator-activated receptor alpha is complexed with the 90-kDa heat shock protein and the hepatitis virus B X-associated protein 2.

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-inducible transcription factor, which belongs to the nuclear receptor superfamily. PPARalpha mediates the carcinogenic effects of peroxisome proliferators in rodents. In humans, PPARalpha plays a fundamental role in regulating energy homeostasis via control of lipid metabolism. To study the possible role of chaperone proteins in the regulation of PPARalpha activity, a monoclonal antibody (mAb) was made against PPARalpha and designated as 3B6/PPAR. The specificity of mAb 3B6/PPAR in recognizing PPARalpha was tested in immunoprecipitations using in vitro translated PPAR subtypes. The mAb 3B6/PPAR recognized PPARalpha, failed to bind to PPARbeta or PPARgamma, and is efficient in both immunoprecipitating and visualizing the receptor on protein blots. The immunoprecipitation of PPARalpha in mouse liver cytosol using mAb 3B6/PPAR has resulted in the detection of two co-immunoprecipitated proteins, which are heat shock protein 90 (hsp90) and the hepatitis B virus X-associated protein 2 (XAP2). The concomitant depletion of PPARalpha in hsp90-depleted mouse liver cytosol was also detected. Complex formation between XAP2 and PPARalpha/FLAG was also demonstrated in an in vitro translation binding assay. hsp90 interacts with PPARalpha in a mammalian two-hybrid assay and binds to the E/F domain. Transient expression of XAP2 co-expressed with PPARalpha resulted in down-regulation of a peroxisome proliferator response element-driven reporter gene activity. Taken together, these results indicate that PPARalpha is in a complex with hsp90 and XAP2, and XAP2 appears to function as a repressor. This is the first demonstration that PPARalpha is stably associated with other proteins in tissue extracts and the first nuclear receptor shown to functionally interact with XAP2.