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Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.

Study Design: Survey study and retrospective review of prospective data., Objectives: To contrast surgeons' perceptions and reported national data regarding the rates of postoperative adverse events following anterior cervical discectomy and fusion (ACDF) and to assess the accuracy of surgeons in predicting the impact of patient factors on such outcomes., Methods: A survey investigating perceived rates of perioperative complications and the perceived effect of patient risk factors on the occurrence of complications following ACDF was distributed to spine surgeons at the Cervical Spine Research Society (CSRS) 2015 Annual Meeting. The equivalent reported rates of adverse events and impacts of patient risk factors on such complications were assessed in patients undergoing elective ACDF from the National Surgical Quality Improvement Program (NSQIP)., Results: There were 110 completed surveys from attending physicians at CSRS (response rate = 44%). There were 18 019 patients who met inclusion criteria in NSQIP years 2011 to 2014. The rates of 11 out of 17 (65%) postoperative adverse events were mildly overestimated by surgeons responding to the CSRS questionnaire in comparison to reported NSQIP data (overestimates ranged from 0.24% to 1.50%). The rates of 2 out of 17 (12%) postoperative adverse events were mildly underestimated by surgeons (range = 0.08% to 1.2%). The impacts of 5 out of 10 (50%) patient factors were overestimated by surgeons (range relative risk = 0.56 to 1.48)., Conclusions: Surgeon estimates of risk factors for and rates of adverse events following ACDF procedures were reasonably nearer to national data. Despite an overall tendency toward overestimation, surgeons' assessments are roughly appropriate for surgical planning, expectation setting, and quality improvement initiatives., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Study Design: Retrospective cohort study., Objective: To determine if International Classification of Diseases (ICD) coding for obesity is biased toward certain subgroups and how potential bias may influence the outcomes of database research in spine., Summary of Background Data: There has been increased use of national databases using administrative data in the spine surgery literature. Past research demonstrates that sensitivity of ICD codes for obesity is poor, but it is unknown if such inaccuracies are systematically biased and if they may bias studies utilizing such data., Methods: Patients who underwent elective posterior lumbar fusion, 2013 to 2016, at a large academic hospital were identified. All ICD codes assigned to the encounter were obtained. Body mass index (BMI) was calculated based on height and weight. The sensitivity of ICD coding for obesity was calculated. Sensitivity was compared for subgroups defined by demographic, comorbidity, intraoperative, and postoperative factors. The association of obesity (as defined by BMI≥30 and ICD coding) with 30-day postoperative adverse events was tested with multivariate regression., Results: The study included 796 patients. The overall sensitivity of ICD coding for obesity was 42.5%. The sensitivity of ICD coding for obesity was significantly higher in patients with greater BMI, diabetes, American Society of Anesthesiologists class≥III, increased length of stay, venous thromboembolism, any adverse event, and major adverse event. Multivariate analysis for determining outcomes of increased risk with obesity as defined by ICD coding included venous thromboembolism, major adverse events, and any adverse events. However, multivariate analysis for determining outcomes of increased risk with obesity defined by BMI did not yield any positive associations., Conclusion: ICD codes for obesity are more commonly assigned to patients with other comorbidities or postoperative complications. Further, use of such nonrandomly assigned ICD codes for obesity has the potential to skew studies to suggest greater associated adverse events than calculated BMI would demonstrate., Level of Evidence: 3.

Study Design: Retrospective cohort comparison study., Objective: To compare perioperative outcomes between navigated and conventional single-level instrumented posterior lumbar fusions in the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) database., Summary of Background Data: Although multiple studies have investigated the accuracy of pedicle screw placement and radiation exposure with navigation, no study has compared perioperative complications between navigated and conventional posterior lumbar fusion. The potential benefits of navigation include improved accuracy of screw placement and reduced surgeon radiation exposure, but this is balanced by potential operative time and surgical site contamination/infection related to this bulky technology., Methods: Patients who underwent navigated or conventional single-level posterior instrumented lumbar fusions were identified in the 2010-2015 NSQIP database. The usage of navigation was characterized. Patient characteristics and comorbidities were compared between the two treatment groups. Propensity score matching was done and comparisons were made for operative time, hospital length of stay, postoperative complications, and 30-day readmissions between the two cohorts., Results: The percentage of navigated cases tended to increase over years studied to approximately 10%. After propensity matching to control potential confounding factors, statistical analysis revealed no significant difference in operative time and for most adverse events including wound infection, return to the operating room, and readmission. There were significantly lower blood transfusions in the navigated cohort (2.84% vs. 7.15%, P < 0.001). Patients who underwent navigated surgery also had a shorter mean hospital length of stay (0.2 day difference, P = 0.016)., Conclusion: The reduced blood loss and mildly reduced hospital length of stay identified for the navigated cases are probably markers of more minimally invasive surgery in the navigated cohort. The current study could not identify other differences in operative time, wound infection, or return to the operating room/readmission between navigated and conventional single level posterior instrumented lumbar cases., Level of Evidence: 3.

Introduction: Hospital stay after posterior spinal fusion (PSF) for adolescent idiopathic scoliosis (AIS) has decreased only modestly over time despite a healthy patient population. The purpose of this study was to evaluate the impact of a novel postoperative pathway on length of stay (LOS) and complications., Methods: A retrospective review of patients undergoing PSF for AIS in 2011 to 2012 was performed at 2 institutions evaluating demographics, preoperative Cobb angles, surgical duration, blood loss, LOS, and postoperative complications. Patients at one center were managed using an accelerated discharge (AD) pathway emphasizing early transition to oral pain medications mobilization with physical therapy 2 to 3 times/d, and discharge regardless of return of bowel function. Expectations were set with the family before surgery for early discharge. Patients at the other center were managed without a standardized pathway., Results: One hundred five patients underwent PSF and were treated by an AD pathway, whereas 45 patients were managed using a traditional discharge (TD) pathway. There was no difference in proximal thoracic and main thoracic Cobb magnitudes and a small difference in thoracolumbar curve magnitudes (35.2±13.0 degrees AD vs. 40.6±11.4 degrees TD, P=0.004) between groups. Surgical time was slightly shorter in AD patients (median 3.1 vs. 3.9 h, P=0.0003) with no difference in estimated blood loss. LOS was 48% shorter in the AD group (2.2 vs. 4.2 d, P<0.0001). There was no difference in readmissions or wound complications between groups., Conclusions: Hospital stay was nearly 50% shorter in patients managed by the AD pathway without any increase in readmissions or early complications., Significance: Discharge after PSF for AIS may be expedited using a coordinated postoperative pathway. No increase in complications was seen using the AD pathway. Earlier discharge may reduce health care costs and allow an earlier return to normalcy for families., Level of Evidence: Level III-case control study.

Study Design: Retrospective cohort study., Objective: To compare short-term morbidity for primary and revision posterior lumbar fusions., Summary of Background Data: Revision lumbar fusions are unfortunately relatively common. Previous studies have described an increased risk of postoperative complications after revision lumbar fusion; however, these studies have been limited by small sample sizes, poor data quality, and/or narrow outcome measures. There is a need to validate these findings using a high-quality, national cohort of patients to have an accurate assessment of the relative risk of revision posterior lumbar fusions compared with primary lumbar fusion., Methods: The prospectively-collected American College of Surgeons National Surgical Quality Improvement Program database was used to identify patients that underwent undergoing primary and revision posterior lumbar fusion from 2005 to 2013. The occurrence of individual and aggregated postoperative complications within 30 days, along with rates of blood transfusion and readmission, were compared between primary and revision procedures using bivariate and multivariate Poisson regression with robust error variance to control for patient and operative characteristics. Operative time and postoperative length of stay were compared between groups using bivariate and multivariate linear regression., Results: Of the 14,873 posterior lumbar fusion procedures that met inclusion criteria, 1287 (8.7%) were revision cases. There were no differences in the rates of 30-day postoperative complications or readmission between primary and revision posterior lumbar fusion using multivariate analysis to control for patient and operative characteristics. Similarly, no significant differences were found for operative time or postoperative length of stay. There was an increased rate of blood transfusion for revision surgery compared with primary surgery (relative risk 1.4, P < 0.001)., Conclusion: This study suggests that revision posterior lumbar fusion does not carry significantly increased risk of complications or readmission compared with a primary posterior lumbar fusion. Patients undergoing revision surgery were more likely to receive a blood transfusion. This information suggests that general health risk stratification for revision procedures can be similar to that considered for primary cases., Level of Evidence: 3.

Ghrelin influences a variety of metabolic functions through a direct action at its receptor, the GhrR (GhrR-1a). Ghrelin knockout (KO) and GhrR KO mice are resistant to the negative effects of high-fat diet (HFD) feeding. We have generated several classes of small-molecule GhrR antagonists and evaluated whether pharmacologic blockade of ghrelin signaling can recapitulate the phenotype of ghrelin/GhrR KO mice. Antagonist treatment blocked ghrelin-induced and spontaneous food intake; however, the effects on spontaneous feeding were absent in GhrR KO mice, suggesting target-specific effects of the antagonists. Oral administration of antagonists to HFD-fed mice improved insulin sensitivity in both glucose tolerance and glycemic clamp tests. The insulin sensitivity observed was characterized by improved glucose disposal with dramatically decreased insulin secretion. It is noteworthy that these results mimic those obtained in similar tests of HFD-fed GhrR KO mice. HFD-fed mice treated for 56 days with antagonist experienced a transient decrease in food intake but a sustained body weight decrease resulting from decreased white adipose, but not lean tissue. They also had improved glucose disposal and a striking reduction in the amount of insulin needed to achieve this. These mice had reduced hepatic steatosis, improved liver function, and no evidence of systemic toxicity relative to controls. Furthermore, GhrR KO mice placed on low- or high-fat diets had lifespans similar to the wild type, emphasizing the long-term safety of ghrelin receptor blockade. We have therefore demonstrated that chronic pharmacologic blockade of the GhrR is an effective and safe strategy for treating metabolic syndrome.

Background: We and others have demonstrated previously that ghrelin receptor (GhrR) knock out (KO) mice fed a high fat diet (HFD) have increased insulin sensitivity and metabolic flexibility relative to WT littermates. A striking feature of the HFD-fed GhrR KO mouse is the dramatic decrease in hepatic steatosis. To characterize further the underlying mechanisms of glucose homeostasis in GhrR KO mice, we conducted both hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI-E) clamps. Additionally, we investigated tissue glucose uptake and specifically examined liver insulin sensitivity., Results: Consistent with glucose tolerance-test data, in HG clamp experiments, GhrR KO mice showed a reduction in glucose-stimulated insulin release relative to WT littermates. Nevertheless, a robust 1st phase insulin secretion was still achieved, indicating that a healthy β-cell response is maintained. Additionally, GhrR KO mice demonstrated both a significantly increased glucose infusion rate and significantly reduced insulin requirement for maintenance of the HG clamp, consistent with their relative insulin sensitivity. In HI-E clamps, both LFD-fed and HFD-fed GhrR KO mice showed higher peripheral insulin sensitivity relative to WT littermates as indicated by a significant increase in insulin-stimulated glucose disposal (Rd), and decreased hepatic glucose production (HGP). HFD-fed GhrR KO mice showed a marked increase in peripheral tissue glucose uptake in a variety of tissues, including skeletal muscle, brown adipose tissue and white adipose tissue. GhrR KO mice fed a HFD also showed a modest, but significant decrease in conversion of pyruvate to glucose, as would be anticipated if these mice displayed increased liver insulin sensitivity. Additionally, the levels of UCP2 and UCP1 were reduced in the liver and BAT, respectively, in GhrR KO mice relative to WT mice., Conclusions: These results indicate that improved glucose homeostasis of GhrR KO mice is characterized by robust improvements of glucose disposal in both normal and metabolically challenged states, relative to WT controls. GhrR KO mice have an intact 1st phase insulin response but require significantly less insulin for glucose disposal. Our experiments reveal that the insulin sensitivity of GhrR KO mice is due to both BW independent and dependent factors. We also provide several lines of evidence that a key feature of the GhrR KO mouse is maintenance of hepatic insulin sensitivity during metabolic challenge.

To define the relationship between the respiratory quotient (RQ) and energy intake (EI) and to determine the impact of spontaneous locomotor activity (LMA) in the development of diet-induced obesity (DIO), we fed C57BL/6 mice a high-fat diet (HFD) for either 4 days or 17 wk and analyzed them using indirect calorimetry. Importantly, changes in body mass during calorimetry (DeltaM(b)) significantly covaried with RQ and EI; adjusting the data for DeltaM(b) permitted an analysis of the energy-balanced state. The 24-h RQ strongly predicted 24-h EI, and the slope of this relationship was diet dependent (HFD or chow) but independent of the HFD feeding period. Early-stage DIO was characterized by dark-period hyperphagia and fat storage, offset by greater light-period lipid oxidation; later stage DIO mice had a milder hyperphagia and lower substrate flexibility. Consequently, whereas 24-h RQ equaled the food quotient of the HFD in both early- and late-stage DIO, the range of RQ values was negatively correlated with, and mostly explained by, 24-h EI only in late-stage DIO. Lean and early-stage DIO mice had similar LMA values that were reduced in late-stage DIO. However, LMA significantly explained variance in total energy expenditure (EE) in only early-stage DIO mice. This indicated that the link between LMA and EE was a transient adaptive response to early DIO, whereas the later loss of LMA did not explain body weight gain in C57BL/6 DIO mice.

The goal of this study was to examine factors that contribute to energy balance in female GHR -/- mice. We measured energy intake, energy expenditure (EE), fuel utilization, body mass (M(b)) changes and physical activity in 17month-old female GHR -/- mice and their age-matched wild type littermates. The GHR -/- mice were smaller, consumed more food per unit M(b), had greater EE per unit M(b) and had an increase in 24-h EE/M(b) that was similar to the increase in their surface-area-to-volume ratio. Locomotor activity (LMA) was reduced in the GHR -/- mice, but the energetic cost associated with their LMA was greater than in wild type controls. Furthermore, M(b) and LMA were independent explanatory covariates of most of the variance in EE, and when adjusted for M(b) and LMA, the GHR -/- mice had higher EE during both the light and dark phases of the daily cycle. Respiratory quotient was lower in GHR -/- mice during the light phase, which indicated a greater utilization of lipid relative to carbohydrate in these mice. Additionally, GHR -/- mice had higher ratios of caloric intake to EE at several intervals during the dark phase, and this effect was greater and more sustained in the final 3h of the dark phase. Therefore, we conclude that GHR -/- mice are able to overcome the substantial energetic challenges of dwarfism through several mechanisms that promote stable M(b). Relative to wild type mice, the GHR -/- mice consumed more calories per unit M(b), which offset the disproportionate increase in their daily energy expenditure. While GHR -/- mice oxidized a greater proportion of lipid during the light phase in order to meet their energy requirements, they achieved greater energy efficiency and storage during the dark phase through a combination of higher energy consumption and lower LMA., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The orexigenic peptide ghrelin has been shown to have prokinetic activity in the gastrointestinal (GI) system of several species, including humans. In this series of experiments, we have evaluated the prokinetic activity of novel, small-molecule ghrelin receptor (GhrR) agonists after parenteral and peroral dosing in mice and rats. Gastric emptying, small intestinal transport, and fecal output were determined after intraperitoneal and intracerebroventricular dosing of GhrR agonists, using ghrelin as a positive control. These same parameters were evaluated after oral gavage dosing of the synthetic agonists. Regardless of dose route, GhrR agonist treatment increased gastric emptying, small intestinal transit, and fecal output. However, fecal output was only increased by GhrR agonist treatment if mice were able to feed during the stimulatory period. Thus, GhrR agonists can stimulate upper GI motility, and the orexigenic action of the compounds can indirectly contribute to prokinetic activity along the entire GI tract. The orexigenic and prokinetic effects of either ghrelin or small-molecule GhrR agonists were selective for the GhrR because they were absent when evaluated in GhrR knockout mice. We next evaluated the efficacy of the synthetic GhrR agonists dosed in a model of opiate-induced bowel dysfunction induced by a single injection of morphine. Oral dosing of a GhrR agonist normalized GI motility in opiate-induced dysmotility. These data demonstrate the potential utility of GhrR agonists for treating gastrointestinal hypomotility disorders.

Stimulation of the ghrelin receptor (GhrR) by ghrelin results in a variety of metabolic changes including increased food intake, fat storage and insulin resistance. Loss of ghrelin signaling is protective against diet-induced obesity, suggesting that ghrelin plays a significant homeostatic role in conditions of metabolic stress. We examined glycemic control in GhrR -/- mice fed a high-fat diet, and used indirect calorimetry to assess fuel substrate usage and energy expenditure. GhrR -/- mice fed a high-fat diet had several measures of greater insulin sensitivity, including: lower fasted blood glucose and plasma insulin, lower %Hb(A1c), lower insulin levels during glucose tolerance tests, and improved performance in hyperinsulinemic-euglycemic and hyperglycemic clamp studies. GhrR -/- mice fed a high-fat diet did not develop hepatic steatosis and had lower total cholesterol, relative to controls. Furthermore, GhrR -/- mice demonstrated a lower intestinal triglyceride secretion rate of dietary lipid. GhrR -/- mice have higher respiratory quotients (RQ), indicating a preference for carbohydrate as fuel. The range of RQ values was wider in GhrR -/- mice, indicating greater metabolic flexibility and insulin sensitivity in these animals. We therefore propose that loss of ghrelin signaling promotes insulin sensitivity and metabolic flexibility, and protects against several fatty diet-induced features of metabolic syndrome due to convergent changes in the intake, absorption and utilization of energy.

Identification of genes and pathways that alter lifespan has allowed for new insights into factors that control the aging process as well as disease. While strong molecular links exist between aging and metabolism, we hypothesize that targeting the mechanisms involved in aging will also give rise to therapeutics that treat other devastating age-related diseases, such as neurodegeneration, cancer, inflammation and cardiovascular disease. Insulin sensitivity, glycemic control and adiposity are not only hallmarks of the major metabolic diseases, type 2 diabetes and obesity, but they also represent significant risk factors for the development of Alzheimer's Disease and cognitive impairment. Insulin/IGF-1 signaling is an important pathway regulating aging and disease in a variety of species, including mammals. Here we describe an important role for the gut-derived peptide ghrelin in upstream signaling through the insulin/IGF-1 pathway and exemplify modulation of ghrelin signaling as an approach to mechanistic treatment of multiple age-related diseases by virtue of its ability to regulate key metabolic functions.

To identify new drug targets for the treatment of obesity, we employed a degenerate reverse transcriptasepolymerase chain reaction technique to isolate novel members of the G-protein coupled receptor superfamily from mouse hypothalamus. One of our clones was found to encode a protein with 90% amino acid identity to human GPR10, which was previously identified as the receptor for prolactin-releasing peptide (PrRP) and has been implicated in lactation, the regulation of food intake and other physiological functions. To investigate the role of GPR10 in food intake and body weight homeostasis, we generated mice carrying a targeted deletion of the GPR10 gene. First, using these knockout animals, we confirmed that GPR10 is the principle receptor for PrRP in the mouse hypothalamus because deletion of GPR10 completely abolished PrRP binding to isolated hypothalamic cell membranes. Second, we investigated the effect of normal and high-fat diets on energy intake, body weight, and glucose homeostasis in wild-type and GPR10 knockout mice. After fasting and refeeding, food intake in knockout animals was unchanged relative to control littermates. However, beginning at 16 wk of age on a normal diet, knockout mice became hyperphagic, obese, and showed significant increases in body fat and the levels of leptin and insulin, as well as decreased glucose tolerance. This metabolic profile was similar to the effect of a high-fat diet on wild-type animals. Our findings provide direct evidence that GPR10 is the receptor for PrRP and that it is involved in the regulation of energy balance in mice. GPR10 knockout mice will also prove useful for investigating other proposed activities for PrRP.

This study reports the characterization of a recombinant adenoviral vector containing a tetracycline-regulatable promoter, driving the bicistronic expression of the human H2 preprorelaxin (hH2) cDNA and enhanced green fluorescent protein, via an internal ribosomal entry site. An hH2 ELISA was used to measure the secreted levels of recombinant hH2 in transfected canine (CF33.Mt) and human (MDA-MB-435) mammary cancer cell lines over a 6-d period; secreted peptide peaked on d 2 and 4 for the canine and human cell types, respectively. An unprocessed hH2 immunoreactive form of approximately 18 kDa was identified by Western blotting analysis and confirmed by mass spectrometry, suggesting that prorelaxin remains unprocessed in these cell types. The biological activity of the adenovirally expressed human prorelaxin was measured in the established human monocytic cell line THP-1 cAMP ELISA and in an in vitro Transwell cell migration system. Exogenous recombinant hH2 and adenovirally-mediated delivery of prorelaxin to CF33.Mt cells conferred a significant migratory action in the cells, compared with controls. Cell proliferation assays were performed to discount the possibility that the effect of relaxin was mitogenic. Thus, we have demonstrated that prorelaxin has the ability to facilitate cell migration processes exclusive of its ability to stimulate cell proliferation. In validating this adenovirus-based system, we have created a potential tool for further exploration of the physiology of relaxin in mammalian systems.

PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-kappa B. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1 beta. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-kappa B and cytokine processing.

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis, NFkappaB activation, and cytokine regulation. In this study we identified a novel human protein, CARD-8, which contains a C-terminal CARD domain with high similarity to the CARD domain of caspase-1/ICE. We demonstrate that CARD-8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1beta generation in the THP-1 monocytic cell line. CARD-8 binds also to ICEBERG and pseudo-ICE, two other recently identified proteins, which bind to the CARD domain of caspase-1 and negatively regulate its activity. Reverse transcriptase-PCR analysis revealed that CARD-8 is expressed mainly in monocytes, placenta, lymph nodes, and spleen. This pattern of expression is consistent with caspase-1 expression in the same cells and tissues. CARD-8 was also found to negatively regulate NF-kappaB activation by TNF-alpha stimulation and by ectopically expressed RICK, suggesting that this protein may control cell survival. Consistent with these results, stable expression of CARD-8 in U937 or THP-1 cells sensitizes the cells to differentiation-induced apoptosis. Overexpression of CARD-8 can also induce apoptosis in transfected cells. The results suggest that CARD-8 represents a new signaling molecule involved in the regulation of caspase-1 and NF-kappaB activation.

The PYRIN domain is a recently identified protein-protein interaction domain that is found at the N terminus of several proteins thought to function in apoptotic and inflammatory signaling pathways. We report here that PYPAF1 (PYRIN-containing Apaf1-like protein 1) is a novel PYRIN-containing signaling protein that belongs to the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signaling proteins. The expression of PYPAF1 is highly restricted to immune cells, and its gene maps to chromosome 1q44, a locus that is associated with the rare inflammatory diseases Muckle-Wells syndrome and familial cold urticaria. To identify downstream signaling partners of PYPAF1, we performed a mammalian two-hybrid screen and identified ASC as a PYRIN-containing protein that interacts selectively with the PYRIN domain of PYPAF1. When expressed in cells, ASC recruits PYPAF1 to distinct cytoplasmic loci and induces the activation of NF-kappaB. Furthermore, coexpression of PYPAF1 with ASC results in a potent synergistic activation of NF-kappaB. These findings suggest that PYPAF1 and ASC function as upstream activators of NF-kappaB signaling.

The CED4/Apaf-1 family of proteins functions as critical regulators of apoptosis and NF-kappaB signaling pathways. A novel human member of this family, called CARD12, was identified that induces apoptosis when expressed in cells. CARD12 is most similar in structure to the CED4/Apaf-1 family member CARD4, and is comprised of an N-terminal caspase recruitment domain (CARD), a central nucleotide-binding site (NBS), and a C-terminal domain of leucine-rich repeats (LRR). The CARD domain of CARD12 interacts selectively with the CARD domain of ASC, a recently identified proapoptotic protein. In addition, CARD12 coprecipitates caspase-1, a caspase that participates in both apoptotic signaling and cytokine processing. CARD12 may assemble with proapoptotic CARD proteins to coordinate the activation of downstream apoptotic and inflammatory signaling pathways., (Copyright 2001 Academic Press.)

A simple method of manipulating neuronal gene expression would greatly facilitate the design of experiments to increase our understanding of and ability to treat diseases of the CNS. However, until recently most transfection methods could only deliver DNA into dividing cells and it was only possible to manipulate neuronal gene expression through the production of transgenic animals. The development of powerful new viral-based gene transfer systems has generated a great deal of research interest in the field of therapeutic gene transfer during the last decade. One of the most powerful and versatile gene delivery systems currently available is the recombinant adenovirus (Ad) vector. These vectors can transfect postmitotic neurons in the CNS, but have not yet been fully evaluated as CNS gene therapy vectors. Brattleboro rats contain a point mutation in the arginine vasopressin (AVP) gene that results in a pathological phenotype characterized by a lack of circulating AVP. This decrease in AVP in turn causes the characteristics signs of diabetes insipidus, with the production of large volumes of dilute urine and a compensatory drinking of large volumes of water (equivalent to the body weight of the rat per day). We have shown that injection of an Ad encoding the arginine vasopressin cDNA into the supraoptic nuclei of the hypothalamus results in the long-term reversal of this pathological phenotype. This was demonstrated by reduced daily water intake and micturition, as well as increased urine osmolality lasting 4 months. The highly characterized Brattleboro rat model of hypothalamic diabetes insipidus, therefore, provides the means to examine noninvasively the efficacy of viral and nonviral gene therapy strategies in the CNS.

Transfection of neurones with an adenoviral vector (Ad) expressing high levels of hsp70i was shown to protect primary hippocampal cultures from heat stress. To investigate one of the molecular mechanisms which may underlie hsp70i's neuroprotective effects we measured luciferase activity in the presence and absence of hsp70i following heat or chemical inactivation. Luciferase activity was recovered to 80% of control levels in the presence of recombinant hsp70i in vitro. Luciferase activity was also maintained in primary hippocampal neurones exposed to a denaturing stress if transfected with Ad-hsp70i. These results support the hypothesis that hsp70i protects neurones from stress by interacting with cytosolic proteins and thereby protecting them from inactivation.

We have developed a tetracycline-regulatable adenoviral transfection system that mediates efficient long-term transfer of genes into neuronal cells in vivo. This system allows gene expression to be switched on, then off, and back on again simply by administering or removing doxycycline from the animals' drinking water. This regulatable adenoviral vector system should be of value in behavioral studies and in vivo studies of neuronal gene function, and may further the development of effective gene therapy strategies in the brain.

The ability of adenovirus (Ad) to transfect most cell types efficiently has already resulted in human gene therapy trials involving the systemic administration of adenoviral constructs. However, because of the complexity of brain function and the difficulty in noninvasively monitoring alterations in neuronal gene expression, the potential of Ad gene therapy strategies for treating disorders of the CNS has been difficult to assess. In the present study, we have used an Ad encoding the arginine vasopressin cDNA (AdAVP) in an AVP-deficient animal model of diabetes insipidus (the Brattleboro rat), which allowed us to monitor chronically the success of the gene therapy treatment by noninvasive assays. Injection of AdAVP into the supraoptic nuclei (SON) of the hypothalamus resulted in expression of AVP in magnocellular neurons. This was accompanied by reduced daily water intake and urine volume, as well as increased urine osmolality lasting 4 months. These data show that a single gene defect leading to a neurological disorder can be corrected with an adenovirus-based strategy. This study highlights the potential of using Ad gene therapy for the long-term treatment of disorders of the CNS.

A transfer system that enabled the efficient introduction of transgenes into neurones and the quantitative control of the expressed transgene would greatly facilitate studies into neuronal gene function. To develop such a system we incorporated the tetracycline (Tet)-responsive On/Off regulatory elements into type-5 adenoviral (Ad) vectors. Regulation of transgene expression following transfection was measured by placing the enhanced green fluorescent protein (EGFP) gene upstream of the Tet regulatory element. The results showed that cultures of primary hippocampal cells could be transfected with very high efficiency (<70%) by the AdTet-On and AdTet-Off systems. Following transfection with the AdTet-On system no EGFP-fluorescent cells could be detected until doxycycline was added. The AdTet-Off system showed the reverse transcriptional regulation, in that the addition of Tet caused EGFP fluorescence to be abolished.

Replication deficient, recombinant adenoviruses (Ads) have been used successfully to transfect several forebrain and brainstem nuclei, but have yet to be demonstrated as useful vectors for transgene delivery in the structurally diverse and highly vascularised nuclei of the hypothalamus. In the present study we have assessed the ability of an Ad expressing the lac-Z gene to transfect cells of the paraventricular nucleus (PVN) of the hypothalamus in vivo. We show that: (1) we can achieve stable expression of the lacZ gene in cells of the magnocellular PVN for at least 2 months; (2) there were no obvious differences in the level of AVP mRNA in the PVNs injected with Ad compared with those injected with vehicle suggesting that Ad treatment is not disrupting normal cellular function in the injection region; (3) the introduction of Ads results in a limited immune response; (4) systemic treatment with cyclosporin dramatically reduces its magnitude. We conclude that Ad vectors represent useful tools for neuroendocrinological and gene therapeutic studies of the hypothalamus.

The novel finding that relaxin has an action on the brain was first published in 1984. Since then, it has been shown that exogenous relaxin affects the release of a number of hypothalamo-pituitary hormones and has a robust pressor action. In this paper, we review the accumulating evidence that relaxin affects the release of oxytocin and vasopressin by an action at the level of the brain. The potential mechanisms of this central action are discussed and the evidence presented for the interaction between relaxin and the forebrain angiotensin-II system. Furthermore, we articulate the possible physiological influences of relaxin on the changes in cardiovascular function that occur during pregnancy.

Experiments were conducted in anesthetized rats to assess the contribution of the brain angiotensin-II system in the relaxin-induced secretion of vasopressin and oxytocin. Intravenous injection of porcine relaxin (5 micrograms) caused a significant (P < 0.05, by analysis of variance) increase in plasma concentrations of both hormones. Peak concentrations of both vasopressin (75.2 +/- 2.9 pmol/liter) and oxytocin (38.4 +/- 1.2 pmol/liter) were observed 1-2.5 min after relaxin injection. Thereafter, concentrations fell significantly (P < 0.05) but remained elevated for a further 25 minutes. Continuous infusion of a specific angiotensin-II receptor antagonist into the lateral cerebral ventricle did not affect baseline levels of either vasopressin or oxytocin, but did significantly reduce (P < 0.05) the relaxin-induced release of both peptides. A significant (P < 0.05) short term increase in both plasma vasopressin and oxytocin occurred 1 min after injection of 5 micrograms relaxin, iv, in angiotensin-II-antagonized rats, but the concentrations of both neuropeptides were significantly (P < 0.05) lower than those observed in the angiotensin-intact relaxin-treated controls. These data suggest that relaxin may act through the central angiotensin-II system to induce the release of vasopressin and oxytocin.