Prosocial effects of an oxytocin metabolite, but not synthetic oxytocin receptor agonists, in a mouse model of autism.

Currently, there are no established pharmaceutical strategies that effectively treat social deficits in autism spectrum disorder (ASD). Oxytocin, a neurohormone that plays a role in multiple types of social behaviors, has been proposed as a possible therapeutic against social impairment and other symptoms in ASD. However, from the standpoint of pharmacotherapy, oxytocin has several liabilities as a standard clinical treatment, including rapid metabolism, low brain penetrance, and activity at the vasopressin (antidiuretic hormone) receptors. The present studies describe findings from a preclinical screening program to evaluate oxytocin receptor (OXTR) agonists and oxytocin metabolites for potential clinical use as more optimal treatments. We first investigated two synthetic oxytocin analogs, TC-OT-39 and carbetocin, using in vitro cell-based assays for pharmacological characterization and behavioral tests in the BALB/cByJ mouse model of ASD-like social deficits. Although both TC-OT-39 and carbetocin selectively activate the OXTR, neither synthetic agonist had prosocial efficacy in the BALB/cByJ model. We next evaluated two oxytocin metabolites: OT(4-9) and OT(5-9). While OT(5-9) failed to affect social deficits, the metabolite OT(4-9) led to significant social preference in the BALB/cByJ model, in a dose-dependent manner. The increased sociability was observed at both 24 h and 12 days following the end of a subchronic regimen with OT(4-9) (2.0 mg/kg). Overall, these results suggest that the prosocial effects of oxytocin could be mediated by downstream activity of oxytocin metabolites, raising the possibility of new pathways to target for drug discovery relevant to ASD.

Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1.

Maternally inherited 15q11-13 chromosomal triplications cause a frequent and highly penetrant type of autism linked to increased gene dosages of UBE3A, which encodes a ubiquitin ligase with transcriptional co-regulatory functions. Here, using in vivo mouse genetics, we show that increasing UBE3A in the nucleus downregulates the glutamatergic synapse organizer Cbln1, which is needed for sociability in mice. Epileptic seizures also repress Cbln1 and are found to expose sociability impairments in mice with asymptomatic increases in UBE3A. This Ube3a-seizure synergy maps to glutamate neurons of the midbrain ventral tegmental area (VTA), where Cbln1 deletions impair sociability and weaken glutamatergic transmission. We provide preclinical evidence that viral-vector-based chemogenetic activation of, or restoration of Cbln1 in, VTA glutamatergic neurons reverses the sociability deficits induced by Ube3a and/or seizures. Our results suggest that gene and seizure interactions in VTA glutamatergic neurons impair sociability by downregulating Cbln1, a key node in the expanding protein interaction network of autism genes.

Reversal of social deficits by subchronic oxytocin in two autism mouse models.

Social deficits are a hallmark feature of autism spectrum disorder (ASD) and related developmental syndromes. Although there is no standard treatment for social dysfunction, clinical studies have identified oxytocin as a potential therapeutic with prosocial efficacy. We have previously reported that peripheral oxytocin treatment can increase sociability and ameliorate repetitive stereotypy in adolescent mice from the C58/J model of ASD-like behavior. In the present study, we determined that prosocial oxytocin effects were not limited to the adolescent period, since C58/J mice, tested in adulthood, demonstrated significant social preference up to 2 weeks following subchronic oxytocin treatment. Oxytocin was also evaluated in adult mice with underexpression of the N-methyl-d-aspartate receptor NR1 subunit (encoded by Grin1), a genetic model of autism- and schizophrenia-like behavior. Subchronic oxytocin had striking prosocial efficacy in male Grin1 knockdown mice; in contrast, chronic regimens with clozapine (66 mg/kg/day) or risperidone (2 mg/kg/day) failed to reverse deficits in sociability. Neither the subchronic oxytocin regimen, nor chronic treatment with clozapine or risperidone, reversed impaired prepulse inhibition in the Grin1 knockdown mice. Overall, these studies demonstrate oxytocin can enhance sociability in mouse models with divergent genotypes and behavioral profiles, adding to the evidence that this neurohormone could have therapeutic prosocial efficacy across a spectrum of developmental disorders.

Repetitive behavior profile and supersensitivity to amphetamine in the C58/J mouse model of autism.

Restricted repetitive behaviors are core symptoms of autism spectrum disorders (ASDs). The range of symptoms encompassed by the repetitive behavior domain includes lower-order stereotypy and self-injury, and higher-order indices of circumscribed interests and cognitive rigidity. Heterogeneity in clinical ASD profiles suggests that specific manifestations of repetitive behavior reflect differential neuropathology. The present studies utilized a set of phenotyping tasks to determine a repetitive behavior profile for the C58/J mouse strain, a model of ASD core symptoms. In an observational screen, C58/J demonstrated overt motor stereotypy, but not over-grooming, a commonly-used measure for mouse repetitive behavior. Amphetamine did not exacerbate motor stereotypy, but had enhanced stimulant effects on locomotion and rearing in C58/J, compared to C57BL/6J. Both C58/J and Grin1 knockdown mice, another model of ASD-like behavior, had marked deficits in marble-burying. In a nose poke task for higher-order repetitive behavior, C58/J had reduced holeboard exploration and preference for non-social, versus social, olfactory stimuli, but did not demonstrate cognitive rigidity following familiarization to an appetitive stimulus. Analysis of available high-density genotype data indicated specific regions of divergence between C58/J and two highly-sociable strains with common genetic lineage. Strain genome comparisons identified autism candidate genes, including Cntnap2 and Slc6a4, located within regions divergent in C58/J. However, Grin1, Nlgn1, Sapap3, and Slitrk5, genes linked to repetitive over-grooming, were not in regions of divergence. These studies suggest that specific repetitive phenotypes can be used to distinguish ASD mouse models, with implications for divergent underlying mechanisms for different repetitive behavior profiles.

Prosocial effects of oxytocin in two mouse models of autism spectrum disorders.

Clinical evidence suggests that oxytocin treatment improves social deficits and repetitive behavior in autism spectrum disorders (ASDs). However, the neuropeptide has a short plasma half-life and poor ability to penetrate the blood-brain barrier. In order to facilitate the development of more bioavailable oxytocinergic compounds as therapeutics to treat core ASD symptoms, small animal models must be validated for preclinical screens. This study examined the preclinical utility of two inbred mouse strains, BALB/cByJ and C58/J, that exhibit phenotypes relevant to core ASD symptoms. Mice from both strains were intraperitoneally administered oxytocin, using either acute or sub-chronic regimens. Acute oxytocin did not increase sociability in BALB/cByJ; however, sub-chronic oxytocin had significant prosocial effects in both BALB/cByJ and C58/J. Increased sociability was observed 24 h following the final oxytocin dose in BALB/cByJ, while prosocial effects of oxytocin emerged 1-2 weeks post-treatment in C58/J. Furthermore, acute oxytocin decreased motor stereotypy in C58/J and did not induce hypoactivity or anxiolytic-like effects in an open field test. This study demonstrates that oxytocin administration can attenuate social deficits and repetitive behavior in mouse models of ASD, dependent on dose regimen and genotype. These findings provide validation of the BALB/cByJ and C58/J models as useful platforms for screening novel drugs for intervention in ASDs and for elucidating the mechanisms contributing to the prosocial effects of oxytocin.

Tumor suppressive activity of prolyl isomerase Pin1 in renal cell carcinoma.

Pin1 specifically recognizes and catalyzes the cis-trans isomerization of phosphorylated-Ser/Thr-Pro bonds, which modulate the stability, localization, and function of numerous Pin1 targets involved in tumor progression. However, the role of Pin1 in cancer remains enigmatic as the gene is located on chromosome 19p13.2, which is a region subject to loss of heterozygosity in several tumors. Since Pin1 protein is frequently under-expressed in kidney cancer, we have explored its role in human clear cell renal cell carcinoma (ccRCC). Here we show evidence for PIN1 gene deletion and mRNA under-expression as a mechanism of Pin1 reduction in ccRCC tumors. We demonstrate that restoration of Pin1 in cell lines found to be deficient in Pin1 protein expression can attenuate the growth of ccRCC cells in soft agar and a xenograft tumor model. Moreover, this ability of Pin1 to negatively influence tumor growth in ccRCC cells may be dependent on the presence of functional p53, which is infrequently mutated in ccRCC. These observations suggest Pin1 may have a mild tumor suppressive role in ccRCC.

Inhibition of hyaluronan synthase-3 decreases subcutaneous colon cancer growth by increasing apoptosis.

Hyaluronan (HA) and hyaluronan synthases (HAS) have been implicated in cancer growth and progression. We previously have shown that HAS3 and HA mediate tumor growth in SW620 colon cancer cells, but the mechanism remains poorly understood. In addition, the effect of HAS3 inhibition on tumor growth with other cells lines has not been explored. We therefore hypothesized that inhibition of HAS3 in highly tumorigenic HCT116 colon cancer cells would decrease tumor growth and that the underlying mechanism would involve altering proliferation and/or apoptosis. HAS3 expression was inhibited by transfection with siRNA; a scrambled sequence served as a control. Stable transfectants were injected into the flanks of nude mice and tumor growth followed for 30 days. Proliferation and apoptosis were then assessed in the harvested tumors. Results were compared using the Students' t-test and ANOVA where appropriate. siRNA transfection decreased HAS3 expression, protein production, and pericellular HA retention, and decreased in vivo tumor growth. Proliferation was unaffected in the HCT116 tumors, but increased slightly in the SW620 tumors. In contrast, HAS3 inhibition significantly increased apoptosis in all tumors. HAS3 inhibition decreases subcutaneous tumor growth by colon cancer cells and significantly increases apoptosis with less effect on proliferation. These data show that HAS3 and HA mediate colon cancer growth by inhibiting apoptosis.

Inhibition of hyaluronan synthase-3 decreases subcutaneous colon cancer growth in mice.

PURPOSE: Hyaluronan and hyaluronan synthases have been implicated in cancer progression. Hyaluronan synthase-3 is up-regulated in metastatic colon cancer cells (SW620), and its expression mediates cellular growth in vitro. We hypothesized that inhibition of hyaluronan synthase-3 would decrease tumor formation and/or alter the pattern of metastasis in mouse models of colon cancer growth. METHODS: Hyaluronan synthase-3 was inhibited in SW620 cells by transfection with small interfering RNA (silenced cells); a scrambled sequence served as a negative control. To study primary tumor growth, transfected cells were injected into the flanks of BALB/c nude mice. To study metastasis, an orthotopic model was used. Metastases were confirmed histologically. Student t test and Fisher exact probability test were used for statistical analysis. RESULTS: Inhibition of hyaluronan synthase-3 significantly decreased subcutaneous tumor growth; tumor weight was 0.94 +/- 0.17 g in the hyaluronan synthase-3-silenced group vs 1.70 +/- 0.26 g in the control scrambled group (P < .01). In contrast, metastases were similar in both groups: liver metastases were present in 22% of the silenced group vs 11% of the scrambled group; lung metastases were present in 6% of the silenced group vs 0% of the scrambled group (P = not significant). CONCLUSION: Inhibition of hyaluronan synthase-3 expression in SW620 colon cancer cells decreases subcutaneous tumor growth in mice, but has less of an effect on lung and liver metastases. This observation suggests that hyaluronan synthase-3 may enhance primary colon cancer growth.

Left paraduodenal hernia causing small bowel obstruction in an adolescent patient.

Internal hernias are an uncommon cause of bowel obstruction, accounting for less than 1% of cases. Paraduodenal hernias, the most common type of internal hernias, are believed to be congenital in origin. They can be asymptomatic, cause chronic abdominal pain, or present with acute intestinal obstruction with strangulation and ischemia. We describe a case of left paraduodenal hernia found in a patient who presented with acute intestinal obstruction.

Assessment of the onset and persistence of amnesia during procedural sedation with propofol.

OBJECTIVES: To assess patients' ability to repeat and recall words presented to them while undergoing procedural sedation with propofol, and correlate their recall with their level of awareness as measured by bispectral index (BIS) monitoring. METHODS: This was a prospective, single-intervention study of consenting adult patients undergoing procedural sedation with propofol between December 28, 2002, and October 31, 2003. BIS monitoring was initiated starting 3 minutes before the procedure and continuing until the patient had regained baseline mental status. At 1-minute intervals during the procedural sedation, until the patient regained baseline mental status at the end of the procedure, a word from a standardized list was read aloud, and the patient was asked to immediately repeat the word to the investigator. The BIS score at the time the word was read and the patient's ability to repeat the word were recorded. After the procedure, the patient was asked to state all of the words from the list that he or she could recall, and to identify the last word recalled from prior to the start of the procedure and the first word recalled from after the procedure was completed. RESULTS: Seventy-five consenting patients were enrolled; one patient was excluded from data analysis for a protocol violation. No serious adverse events were noted during the procedural sedations. The mean (+/-standard deviation) time of data collection was 16.4 minutes (+/-7.1; range 5 to 34 minutes). The mean initial (preprocedure) BIS score was 97.1 (+/-2.3; range 92 to 99). The mean lowest BIS score occurring during these procedural sedations was 66.9 (+/-14.4; range 33 to 91). The mean lowest BIS score corresponding to the ability of the patient to immediately repeat words read from the list was 77.1 (95% CI = 74.3 to 80.0). The mean highest BIS score corresponding to the inability to repeat words was 81.5 (95% CI = 78.1 to 84.8). The mean BIS score corresponding to the last word recalled from prior to the initiation of the sedation was 96.7 (+/-2.4; range 84 to 98). The mean BIS score corresponding to the first word recalled after the procedure was completed was 91.2 (95% CI = 88.1 to 94.3). All patients recalled at least one word that had been read to them during the protocol. The mean lowest BIS score for any recalled word was 91.5 (+/-11.1; range 79 to 98), and no words were recalled when the corresponding BIS score was less than 90. CONCLUSIONS: There is a range of BIS scores during which sedated patients are able to repeat words read to them but are not able to subsequently recall these words. Furthermore, patients had no recall of words repeated prior to procedural sedation in BIS ranges associated with recall after procedural sedation, suggestive of retrograde amnesia.