Differential expression of small bowel TGFß1 and TGFß3 characterizes intestinal strictures in patients with fibrostenotic Crohn's disease.

Small bowel strictures remain a debilitating consequence of Crohn's disease and contribute to poor outcomes for patients. Recently, TGFß has been identified as an important driver of intestinal fibrosis. We studied the localization of TGFß isoforms in ileal strictures of patients with Crohn's disease using in situ hybridization to understand TGFß's role in stricture formation. The mucosa of strictures was characterized by higher TGFß1 while the stricture submucosa showed higher TGFß3 compared to normal ileum from patients without Crohn's disease (p = 0.02 and p = 0.044, respectively). We correlated these findings with single-cell transcriptomics which demonstrated that TGFß3 transcripts overall are very rare, which may partially explain why its role in intestinal fibrosis has remained unclear to date. There were no significant differences in fibroblast or B cell TGFß1 and/or TGFß3 expression in inflamed vs. noninflamed ileum. We discuss the implications of these findings for therapeutic development strategies to treat patients with fibrostenotic Crohn's disease.

A Phase 1a Study to Evaluate Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of RO7303509, an Anti-TGFß3 Antibody, in Healthy Volunteers.

INTRODUCTION: Transforming growth factor beta (TGFß) cytokines (TGFß1, TGFß2, and TGFß3) play critical roles in tissue fibrosis. However, treatment with systemic pan-TGFß inhibitors have demonstrated unacceptable toxicities. In this study, we evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of RO7303509, a high-affinity, TGFß3-specific, humanized immunoglobulin G1 monoclonal antibody, in healthy adult volunteers (HVs). METHODS: This phase 1a, randomized, double-blind trial included six cohorts for evaluation, with each cohort receiving single doses of placebo or RO7303509, administered intravenously (IV; 50 mg, 150 mg, 240 mg) or subcutaneously (SC; 240 mg, 675 mg, 1200 mg). The frequency and severity of adverse events (AEs) and RO7303509 serum concentrations were monitored throughout the study. We also measured serum periostin and cartilage oligomeric matrix protein (COMP) by immunoassay and developed a population pharmacokinetics model to characterize RO7303509 serum concentrations. RESULTS: The study enrolled 49 HVs, with a median age of 39 (range 18-73) years. Ten (27.8%) RO7303509-treated subjects reported 24 AEs, and six (30.8%) placebo-treated subjects reported six AEs. The most frequent AEs related to the study drug were injection site reactions and infusion-related reactions. Maximum serum concentrations (Cmax) and area under the concentration-time curve from time 0 to infinity (AUC0-inf) values for RO7303509 appeared to increase dose-proportionally across all doses tested. Serum concentrations across cohorts were best characterized by a two-compartment model plus a depot compartment with first-order SC absorption kinetics. No subjects tested positive for anti-drug antibodies (ADAs) at baseline; one subject (2.8%; 50 mg IV) tested positive for ADAs at a single time point (day 15). No clear pharmacodynamic effects were observed for periostin or COMP upon TGFß3 inhibition. CONCLUSION: RO7303509 was well tolerated at single SC doses up to 1200 mg in HVs with favorable pharmacokinetic data that appeared to increase dose-proportionally. TGFß3-specific inhibition may be suitable for development as a chronic antifibrotic therapy. TRIAL REGISTRATION: ISRCTN13175485.

Utilizing PK and PD Biomarkers to Guide the First-in-Human Starting Dose Selection of MTBT1466A: A Novel Humanized Monoclonal Anti-TGFß3 Antibody for the Treatment of Fibrotic Diseases.

MTBT1466A is a high-affinity TGFß3-specific humanized IgG1 monoclonal antibody with reduced Fc effector function, currently under investigation in clinical trials as a potential anti-fibrotic therapy. Here, we characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of MTBT1466A in mice and monkeys and predicted the PK/PD of MTBT1466A in humans to guide the selection of the first-in-human (FIH) starting dose. MTBT1466A demonstrated a typical IgG1-like biphasic PK profile in monkeys, and the predicted human clearance of 2.69 mL/day/kg and t1/2 of 20.4 days are consistent with those expected for a human IgG1 antibody. In a mouse model of bleomycin-induced lung fibrosis, changes in expression of TGFß3-related genes, serpine1, fibronectin-1, and collagen 1A1 were used as PD biomarkers to determine the minimum pharmacologically active dose of 1 mg/kg. Unlike in the fibrosis mouse model, evidence of target engagement in healthy monkeys was only observed at higher doses. Using a PKPD-guided approach, the recommended FIH dose of 50 mg, IV, provided exposures that were shown to be safe and well tolerated in healthy volunteers. MTBT1466A PK in healthy volunteers was predicted reasonably well using a PK model with allometric scaling of PK parameters from monkey data. Taken together, this work provides insights into the PK/PD behavior of MTBT1466A in preclinical species, and supports the translatability of the preclinical data into the clinic.

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding.

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-κB (NF-kB) and inhibitor κB (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi.

Little is known about the role of oxytocin (OT) in colostrum during early gut colonization. We previously showed that transient OT receptor (OTR) expression on newborn rat enterocytes coincides with the milk-suckling period, and that OT activates endoplasmic reticulum stress sensors in cultured enterocytes. Here, we explored whether colostrum-OT attenuates stress in newborn villi primed and unprimed by colostrum by measuring levels of stress markers including BiP (an ER chaperone), eIF2a (translation initiation factor), and pPKR (eIF2a kinase). We also measured two inflammation-signaling proteins NF-κB and its inhibitor IκB. To test the impact of colostrum on autophagy, we measured a marker of autophagy initiation, LC3A. Colostrum increased inactive p-eIF2a, p-PKR and IκB and reduced p-IκB, BiP and LC3A. LPS increased and OT decreased p-IkB. BiP (GRP78) was higher in unprimed than primed villi. Together, these data suggest that colostrum OT attenuates the impact of inflammation on postnatal gut villi and that OT enhances autophagy to protect against amino acid insufficiency-induced stress during the interval between birth and the first feeding.

Oxytocin opposes effects of bacterial endotoxin on ER-stress signaling in Caco2BB gut cells.

BACKGROUND: The neuropeptide neuromodulator and hormone oxytocin (OT) activates signaling pathways involved in mRNA translation in response to endoplasmic reticulum stress and reduces inflammation associated with experimental colitis in rats. The anti-inflammatory effects of OT may serve a vital role in the development, survival and function of newborn-type enterocytes during microbial gut colonization, which coincides with the milk suckling period when OT receptor expression peaks in the gut. Furthermore, mice deficient in the OT receptor have abnormal gut structure and function, underscoring OT's developmental importance. METHODS: We tested the effect of OT upon lipopolysaccharide (LPS)-induced markers of the inflammatory response in Caco2BB gut cells in vitro using automated immunocapillary electrophoresis. RESULTS: We demonstrate that OT suppresses NF-κB signaling and presumably inflammatory transcriptional programs, which are unleashed by LPS through the modulation of IκB. We show that OT counteracts LPS-elicited silencing of the unfolded protein response, a pathway limiting endoplasmic reticulum stress by suppressing protein translation. OT selectively activates dsRNA-activated kinase (PKR), X-box binding protein 1 (XBP1), immunoglobulin binding protein (BiP), A20 (TNFα-induced protein 3) and inositol requiring enzyme 1a (IRE1a). OT inactivates eukaryotic translation initiation factor 2a (eIF2a) without significant activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK). CONCLUSIONS: Mild, preemptive stimulation of endoplasmic reticulum stress sensors by OT may precondition newborn enterocytes to resist apoptosis associated with inflammation and may support their differentiation and development by modulating cellular metabolism. GENERAL SIGNIFICANCE: OT may protect enterocytes and other cell types, such as neurons, from stress-related complications during postnatal development.

Family Nurture Intervention in the Neonatal Intensive Care Unit improves social-relatedness, attention, and neurodevelopment of preterm infants at 18 months in a randomized controlled trial.

BACKGROUND: Preterm infants are at high risk for adverse neurodevelopmental and behavioral outcomes. Family Nurture Intervention (FNI) in the Neonatal Intensive Care Unit (NICU) is designed to counteract adverse effects of separation of mothers and their preterm infants. Here, we evaluate effects of FNI on neurobehavioral outcomes. METHODS: Data were collected at 18 months corrected age from preterm infants. Infants were assigned at birth to FNI or standard care (SC). Bayley Scales of Infant Development III (Bayley-III) were assessed for 76 infants (SC, n = 31; FNI, n = 45); the Child Behavior Checklist (CBCL) for 57 infants (SC, n = 31; FNI, n = 26); and the Modified Checklist for Autism in Toddlers (M-CHAT) was obtained for 59 infants (SC, n = 33; FNI, n = 26). RESULTS: Family Nurture Intervention significantly improved Bayley-III cognitive (p = .039) and language (p = .008) scores for infants whose scores were greater than 85. FNI infants had fewer attention problems on the CBCL (p < .02). FNI improved total M-CHAT scores (p < .02). Seventy-six percent of SC infants failed at least one of the M-CHAT items, compared to 27% of FNI infants (p < .001). In addition, 36% of SC infants versus 0% of FNI infants failed at least one social-relatedness M-CHAT item (p < .001). CONCLUSIONS: Family Nurture Intervention is the first NICU intervention to show significant improvements in preterm infants across multiple domains of neurodevelopment, social-relatedness, and attention problems. These gains suggest that an intervention that facilitates emotional interactions between mothers and infants in the NICU may be key to altering developmental trajectories of preterm infants.

Family nurture intervention improves the quality of maternal caregiving in the neonatal intensive care unit: evidence from a randomized controlled trial.

OBJECTIVE: This study assessed the impact of Family Nurture Intervention (FNI) on the quality of maternal caregiving behavior (MCB) while in the neonatal intensive care unit (NICU). FNI is a randomized controlled trial conducted in a high-acuity NICU to facilitate an emotional connection between mothers and their premature infants. FNI begins shortly after birth, continues until discharge, and involves mother/infant calming sessions that include scent cloth exchange, vocal soothing and emotion expression, eye contact, skin-to-skin and clothed holding, and family-based support sessions. METHODS: Maternal caregiving behavior was coded during a single holding and feeding session (∼30 min) in the NICU before discharge at approximately 36 weeks gestational age (GA). Sixty-five mothers and their premature infants (34 male, 31 female; 26-34 wk GA) were included in these analyses (FNI, n = 35; standard care [SC], n = 30). RESULTS: Relative to mothers in the SC condition, those in the FNI group showed significantly higher quality MCB, which remained significant when controlling for birth order, twin status, maternal depression, and maternal anxiety. CONCLUSION: This is the first study to demonstrate that in-unit MCB can be enhanced by a hospital-based intervention. FNI provides a new rationale for integrating nurture-based interventions into standard NICU care.

Interneuron precursor transplants in adult hippocampus reverse psychosis-relevant features in a mouse model of hippocampal disinhibition.

GABAergic interneuron hypofunction is hypothesized to underlie hippocampal dysfunction in schizophrenia. Here, we use the cyclin D2 knockout (Ccnd2(-/-)) mouse model to test potential links between hippocampal interneuron deficits and psychosis-relevant neurobehavioral phenotypes. Ccnd2(-/-) mice show cortical PV(+) interneuron reductions, prominently in hippocampus, associated with deficits in synaptic inhibition, increased in vivo spike activity of projection neurons, and increased in vivo basal metabolic activity (assessed with fMRI) in hippocampus. Ccnd2(-/-) mice show several neurophysiological and behavioral phenotypes that would be predicted to be produced by hippocampal disinhibition, including increased ventral tegmental area dopamine neuron population activity, behavioral hyperresponsiveness to amphetamine, and impairments in hippocampus-dependent cognition. Remarkably, transplantation of cells from the embryonic medial ganglionic eminence (the major origin of cerebral cortical interneurons) into the adult Ccnd2(-/-) caudoventral hippocampus reverses these psychosis-relevant phenotypes. Surviving neurons from these transplants are 97% GABAergic and widely distributed within the hippocampus. Up to 6 mo after the transplants, in vivo hippocampal metabolic activity is lowered, context-dependent learning and memory is improved, and dopamine neuron activity and the behavioral response to amphetamine are normalized. These findings establish functional links between hippocampal GABA interneuron deficits and psychosis-relevant dopaminergic and cognitive phenotypes, and support a rationale for targeting limbic cortical interneuron function in the prevention and treatment of schizophrenia.

Oxytocin modulates markers of the unfolded protein response in Caco2BB gut cells.

We have shown that oxytocin receptor (OTR) expression in neonatal rat enterocytes is robust from birth to weaning, but OTR function during this period is unknown. We previously reported that oxytocin (OT) stimulation of Caco2BB cells (enterocytes in vitro) inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling. The unfolded protein response (UPR) is known to protectively reduce translation during endoplasmic reticulum (ER) stress. Because the mTORC1 pathway is linked to cellular stress, we investigated markers of UPR in OT-stimulated Caco2BB cells. We report that OT modulates several factors involved in sensing and translation of ER stress. High OT (62.5 nM) reduced translation initiation factor 4E-BP1 phosphorylation (Ser65), which is known to inhibit cap-dependent translation via its rate-limiting eukaryotic translation initiation factor 4E (eIF4E). Importantly, high OT increased phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) phospho-Ser51, which inhibits eIF2a. High OT also increased protein kinase RNA-like endoplasmic reticulum kinase phosphorylation, a sensor of ER stress and a kinase of eIF2a. Both high and low OT activated inositol requiring enzyme1 (IRE1), which generates the transcription factor X-box binding protein 1 (XBP1) and induces the UPR. We also show that OT modulates XBP1 splicing and induces tribbles 3 (TRIB3; a negative regulator of Akt and protein involved in autophagy) and immunoglobulin binding protein (BiP; ER-chaperone). Taken together, these results indicate that OT modulates sensors of ER stress and autophagy. These findings support our hypothesis that transiently elevated OTR expression in neonatal gut may serve a protective function during a critical postnatal developmental period.

Randomized controlled trial of Family Nurture Intervention in the NICU: assessments of length of stay, feasibility and safety.

BACKGROUND: While survival rates for preterm infants have increased, the risk for adverse long-term neurodevelopmental and behavioral outcomes remains very high. In response to the need for novel, evidence-based interventions that prevent such outcomes, we have assessed Family Nurture Intervention (FNI), a novel dual mother-infant intervention implemented while the infant is in the Neonatal Intensive Care Unit (NICU). Here, we report the first trial results, including the primary outcome measure, length of stay in the NICU and, the feasibility and safety of its implementation in a high acuity level IV NICU. METHODS: The FNI trial is a single center, parallel-group, randomized controlled trial at Morgan Stanley Children's Hospital for mothers and their singleton or twin infants of 26-34 weeks gestation. Families were randomized to standard care (SC) or (FNI). FNI was implemented by nurture specialists trained to facilitate affective communication between mother and infant during specified calming interactions. These interactions included scent cloth exchange, sustained touch, vocal soothing and eye contact, wrapped or skin-to-skin holding, plus family-based support interactions. RESULTS: A total of 826 infants born between 26 and 34 weeks during the 3.5 year study period were admitted to the NICU. After infant and mother screening plus exclusion due to circumstances that prevented the family from participating, 373 infants were eligible for the study. Of these, we were unable to schedule a consent meeting with 56, and consent was withheld by 165. Consent was obtained for 150 infants from 115 families. The infants were block randomized to groups of N = 78, FNI and N = 72, SC. Sixteen (9.6%) of the randomized infants did not complete the study to home discharge, 7% of those randomized to SC and 12% of FNI infants. Mothers in the intervention group engaged in 3 to 4 facilitated one- to two-hour sessions/week. Intent to treat analyses revealed no significant difference between groups in medical complications. The mean length of stay was not significantly affected by the intervention. CONCLUSION: There was no significant effect demonstrated with this intervention amount on the primary short-term outcome, length of stay. FNI can be safely and feasibly implemented within a level IV NICU. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01439269.

Oxytocin modulates mTORC1 pathway in the gut.

Our recent findings of a weaning-related pattern of oxytocin (OT) and OT receptor (OTR) expression in the rat enteric nervous system and in villus-crypt enterocytes, together with the known high level and stability of OT in breast milk support that OT may play a role in gut function and development. We previously described a biphasic dose-response of the PI3K/Akt pathway in gut cells treated with OT. Activation peaked at 62.5 nM OT (30 min) and coincided with OTR internalization. Here we use automated Western blotting to further explore OT-elicited changes in Akt and pAkt(T308), as well as in downstream substrates p70 S6 kinase-1 (S6K1) and eIF-4E binding protein 1 (4E-BP1). Relative to fresh growth medium (FGM) alone, our results showed OT in FGM reduced the abundance and phosphorylation of S6K1 and the phosphorylation of 4E-BP1, both substrates of mammalian target of rapamycin complex 1 (mTORC1). Phosphorylation of mTORC1 regulator, Raptor(S792), was increased by high and low OT concentrations, with predicted inhibitory effects on mTORC1. OT thus downregulates anabolic effects induced by FGM activity catalyzed by mTORC1. OT is a regulator of the PI3K/Akt/mTORC1 pathway in Caco2BB cells and may modulate translation in gut cells.

Effects of eicosapentaenoic acid and docosahexaenoic acid on low-density lipoprotein cholesterol and other lipids: a review.

In this exploratory, hypothesis-generating literature review, we evaluated potentially differential effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), and non-HDL-C in published studies of ω-3 fatty acid supplementation or prescription ω-3 fatty acid ethyl esters. Placebo-adjusted changes in mean lipid parameters were compared in randomized, controlled trials in subjects treated for ≥ 4 weeks with DHA or EPA. Of 22 studies identified, 6 compared DHA with EPA directly, 12 studied DHA alone (including 14 DHA-treated groups), and 4 examined EPA alone. In studies directly comparing EPA with DHA, a net increase in LDL-C of 3.3% was observed with DHA (DHA: +2.6%; EPA: -0.7%). In such head-to-head comparative studies, DHA treatment was associated with a net decrease in TG by 6.8% (DHA: -22.4%; EPA: -15.6%); a net increase in non-HDL-C by 1.7% (DHA: -1.2%; EPA -2.9%); and a net increase in HDL-C by 5.9% (DHA: +7.3%; EPA: +1.4%). Increases in LDL-C were also observed in 71% of DHA-alone groups [with demonstrated statistical significance (P < .05) in 67% (8 of 12) DHA-alone studies] but not in any EPA-alone studies. Changes in LDL-C significantly correlated with baseline TG for DHA-treated groups. The range of HDL-C increases documented in DHA-alone vs EPA-alone studies further supports the fact that HDL-C is increased more substantially by DHA than EPA. In total, these findings suggest that DHA-containing supplements or therapies were associated with more significant increases in LDL-C and HDL-C than were EPA-containing supplements or therapies. Future prospective, randomized trials are warranted to confirm these preliminary findings, determine the potential effects of these fatty acids on other clinical outcomes, and evaluate the generalizability of the data to larger and more heterogeneous patient populations.

Cyclin D2 is critical for intermediate progenitor cell proliferation in the embryonic cortex.

Expression of cyclins D1 (cD1) and D2 (cD2) in ventricular zone and subventricular zone (SVZ), respectively, suggests that a switch to cD2 could be a requisite step in the generation of cortical intermediate progenitor cells (IPCs). However, direct evidence is lacking. Here, cD1 or cD2 was seen to colabel subsets of Pax6-expressing radial glial cells (RGCs), whereas only cD2 colabeled with Tbr2. Loss of IPCs in cD2(-/-) embryonic cortex and analysis of expression patterns in mutant embryos lacking cD2 or Tbr2 indicate that cD2 is used as progenitors transition from RGCs to IPCs and is important for the expansion of the IPC pool. This was further supported by the laminar thinning, microcephaly, and selective reduction in the cortical SVZ population in the cD2(-/-)cortex. Cell cycle dynamics between embryonic day 14-16 in knock-out lines showed preserved parameters in cD1 mutants that induced cD2 expression, but absence of cD2 was not compensated by cD1. Loss of cD2 was associated with reduced proliferation and enhanced cell cycle exit in embryonic cortical progenitors, indicating a crucial role of cD2 for the support of cortical IPC divisions. In addition, knock-out of cD2, but not cD1, affected both G(1)-phase and also S-phase duration, implicating the importance of these phases for division cycles that expand the progenitor pool. That cD2 was the predominant D-cyclin expressed in the human SVZ at 19-20 weeks gestation indicated the evolutionary importance of cD2 in larger mammals for whom expansive intermediate progenitor divisions are thought to enable generation of larger, convoluted, cerebral cortices.

Cyclin D1 in excitatory neurons of the adult brain enhances kainate-induced neurotoxicity.

G1-phase cyclin D1 (cD1) expression has been documented in post-mitotic neurons undergoing apoptosis, leading others to propose that attempted cell cycle re-entry may induce cell death. Here, cD1 immunoreactivity was found in a subpopulation of healthy excitatory neurons throughout the brain. Most striking was the selective cD1 expression in hippocampal pyramidal neurons, an especially vulnerable cell group. Seizure threshold, cD1 induction and CA1 neuron death were examined following application of kainate (KA) or pentylenetetrazole (PTZ) in cD1 heterozygous (+/-) and wildtype mice to determine whether baseline cD1 correlates with pathology. cD1+/- mice displayed resistance to KA, but not PTZ-induced seizures and had reduced or equivalent cytotoxicity respectively, compared with wildtype. KA administration, but not PTZ, induced cD1 expression. These findings suggest that basal cD1 expression may render hippocampal circuits more susceptible to particular epileptogenic agents and excitotoxic cell death, though cD1 is not a direct precipitant in apoptosis.

Selective cortical interneuron and GABA deficits in cyclin D2-null mice.

In contrast to cyclin D1 nulls (cD1-/-), mice without cyclin D2 (cD2-/-) lack cerebellar stellate interneurons; the reason for this is unknown. In the present study in cortex, we found a disproportionate loss of parvalbumin (PV) interneurons in cD2-/- mice. This selective reduction in PV subtypes was associated with reduced frequency of GABA-mediated inhibitory postsynaptic currents in pyramidal neurons, as measured by voltage-clamp recordings, and increased cortical sharp activity in the EEGs of awake-behaving cD2-/- mice. Cell cycle regulation was examined in the medial ganglionic eminence (MGE), the major source of PV interneurons in mouse brain, and differences between cD2-/- and cD1-/- suggested that cD2 promotes subventricular zone (SVZ) divisions, exerting a stronger inhibitory influence on the p27 Cdk-inhibitor (Cdkn1b) to delay cell cycle exit of progenitors. We propose that cD2 promotes transit-amplifying divisions in the SVZ and that these ensure proper output of at least a subset of PV interneurons.

The anaphase-promoting complex coordinates initiation of lens differentiation.

Lens development requires the precise coordination of cell division and differentiation. The mechanisms by which the differentiation program is initiated after cell cycle arrest remains not well understood. Cyclin-dependent kinase inhibitors (CKIs), such as p15 and p21, have been suggested to be critical components that inhibit G1 progression and therefore, their activation is necessary for quiescence and important for the onset of differentiation. Regulation of p15 and p21 is principally governed by transforming growth factor (TGF)-beta-signaling pathway. We have identified that Cdh1/APC, a critical ubiquitin protein ligase, plays an important role in regulating lens differentiation by facilitating TGF-beta-induced degradation of SnoN, a transcriptional corepressor that needs to be removed for transcriptional activation of p15 and p21. The depletion of Cdh1 by RNA interference attenuates the TGF-beta-mediated induction of p15 and p21 and significantly blocks lens differentiation. Expression of nondegradable SnoN also noticeably attenuates lens induction. Furthermore, we have shown that Cdh1 and SnoN form a complex at the onset of lens differentiation. In vivo histological analysis confirms our biochemical and genetic results. Thus, Cdh1/APC is crucial to the coordination of cell cycle progression and the initiation of lens differentiation through mediating TGF-beta-signaling-induced destruction of SnoN.

Differences in cyclin D2 and D1 protein expression distinguish forebrain progenitor subsets.

Regulation of neural proliferation is an essential component of brain formation and is driven by both intrinsic cell cycle and extrinsic growth and trophic molecules. Among the cell cycle proteins, understanding of the relative roles of the G1-phase active cyclins D2 and D1 (cD2 and cD1) has been hampered by lack of data regarding their expression patterns. In this study, cD2 immunoreactivity was examined in the neocortex, ganglionic eminences/striatum, and hippocampal formation from embryonic day 12.5 until postnatal day 60 to more precisely characterize the expression of this protein during forebrain development. The localization of cD1 was also immunohistologically mapped for comparison. Throughout forebrain development, both overlapping and nonoverlapping protein expression of these cyclins suggests the presence of shared and unique cell cycle requirements for neurogenesis that distinguishes progenitor pools.

Inducible nitric oxide synthase contributes to gender differences in ischemic brain injury.

Estrogens have antiinflammatory actions and protect the brain from ischemic injury. Cerebral ischemia is accompanied by an inflammatory reaction that contributes to the tissue damage, an effect mediated in part by toxic amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS). Therefore, estrogens may protect the female brain by modulating postischemic iNOS expression. To test this hypothesis, we studied whether iNOS plays a role in the mechanisms of the reduced susceptibility to ischemic injury observed in female mice. The middle cerebral artery was occluded for 20 mins using an intraluminal filament in C57Bl/6 mice, and infarct volume was assessed 3 days later in cresyl violet-stained sections. Infarcts were 53% smaller in female mice than in males (P < 0.05), a reduction abolished by ovariectomy (OVX) and reinstated by estrogen replacement. In normal female mice, postischemic iNOS mRNA was lower than in males (P < 0.05). Ovariectomy increased iNOS mRNA after ischemia and estrogen replacement blocked this effect. Furthermore, the iNOS inhibitor aminoguanidine reduced infarct volume in male, but not in female, mice. Similarly, male iNOS-null mice had smaller infarcts than wild-type mice, but female iNOS nulls were not protected. Ovariectomy and OVX with estrogen replacement did not affect infarct volume in iNOS-null female mice. The findings suggest that the neuroprotection conferred by estrogens is, in part, related to attenuation of iNOS expression. Such attenuation could result from the potent antiinflammatory effects of estrogens that downregulate iNOS expression via transcriptional or posttranscriptional mechanisms.

Crooked tail (Cd) model of human folate-responsive neural tube defects is mutated in Wnt coreceptor lipoprotein receptor-related protein 6.

A cranial neural tube defect in Crooked tail (Cd) mice is prevented with prenatal dietary folic acid Cd positional cloning reveals a missense mutation of a highly conserved amino acid in the low density lipoprotein receptor-related protein 6 (Lrp6), a coreceptor required for Wnt canonical signaling. Molecular modeling predicts that Lrp6(Cd) alters a hinge region of the second YWTD beta-propeller domain. Mutant LRP6 binds to Wnt and Dickkopf1 (Dkk1) but not Mesd1, and Dkk1 cannot antagonize Wnt in Cd/Cd cells, resulting in hyperactivity. NIH 3T3 cells transfected with a mutant Lrp6 plasmid resist Dkk1 antagonism much like Cd/+ cells, confirming the significance of the mutation. The Lrp6 mutation in Cd mice provides evidence for a functional connection between Wnt signaling and folate rescue of neural tube defects.