Disruption of basal forebrain cholinergic neurons after traumatic brain injury does not compromise environmental enrichment-mediated cognitive benefits.

Environmental enrichment (EE) attenuates traumatic brain injury (TBI)-induced loss of medial septal (MS) choline acetyltransferase (ChAT)-cells and enhances spatial learning and memory vs. standard (STD) housing. Whether basal forebrain cholinergic neurons (BFCNs) are important mediators of EE-induced benefits after TBI requires further investigation. Anesthetized female rats were randomly assigned to intraseptal infusions of the immunotoxin 192-IgG-saporin (SAP; 0.22 µg in 1.0 µL) or vehicle (VEH; 1.0 µL IgG) followed immediately by a cortical impact (2.8 mm deformation depth at 4 m/s) or sham injury and divided into EE and STD housing. Spatial learning and memory retention were assessed on post-operative days 14-19. MS ChAT+ cells were quantified at 3 weeks. SAP significantly reduced ChAT+ cells in both the EE and STD groups. Cognitive performance was improved in the EE groups, regardless of VEH or SAP infusion, vs. the STD-housed groups (p's < 0.05). No cognitive differences were revealed between the TBI + EE + SAP and TBI + EE + VEH groups (p > 0.05) or between the TBI + STD + SAP and TBI + STD + VEH groups (p > 0.05). These data show that despite significant MS ChAT+ cell loss, the EE-mediated benefit in cognitive recovery is not compromised.

Low UBE4B expression increases sensitivity of chemoresistant neuroblastoma cells to EGFR and STAT5 inhibition.

Neuroblastoma is the most common malignancy in infants. Overexpression of the epidermal growth factor receptor (EGFR) in neuroblastoma tumors underlies resistance to chemotherapeutics. UBE4B, an E3/E4 ubiquitin ligase involved in EGFR degradation, is located on chromosome 1p36, a region in which loss of heterozygosity is observed in approximately one-third of neuroblastoma tumors and is correlated with poor prognosis. In chemoresistant neuroblastoma cells, depletion of UBE4B yielded significantly reduced cell proliferation and migration, and enhanced apoptosis in response to EGFR inhibitor, Cetuximab. We have previously shown that UBE4B levels are inversely correlated with EGFR levels in neuroblastoma tumors. We searched for additional targets of UBE4B that mediate cellular alterations associated with tumorogenesis in chemoresistant neuroblastoma cells depleted of UBE4B using reverse phase protein arrays. The expression of STAT5a, an effector protein downstream of EGFR, doubled in the absence of UBE4B, and verified by quantitative immunoblotting. Chemoresistant neuroblastoma cells were treated with SH-4-54, a STAT5 inhibitor, and observed insignificant effects on cell proliferation, migration, and apoptosis. However, SH-4-54 significantly enhanced the anti-proliferative and anti-migratory effects of Cetuximab in naïve SK-N-AS neuroblastoma cells. Interestingly, in UBE4B depleted SK-N-AS cells, SH-4-54 significantly potentiated the effect of Cetuximab rendering cells increasingly sensitive an otherwise minimally effective Cetuximab concentration. Thus, neuroblastoma cells with low UBE4B levels were significantly more sensitive to combined EGFR and STAT5 inhibition than parental cells. These findings may have potential therapeutic implications for patients with 1p36 chromosome LOH and low tumor UBE4B expression.

Regulation of Somatostatin Receptor 2 Trafficking by C-Tail Motifs and the Retromer.

The Gi-coupled somatostatin receptor 2 (SST2) is a G protein-coupled receptor (GPCR) that mediates many of somatostatin's neuroendocrine actions. Upon stimulation, SST2 is rapidly internalized and transported to early endosomes before being recycled to the plasma membrane. However, little is known about the intracellular itinerary of SST2 after it moves to the early endosomal compartment or the cytoplasmic proteins that regulate its trafficking. As postsynaptic density protein/discs large 1/zonula occludens-1 (PDZ) domain interactions often regulate the trafficking and signaling potential of GPCRs, we examined the role of the SST2 PDZ ligand and additional C-terminal residues in controlling its intracellular trafficking. We determined that SST2 can recycle to the plasma membrane via multiple pathways, including a LAMP1/Rab7-positive late endosome to the trans-Golgi network (TGN) pathway. Trafficking from the late endosome to the TGN is often regulated by the retromer complex of endosomal coat proteins, and disrupting the retromer components sorting nexins 1/2 inhibits the budding of SST2 from late endosomes. Moreover, trafficking through the late endosomal/TGN pathway is dependent on an intact PDZ ligand and C-terminal tail, as truncating either the 3 or 10 C-terminal amino acids of SST2 alters the pathway through which it recycles to the plasma membrane. Moreover, addition of these amino acids to a heterologous receptor is sufficient to redirect it from a degradation pathway to a recycling itinerary. Our results demonstrate that endosomal trafficking of SST2 is dependent on numerous regulatory mechanisms controlled by its C terminus and the retromer machinery.

Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies.

Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) cytosol. We observed that inward and outward budding from the MVB membrane are mechanistically distinct but may be linked, such that inhibition of inward budding triggers a re-routing of cargo from inward to outward budding vesicles, without affecting the number of vesicles that bud outwardly from MVBs.

Systemic administration of donepezil attenuates the efficacy of environmental enrichment on neurobehavioral outcome after experimental traumatic brain injury.

BACKGROUND: The acetylcholinesterase inhibitor (AChEI) donepezil (DON) is recommended as a potential treatment for cognition after clinical traumatic brain injury (TBI) and therefore may be prescribed as an adjunct therapy during rehabilitation. However, a dose-response study evaluating DON after a controlled cortical impact (CCI) injury in rats did not reveal cognitive benefits. OBJECTIVE: The aim of this study was to determine the effect of DON on behavioral and histological outcome when combined with environmental enrichment (EE), a preclinical model of neurorehabilitation. It was hypothesized that the combined treatments would produce a synergistic effect yielding improved recovery over neurorehabilitation alone. METHODS: Isoflurane-anesthetized adult male rats received a CCI or sham injury and then were randomly assigned to EE or standard (STD) housing plus systemic injections of DON (0.25 mg/kg) or vehicle (VEH; 1.0 mL/kg saline) once daily for 19 days beginning 24 hr after injury. Function was assessed by established motor and cognitive tests on post-injury days 1-5 and 14-19, respectively. Cortical lesion volume was quantified on day 19. RESULTS: DON was ineffective when administered alone. In contrast, EE conferred significant motor and cognitive benefits, and reduced cortical lesion volume vs. STD (p < 0.05). Combining the therapies weakened the efficacy of rehabilitation as revealed by diminished motor and cognitive recovery in the TBI+EE+DON group vs. the TBI+EE+VEH group (p < 0.05). CONCLUSION: These data replicate previous findings showing that EE is beneficial and DON is ineffective after CCI and add to the literature a novel and unpredicted finding that supports neither the hypothesis nor the use of DON for TBI. Investigation of other AChEIs after CCI injury is necessary to gain further insight into the value of this therapeutic strategy.