An unusual protease ␥-secretase requires functional presenilins and cleaves substrates (e.g. amyloid -protein precursor and Notch) with very loose amino acid sequence specificity within the transmembrane region. Here we report that ErbB4, a tyrosine kinase receptor for neuregulins, is a substrate for presenilin-dependent ␥-secretase. Our studies show that constitutive ectodomain shedding of full-length ErbB4 yields the ϳ80-kDa membrane-associated C-terminal fragment (B4-CTF). Subsequent intramembrane cleavage of the B4-CTF was inhibited in the cells devoid of functional presenilins or by treatment of cells with a ␥-secretase inhibitor, leading to enhanced accumulation of B4-CTF. Furthermore, an in vitro ␥-secretase assay demonstrated that the intracellular domain of ErbB4 (B4-ICD) was produced and subsequently released into the soluble fraction in a presenilin-dependent manner. We have also shown that ectopically expressed B4-ICD is localized to the nucleus, suggesting that the presenilin-dependent cleavage of ErbB4 generates the soluble B4-ICD that functions in the nucleus presumably at transcriptional level. Our study indicates that ErbB4 represents a first receptor tyrosine kinase that undergoes intramembrane proteolysis and may mediate a novel signaling function independent of its canonical role as a receptor tyrosine kinase. Our studies also support the idea that presenilins play a generic role in intramembrane cleavage of selected type I membrane proteins.ErbB4 is a type I membrane receptor tyrosine kinase, which belongs to the epidermal growth receptor family and mediates response to multiple growth factors, including neuregulins (reviewed in Refs. 1-3). ErbB4 has been implicated in many important biological and pathological processes, such as cardiovascular, mammary gland, and neural development, as well as malignancy and heart disease (1-3).Presenilins (PS1 and PS2), 1 gene products of the major earlyonset familial Alzheimer's disease genes (reviewed in Refs. 4 -6), are required for the activity of ␥-secretase, an unusual aspartyl protease that cleaves substrates within the predicted transmembrane region (7; reviewed in Refs. 8 -10). Two of the characteristics of ␥-secretase include a lack of requirement for specific amino acid target sequences within the transmembrane domain and a requirement for ectodomain shedding to produce membrane-anchored truncated C-terminal derivatives (10, 11). These observations imply that the presenilins may also be involved in the intramembrane cleavage of other type I membrane proteins. The ␥-secretase cleavage of amyloid -protein precursor (APP) is a critical rate-limiting step toward the production of amyloid -peptide (A) in Alzheimer's disease (6). In addition to APP, transmembrane cleavage of Notch, which releases the Lin-12/Notch intracellular domain, plays a pivotal role in cell fate determination (12, 13). Ectopically expressed intracellular domains of APP (AICD) and Notch (NICD) appear to be localized in the nucleus and participate in gene transcription (12-20)...
Neurogenesis in the adult mammalian nervous system is now well established in the subventricular zone of the anterolateral ventricle and subgranular zone of the hippocampus. In these regions, neurons are thought to arise from neural stem cells, identified by their expression of specific intermediate filament proteins (nestin, vimentin, GFAP) and transcription factors (Sox2). In the present study, we show that in adult rat and mouse, the circumventricular organs (CVOs) are rich in nestin + , GFAP + , vimentin + cells which express Sox2 and the cell cycle-regulating protein Ki67. In culture, these cells proliferate as neurospheres and express neuronal (doublecortin + , β-tubulin III + ) and glial (S100β + , GFAP + , RIP + ) phenotypic traits. Further, our in vivo studies using bromodeoxyuridine show that CVO cells proliferate and undergo constitutive neurogenesis and gliogenesis. These findings suggest that CVOs may constitute a heretofore unknown source of stem/progenitor cells, capable of giving rise to new neurons and/or glia in the adult brain.
The mechanism of copper (Cu) neurotoxicity was studied in the RCSN-3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu± dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu±dopamine complex mediates the uptake of 64 CuSO 4 into the Rau  lCaviedes substantia nigra-clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 mM) (63%, p , 0.001) and nomifensine (2 mM) (77%, p , 0.001). Copper sulfate (1 mM) alone was not toxic to RCSN-3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p , 0.001) which inhibits DPNH and TPNH (DT)-diaphorase. Electron spin resonance (ESR) spectrum of the 5,5-dimethylpyrroline-N-oxide (DMPO) spin trap adducts showed the presence of a C-centered radical when incubating cells with dopamine, CuSO 4 and dicoumarol. A decrease in the expression of CuZn-superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN-3 cells were treated with CuSO 4 , dopamine, or CuSO 4 and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO 4 , dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO 4 increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu±dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine-dependent Cu uptake; and (iii) one-electron reduction of aminochrome.
Production of new neurons throughout adulthood has been well characterized in two brain regions, the subventricular zone (SVZ) of the anterolateral ventricle and the subgranular zone (SGZ) of the hippocampus. The neurons produced from these regions arise from neural stem cells (NSCs) found in highly regulated stem cell niches. We recently showed that midline structures called circumventricular organs (CVOs) also contain NSCs capable of neurogenesis and/or astrogliogenesis in vitro and in situ [3]. The present study demonstrates that NSCs derived from two astrogliogenic CVOs, the median eminence and organum vasculosum of the lamina terminalis of the Nestin-GFP mouse, possess the potential to integrate into the SVZ and differentiate into cells with a neuronal phenotype. These NSCs, following expansion and BrdU-labeling in culture and heterotopic transplantation into a region proximal to the SVZ in adult mice, migrate caudally to the SVZ and express early neuronal markers (TUC-4, PSA-NCAM) as they migrate along the rostral migratory stream. CVO-derived BrdU + cells ultimately reach the olfactory bulb where they express early (PSA-NCAM) and mature (NeuN) neuronal markers. Collectively, these data suggest that although NSCs derived from the ME and OVLT CVOs are astrogliogenic in situ, they produce cells phenotypic of neurons in vivo when placed in a neurogenic environment. These findings may have implications for neural repair in the adult brain.
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