Faculty Bibliography

A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf(mi), with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf(mi)/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf(mi)/Mitf(mi) embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration

Excitatory synaptic currents in the mammalian brain are typically mediated by the neurotransmitter glutamate, acting at AMPA receptors. We used immunocytochemistry to investigate the distribution of AMPA receptor-binding protein (ABP) in the cerebral neocortex. ABP was most prominent in pyramidal neurons, although it was also present (at lower levels) in interneurons. ABP and its putative binding partners, the GluR2/3 subunits of the AMPA receptor, exhibited prominent cellular colocalization. Under appropriate processing conditions, colocalization could also be documented in puncta, many of which could be recognized as dendritic spines. However, a sizable minority of GluR2/3-positive puncta were immunonegative for ABP. Because glutamate receptor-interacting protein (GRIP) may also anchor GluR2, we studied the relative distribution of ABP and GRIP. There was extensive colocalization of these two antigens at the cellular level, although GRIP, unlike ABP, was strongest in nonpyramidal neurons. Different parts of a single dendrite could stain selectively for ABP or GRIP. To further characterize this heterogeneity, we investigated punctate staining of neuropil using synaptophysin and the membrane tracer DiA to identify probable synapses. Some puncta were comparably positive for both ABP and GRIP, but the majority were strongly positive for one antigen and only weakly positive or immunonegative for the other. This heterogeneity could be seen even within adjacent spines of a single dendrite. These data suggest that ABP may act as a scaffold for AMPA receptors either in concert with or independently from GRIP

Recent studies have revealed that trafficking of AMPA receptors may be responsible for some forms of synaptic plasticity. For example, LTD is mediated at least in part by endocytosis of AMPA receptors from the synaptic plasma membrane, and LTP can involve insertion of AMPARs into the synaptic membrane by exocytosis. The PDZ proteins ABP/GRIP and PICK1 in addition to the ATPase NSF bind to the C-terminal tail of the GluR2 subunit and are thought to play roles in these processes. NSF is a well characterized molecular chaperone which disrupts SNARE complexes upon ATP hydrolysis as a crucial step in membrane fusion events. alpha-SNAP is a required co-factor for this reaction. PICK1 may function to traffic AMPA receptors to or from the plasma membrane. We studied the interactions between GluR2, PDZ proteins, NSF and alpha/beta-SNAPs in an isolated system of purified, bacterially expressed components. The GluR2-NSF interaction in the absence if other proteins is sensitive to ATP hydrolysis, and the GluR2-PICK1 complex has a much higher affinity for NSF than GluR2 alone. The GluR2-PICK1 interaction requires non-PDZ contacts within the NSF binding site. Both alpha- and beta-SNAP bind directly to PICK1. We demonstrate association of PICK1 with NSF and SNAPs in brain. ATP hydrolysis by NSF will disrupt the PICK1-GluR2 complex when alpha-SNAP is bound, and this is inhibited by the binding of beta-SNAP. We propose a model whereby NSF and SNAPs modulate levels of AMPA receptor at the synaptic membrane by regulating interaction of PICK1 with GluR2

Establishment of mice deficient in neuropeptide FF (NPFF) was achieved using homologous recombination of the entire NPFF precursor coding region in embryonic stem cells. The disruption of the NPFF locus was confirmed by Southern blotting and PCR. The absence of NPFF precursor mRNA expression in null mutant mice was confirmed using in situ hybridization and RT-PCR. The expression of related peptide precursor mRNAs such as prolactin releasing peptide (PrRP) mRNA and RFamide related peptide (RFRP) mRNA was not affected in the NPFF null mutant mice. Immunohistochemical analysis of NPFF in the CNS of null mutant mice revealed weakly NPFF-positive fibers in the nucleus tractus solitarius. This result may be due to antibody crossreactivity to closely related RFamide peptides such as RFRP-1 and RFRP-3. In heterozygous mice, variable levels of NPFF-immunoreactivity were observed in the CNS, suggesting a complex regulation of RFamide peptide expression. Interestingly, wild type mice were observed to contain an abundant variant form of NPFF mRNA that retains the first intron. Whether this NPFF splice variant excerts a regulatory role in the expression of the NPFF or generates additional peptides remains to be elucidated. Characterization of the behavioural phenotype of the NPFF null mutant mice is in progress

The regulation of AMPA-receptor (AMPAR) transport to synapses may be involved in the modulation of synaptic strength. We describe the existence of a prominent intracellular pool of the Ca2+-impermeable AMPAR subunit GluR2. The intracellular form of GluR2 is characterised by the presence of high-mannose type glycans, which are linked to proteins traversing the early part of the secretory pathway, i.e the ER, the intermediate compartment and the cis-Golgi. The existence of this GluR2 pool is based on subcellular fractionations, sedimentation on sucrose gradients, de-glycosylation and cell surface crosslinking experiments. An ER localisation is suggested by the co-fractionation and co-sedimentation with the ER marker calnexin. Interestingly, the subcellular localisation and ER export kinetics of GluR2 are strikingly different from GluR1, a receptor subunit that oligomerises with GluR2 to form a functional AMPAR. Since this form of GluR2 is unexpectedly stable (t1/2apprx24 hrs) it may represent an intracellular GluR2 reserve pool

OBJECTIVE: Few molecular targets are both consistently and selectively expressed in a majority of central nervous system (CNS) neoplasms. Receptor tyrosine kinases have been implicated in brain tumor oncogenesis. We previously isolated one such receptor, discoidin domain receptor-1 (DDR1), from high-grade pediatric brain tumors. Here, we analyze the cellular origin and distribution of DDR1 expression in human brain tumors and its expression in tumor cells relative to surrounding brain. METHODS: By use of a digoxigenin-labeled DDR1 riboprobe, we investigated the expression of DDR1 messenger ribonucleic acid in a prospective series of 30 resected human primary and metastatic brain neoplasms, nonneoplastic human brain, and mouse embryonic brain, as well as a mouse glioblastoma model, by in situ hybridization. RESULTS: All the high-grade primary brain and metastatic brain tumors showed unequivocal, intense DDR1 expression within the majority of tumor cells, whereas expression was not observed in hyperplastic tumor blood vessels, normal brain blood vessels, inflammatory cells, or in the normal brain tissue that surrounded the tumor. Receptor expression was limited to tumor cells located within solid tumor tissue. Overall, 27 of 29 resected CNS tumors exhibited tumor cell-specific DDR1 expression, whereas one specimen composed of isolated glioblastoma cells within invaded brain parenchyma showed no detectable staining for this receptor. DDR1 was also expressed preferentially in the ventricular zone (a region of highly proliferating precursor cells) of mice at embryonic Day 15.5. CONCLUSION: We found that DDR1 is consistently expressed in all high-grade brain neoplasms studied and is selective for tumor cells in the specimens analyzed. The expression of DDR1 by tumor cells of CNS neoplasms and by primitive cells of the embryonic ventricular zone suggests that DDR1 is a potentially useful marker of tumor cells within the CNS

The mechanisms by which the NMDA receptor (NMDAR) induces excitotoxicity were investigated using a novel assay. We quantitated the capacity of wild type and mutant receptors for cell killing in CHO cells and cultured cortical neurons by measuring the activity of a co-transfected firefly luciferase expression plasmid. NR1 subunit pore mutations that block Ca(2+) influx, and deletion of the NR1 cytoplasmic C-terminal domain, which functions in Ca(2+) regulation of receptor currents, decreased NMDAR mediated cell killing. We also transfected the NR1 pore mutants and C-terminal truncations in the presence of co-expressed exogenous wild type subunits. The pore and C-terminal truncation mutants acted in a dominant negative fashion and increased the survival of NMDAR-expressing CHO cells. Although physiological studies of similar NMDA receptor mutants have been carried out in heterologous cell lines, their functions in neurons remain relatively unknown. We show that expression of pore mutants and specific C terminal truncation mutants in cultured cortical neurons also exerts dominant negative function and protects these primary cells from endogenous receptor induced excitotoxic death. These results implicate positive actions of the selectivity filter and of the NR1 C-terminal domain in a Ca(2+)-dependent mechanism for NMDAR excitotoxicity. They also indicate that the mutant receptors which show diminished excitotoxicity and dominant negative action in heterologous cells can co-assemble with endogenous subunits in primary neurons and block NMDAR-dependent excitotoxic death

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate

The expression of the dopachrome tautomerase gene (Dct) and its protein product, tyrosinase-related protein-2, was studied in the cultured, phorbol-ester-dependent murine melanocyte cell line melan-a. Increased cell density was found to stimulate Dct expression both in cells stably transfected with a Dct promoter-lacZ construct and endogenously in nontransfected cells. Increased Dct expression under these conditions corresponds to increased tyrosinase-related protein-2 production. Tyrosinase-related protein-2 was found to exist in two distinct glycoforms with different endoglycosidase sensitivities. Density-dependent expression of tyrosinase-related protein-2 was independent of time of cell growth, cell proliferation, and soluble factors, implying that cell-cell contact is the important determinant governing increased Dct expression under these conditions. Tyrp1 gene expression and tyrosinase-related protein-1 production were also induced under similar conditions. The results show that cell-cell contact between melanocytes induces a coordinated response at both transcriptional and nontranscriptional levels that induces production of the tyrosinase-related proteins that have a significant role in melanization