Ubiquitin-positive, tau- and alpha-synuclein-negative inclusions are hallmarks of frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Although the identity of the ubiquitinated protein specific to either disorder was unknown, we showed that TDP-43 is the major disease protein in both disorders. Pathologic TDP-43 was hyper-phosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments and was recovered only from affected central nervous system regions, including hippocampus, neocortex, and spinal cord. TDP-43 represents the common pathologic substrate linking these neurodegenerative disorders.
Frontotemporal lobar degeneration with ubiquitinpositive inclusions (FTLD-U) is a common neuropathological subtype of frontotemporal dementia. Although this subtype of frontotemporal dementia is defined by the presence of ubiquitin-positive but tauand ␣-synuclein-negative inclusions, it is unclear whether all cases of FTLD-U have the same underlying pathogenesis. Examination of tissue sections from FTLD-U brains stained with anti-ubiquitin antibodies revealed heterogeneity in the morphological characteristics of pathological inclusions among subsets of cases. Three types of FTLD-U were delineated based on morphology and distribution of ubiquitin-positive inclusions. To address the hypothesis that FTLD-U is pathologically heterogeneous, novel monoclonal antibodies (mAbs) were generated by immunization of mice with high molecular mass (M r > 250 kd) insoluble material prepared by biochemical fractionation of FTLD-U brains. Novel mAbs were identified that immunolabeled all of the ubiquitin-positive inclusions in one subset of FTLD-U cases, whereas other mAbs stained the ubiquitin-positive inclusions in a second subset of cases. These novel mAbs did not stain inclusions in other neurodegenerative disorders, including tauopathies and ␣-synucleinopathies. Therefore, ubiquitin immunohistochemistry and the immunostaining properties of the novel mAbs generated here suggest that FTLD-U is pathologically heterogeneous. Identification of the disease proteins recognized by these mAbs will further advance understanding of molecular substrates of FTLD-U neurodegenerative pathways.
Neurotrophins act through their cognate receptors to promote the differentiation and/or survival of neuronal progenitor cells, immature neurons, and other cells. Here, we examined the effects of nerve growth factor (NGF) and its cognate receptor (Trk or TrkA) on the survival of a common childhood brain tumor, i.e., medulloblastoma, a tumor that resembles CNS neuroepithelial progenitor cells. To do this, we engineered two human medulloblastoma cell lines (i.e., D283MED and DAOY cells) to express human TrkA using a retroviral expression vector. Surprisingly, NGF-treated medulloblastoma cells expressing the TrkA receptor (D283trk and DAOYtrk cells) grown in the presence or absence of serum underwent massive apoptosis, but similar treatment did not induce apoptosis in wild-type uninfected cells, cells expressing an empty vector, or cells expressing the TrkC receptor. Furthermore, D283MED cells engineered to express the human p75 NGF receptor (D283p75) also did not undergo apoptosis. Significantly, NGF-induced apoptosis in D283trk and DAOYtrk cells can be inhibited by anti-NGF antibodies and by K-252a, an inhibitor of TrkA tyrosine phosphorylation and mimicked by high concentrations of NT3. Because NGF treatment primarily eliminated D283trk cells from the S phase of the cell cycle, this form of NGF-mediated apoptosis is cell cycle-dependent. These findings suggest that a NGF/TrkA signal transduction pathway could activate apoptotic cell death programs in CNS neuroepithelial progenitor cells and in childhood brain tumors.
Activation of the neurotrophin receptor TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events leading to neuronal di erentiation in vitro and might play an important role in the di erentiation of favorable neuroblastomas (NB) in vivo. To study TrkA signal transduction pathways and their e ects on di erentiation in NB, we stably expressed wild-type TrkA and ®ve di erent TrkA mutants in the NGF unresponsive human NB cell line SH-SY5Y. Resulting clones were characterized by TrkA mRNA and protein expression, and by autophosphorylation of the receptor. Introduction of wild-type TrkA restored NGF responsiveness of SH-SY5Y cells, as demonstrated by morphological di erentiation, activation of mitogenactivated protein kinases (MAPK) and induction of immediate-early genes. Expression of TrkA in the absence of NGF resulted in growth inhibition of transfectants compared to parental cells, whereas NGFtreatment increased their proliferation rate. Analysis of downstream signal transduction pathways indicated that NGF-induced di erentiation was dependent on TrkA kinase activity. Our data suggest that several redundant pathways are present further downstream, but activation of the RAS/MAPK signaling pathway seems to be of major importance for NGF mediated di erentiation of NB cells. Our results also show that the signaling e ector SH2-B is a substrate of NGF-mediated Trk signaling in NB, whereas it is not activated by NGF in rat pheochromocytoma PC12 cells. This might explain the di erences we observed in TrkA signaling between neuroblastoma and PC12 cells. Further insight into TrkA signaling may suggest new options for the treatment of NB. Oncogene (2000) 19, 2043 ± 2051.Keywords: neuroblastoma; tyrosine kinase receptors; neurotrophins; signal transduction; di erentiation IntroductionNeuroblastoma is one of the most common pediatric neoplasms and is derived from the neural crest. Neurotrophic factors and their tyrosine kinase receptors (Trks) play an important role in the pathogenesis, biology and clinical behavior of NB (Brodeur, 1993). Observations from several independent studies suggest that high expression of the neurotrophin receptor TrkA is present in NB with favorable biological features and highly correlated with patient survival (Nakagawara et al., 1993;Kogner et al., 1993). Activation of TrkA by its ligand NGF leads to survival and di erentiation of TrkA expressing cells in vitro (Klein et al., 1991) and might play an important role in the regression or di erentiation of NB in vivo.Signal transduction pathways used by TrkA have been studied mainly in the PC12 rat pheochromocytoma cell line. Following NGF binding, TrkA receptors rapidly become phosphorylated on tyrosine residues, and their tyrosine kinase domain is activated (Klein et al., 1991). Phosphorylated tyrosine residues in the TrkA cytoplasmic domain serve as anchors for binding downstream signaling molecules (Schlessinger and Ullrich, 1992). Proteins known to become phosphorylated and activated in response to NGF include phosph...
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