TRK-fused Gene Protein Localization Is Prominent in Serotonergic and Noradrenergic Cell Groups, and Some Lower Motor Neurons in the Corticospinal Tract of the Rat Brainstem.

The TRK-fused gene (TFG) is reported to be involved in the regulation of cell size, apoptosis, cell growth, ER-Golgi protein secretion, NF-κß pathway signaling, the ubiquitin-proteasome system, and pancreatic ß-cell mass and function. TFG mutations were reported in some neurodegenerative diseases affecting sensory and motor functions. However, the function of TFG in the nervous system and how TFG mutations lead to neurodegeneration remain unclear. In this study, we employed double immunohistochemistry to investigate the details of TFG localization patterns in monoaminergic and cholinergic neurons in the brainstem. Intense TFG immunoreactivity was observed in the dorsal raphe nucleus, the locus coeruleus, and the ventral horn of the spinal cord. TFG immunoreactivity was observed in some serotonergic neurons in all B1-B9 cell groups, and some noradrenergic neurons in all A1-A7 cell groups in the rat brainstem, while no immunoreactivity was observed in the dopaminergic neurons in A8-A10 cell groups. TFG immunoreactivity was observed in all ChAT-positive motor nuclei in the lower corticospinal tract of the rat brainstem.

Differences in Gene Expression Profiles and Phenotypes of Differentiated SH-SY5Y Neurons Stably Overexpressing Mitochondrial Ferritin.

Mitochondrial ferritin (FtMt) is an iron-transport protein with ferroxidase properties localized to mitochondria. Levels are generally low in all tissues, while increasing the expression of FtMt in neuronal-like cells has been shown to be protective. To determine whether FtMt has potential as a therapeutic approach, there remains the question of how much FtMt is protective. To address this issue, we transfected SH-SY5Y neuroblastoma cells with a FtMt expression plasmid and isolated cell lines with stable expression of FtMt at high, medium and low levels. Using these cell lines, we examined effects of FtMt on neuronal phenotype, neuroprotective activity and gene expression profiles. The phenotypic properties of high, medium and low FtMt expressors were compared with native untransfected SH-SY5Y cells after differentiation with retinoic acid to a neuronal phenotype. Overexpression of FtMt, even in low expressing cells, showed significant protection from oxidative stress induced by hydrogen peroxide or cobalt chloride. Higher levels of FtMt expression did not appear to offer greater protection, and did not have toxic consequences to cells, even though there were significantly more aggregated mitochondria in the highest expressing clone. The phenotypes differed between cell clones when assessed by cell growth, neurite outgrowth, and expression of neuronal proteins including those associated with neurodegenerative diseases. Microarray analysis of high, medium and negative FtMt-expressing cells identified different patterns of expression of certain genes associated with oxidative stress and neuronal development, amongst others. Validation of microarray analyses was carried out by real time polymerase chain reaction. The results showed significant differences in expression of thioredoxin-interacting protein (TXNIP) and microsomal glutathione transfer-1 (MGST-1), which can have critical roles in the regulation of oxidative stress. Differences in expression of calcitonin-related polypeptide alpha (CALCA), growth differentiation factor-15 (GDF-15) and secretogranin II (SCG2) were also observed. Our findings indicate that even low levels of increased FtMt expression can be protective possibly by alterations of some oxidative stress-related and growth factor genes, while high levels of expression did not appear to offer greater protection from oxidative stress or induce significant toxicity in cells. These experiments provide supporting data that increasing FtMt might be a feasible strategy for therapeutics in certain neurodegenerative and neurological diseases.

Upregulation of mitochondrial ferritin by proinflammatory cytokines: implications for a role in Alzheimer's disease.

Studies have shown an increased expression of mitochondrial ferritin (FtMt) and an antioxidant role for the protein in the brains of Alzheimer's disease (AD) patients. However, little information is available concerning the role of FtMt in other AD pathologies, including inflammation and amyloidogenesis. Therefore, we investigated the regulation and function of FtMt in inflammation and amyloidogenesis. FtMt protein expression was increased by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin 6 (IL-6), whereas FtMt mRNA levels were increased by TNF-α but not by IL-1ß or IL-6 in IMR-32 cells. The transcription factor nuclear factor-κB (NF-κB) inhibitor, Bay 11-7082, suppressed this TNF-α-induced FtMt expression. FtMt overexpression increased NF-κB activity and translocation of p65 into the nucleus in HEK293 cells. Conversely, knockdown of FtMt attenuated TNF-α-induced NF-κB activity. Overexpression of FtMt inhibited TNF-α-induced apoptosis in the cell culture. FtMt overexpression reduced iron-mediated expression of amyloid-ß protein precursor and decreased NF-κB-dependent increases in ß- and γ-secretase, leading to decreased amyloid-ß production. Our data provide new insights into the mechanism underlying the regulation of FtMt expression by proinflammatory cytokines and indicate further roles for FtMt in AD.

Immunohistochemical Localization of an Isoform of TRK-Fused Gene-Like Protein in the Rat Retina.

The TRK-fused gene (TFG) was originally identified in chromosome translocation events, creating a pair of oncogenes in some cancers, and was recently demonstrated as the causal gene of hereditary motor and sensory neuropathy with proximal dominant involvement. Recently, we cloned an alternative splicing variant of Tfg from a cDNA library of the rat retina, tentatively naming it retinal Tfg (rTfg). Although the common form of Tfg is ubiquitously expressed in most rat tissues, rTfg expression is localized to the central nervous system. In this study, we produced an antibody against an rTFG-specific amino acid sequence and used it to examine the localization of rTFG-like protein in the rat retina by immunohistochemistry and Western blots. Western blot analysis showed that the antibody detected a single band of 24 kDa in the rat retina. When we examined rTFG recombinant protein, the antibody detected two bands of about 42 kDa and 24 kDa. The results suggest that the 24 kDa rTFG-like protein is a fragment of rTFG. In our immunohistochemical studies of the rat retina, rTFG-like immunoreactivity was observed in all calbindin D-28K-positive horizontal cells and in some syntaxin 1-positive amacrine cells (ACs). In addition, the rTFG-like immunopositive ACs were actually glycine transporter 1-positive glycinergic or glutamate decarboxylase-positive GABAergic ACs. Our findings indicate that this novel 24 kDa rTFG-like protein may play a specific role in retinal inhibitory interneurons.

Mitochondrial ferritin in neurodegenerative diseases.

Mitochondrial ferritin (FtMt) is a novel protein encoded by an intronless gene mapped to chromosome 5q23.1. Ferritin is ubiquitously expressed; however, FtMt expression is restricted to specific tissues such as the testis and the brain. The distribution pattern of FtMt suggests a functional role for this protein in the brain; however, data concerning the roles of FtMt in neurodegenerative diseases remain scarce. In the human cerebral cortex, FtMt expression was increased in Alzheimer's disease patients compared to control cases. Cultured neuroblastoma cells showed low-level expression of FtMt, which was increased by H2O2 treatment. FtMt overexpression showed a neuroprotective effect against H2O2-induced oxidative stress and Aß-induced neurotoxicity in neuroblastoma cells. FtMt expression was also detected in dopaminergic neurons in the substantia nigra and was increased in patients with restless legs syndrome, while FtMt had a protective effect against cell death in a neuroblastoma cell line model of Parkinson's disease. FtMt is involved in other neurodegenerative diseases such as age-related macular degeneration (AMD), with an FtMt gene mutation identified in AMD patients, and Friedreich's ataxia, which is caused by a deficiency in frataxin. FtMt overexpression in frataxin-deficient cells increased cell resistance to H2O2 damage. These results implicate a neuroprotective role of FtMt in neurodegenerative diseases.

Kainic acid induces expression of caveolin-1 in activated microglia in rat brain.

Caveolin-1, a major constituent of caveolae, has been implicated in endocytosis, signal transduction and cholesterol transport in a wide variety of cells. In the present study, the expression of caveolin-1 was examined by immunohistochemistry in rat brain with or without systemic injection of kainic acid (KA). Caveolin-1 immunoreactivity was observed in capillary walls in brains of control rats. From one to seven days after KA injection, caveolin-1 immunoreactivity appeared in activated microglia in the cerebral cortex, hippocampus and other brain regions. The strongest immunoreactivity of microglia was seen after 3 days after KA administration. The expression of caveolin-1 was confirmed by RT-PCR and Western blot analysis, respectively. The induction of caveolin-1 expression in microglia activated in response to kainic acid administration suggests its possible role in a modulation of inflammation.

Immunohistochemical Mapping of TRK-Fused Gene Products in the Rat Brainstem.

The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It was since reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. As shown in the accompanying paper, we produced an antibody to rat TFG and used it to localize TFG to selected neurons in specific regions. In the present study, we mapped the TFG-positive neurons in the brainstem, cerebellum, and spinal cord of rats. In the brainstem, neurons intensely positive for TFG were distributed in the raphe nuclei, the gigantocellular reticular nucleus, the reticulotegmental nucleus of the pons, and some cranial nerve nuclei such as the trigeminal nuclei, the vestibulocochlear nuclei, and the dorsal motor nucleus of the vagus. Purkinje cells in the cerebellum and motor neurons in the spinal anterior horn were also positive for TFG. These results provide fundamental data for studying the functions of TFG in the brain.

Mapping of FGF1 in the Medulla Oblongata of Macaca fascicularis.

FGF1 is highly expressed in neurons and it has been proposed to play a role in the neuroprotection and in regeneration. Low FGF1 expression in neurons has been linked to increased vulnerability in cholinergic neurons. Previous reports have shown that the expression of FGF1 in rat brain is localized to the cholinergic nuclei of the medulla oblongata, with low ratio of neurons positive for FGF1 in the dorsal motor nucleus of the vagus (DMNV). The role of FGF1 in the primate brain has yet to be clarified. In this study, we mapped FGF1 immunoreactivity in the medulla oblongata of cynomolgus monkey brainstems. Our results demonstrated that FGF1 immunoreactivity follows the pattern of distribution of cholinergic nuclei in the medulla oblongata; with strong localization of FGF1 to cholinergic neurons of the hypoglossal nucleus, the facial nucleus and the nucleus ambiguus. In contrast, the DMNV shows markedly lower FGF1 immunoreactivity. Localization of FGF1 to cholinergic neurons was only observed in the lateral region of the DMNV, with higher immunoreactivity in the rostral ventral-lateral region of the DMNV. These findings are consistent with the distribution of FGF1 immunoreactivity in previous studies of the rat brain.

Induction of protective immunity by vaccination with wild-type apo superoxide dismutase 1 in mutant SOD1 transgenic mice.

Vaccinations targeting extracellular superoxide dismutase 1 (SOD1) mutants are beneficial in mouse models of amyotrophic lateral sclerosis (ALS). Because of its misfolded nature, wild-type nonmetallated SOD1 protein (WT-apo) may have therapeutic application for vaccination of various SOD1 mutants. We compared the effects of WT-apo to those of a G93A SOD1 vaccine in low-copy G93A SOD1 transgenic mice. Both SOD1 vaccines induced antibody against G93A SOD1 and significantly delayed disease onset compared with saline/adjuvant controls. WT-apo SOD1 significantly extended the life span of vaccinated mice. The vaccines potentiated TH2 deviation in the spinal cord as determined by the ratio of interleukin-4 to interferon-γ (IFNγ) or tumor necrosis factor and induced C1q deposition around motor neurons. Transgenic mice had abundant microglial expression of signal transducers and activators of transcription 4, an activator of transcription of IFNγ, in the spinal cord implicating IFNγ in the pathogenesis. On the other hand, the sera from G93A SOD1-vaccinated mice showed higher IFNγ or tumor necrosis factor and yielded a lower IgG1/IgG2c ratio than the sera from WT-apo-vaccinated mice. These results indicate that the TH1/TH2 milieu is affected by specific vaccinations and that antigenicity might counteract beneficial effects by enhancing TH1 immunity. Thus, because of its lower TH1 induction, WT-apo may be a therapeutic option and have broader application in ALS associated with diverse SOD1 mutations.