Patricia Crisanti

The Expression of Thyrotropin Receptor in the Brain1

The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypot...The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypothalamic nuclei) and was mostly colocalized with neuron-specific enolase. In astrocytes, this mRNA was detected in the ependymal cell layer and the subependymal zone, and several isolated cells were also found in the brain parenchyma. We also detected TSH receptor mRNA and protein in primary cultured human astrocytes. The protein was detected as well in both rat and human brain cryoslices. Together, these findings clearly demonstrate the expression of the TSH receptor gene in the brain in both neuronal cells and astrocytes.

The Zinc Finger Transcription Factor, MOK2, Negatively Modulates Expression of the Interphotoreceptor Retinoid-binding Protein Gene, IRBP*

The human and murine MOK2 orthologue genes encode Krüppel/TFIIIA-related zinc finger proteins, which are factors able to recognize both DNA and RNA through their zinc finger motifs. MOK2 proteins have been shown to bind to the same 18-base pair (bp)-specific sequence in duplex DNA. This MOK2-binding site was found within introns 7 and 2 of human PAX3 and interphotoreceptor retinoid-binding protein (IRBP) genes, respectively. As these two genes are expressed in the brain asMOK2, we have suggested that PAX3 andIRBP genes are two potentially important target genes for the MOK2 protein. In this study, we focused our attention onIRBP as a potential MOK2 target gene. Sequence comparison and binding studies of the 18-bp MOK2-binding sites present in intron 2 of human, bovine, and mouse IRBP genes show that the 3′-half sequence is the essential core element for MOK2 binding. Very interestingly, 8-bp of this core sequence are found in a reverse orientation, in the IRBP promoter. We demonstrate that MOK2 can bind to the 8-bp sequence present in the IRBP promoter and repress its transcription when transiently overexpressed in retinoblastoma Weri-RB1 cells. In the IRBP promoter, it appears that the TAAAGGCT MOK2-binding site overlaps with the photoreceptor-specific CRX-binding element. We suggest that MOK2 represses transcription by competing with the cone-rod homeobox protein (CRX) for DNA binding, thereby decreasing transcriptional activation by CRX. Furthermore, we show that Mok2 expression in the developing mouse and in the adult retina seems to be concordant with IRBP expression.

Potassium channel ether à go-go mRNA expression in the spiral ligament of the rat

Identification of the K+ transporters located in the lateral wall of the cochlea is essential for a better understanding of the mechanisms by which a positive endocochlear potential and a high K+ concentration are achieved in endolymph. In this study, we have determined the distribution of the K+ channel rat ether à go-go (eag) mRNA in the cochlea. After reverse transcription of adult rat cochlear tissues, cDNA was amplified with primers specific to eag channel. The eag mRNA was localized in cochlear tissues by in situ hybridization using specific oligonucleotide probes tailed with digoxigenin conjugated UTP. Eag mRNA was detected in the organ of Corti but mainly in the fibrocytes of the spiral ligament but not in spiral prominence or in stria vascularis. The expression pattern of rat eag transcript in spiral ligament is complementary to the Na+,K+-ATPase distribution in the cochlear lateral wall. The localization of eag mRNA suggests that eag potassium channel may be produced in the corresponding cells. Considering the importance of the K+ gradient in the cochlea, the result reported here suggests that eag channel may play a role in the control of K+ fluxes in the spiral ligament.

Protein kinase C-ζ mediates retinal degeneration in response to TNF

Tumor necrosis factor-alpha (TNF) has been implicated in retinal ganglion cells (RGC) degeneration in glaucoma. Atypical protein kinase C (PKC) zeta is involved in cell protection against various stresses. The aim of this study was to investigate the potential proapoptotic effects of intravitreal injections of TNF with or without PKCzeta specific inhibitor on the rat retina. TNF was injected in the vitreous of rat eyes alone or in combination with specific PKCzeta inhibitor. PKCzeta and NF-kappaB were studied by immunohistochemistry and western-blotting analysis on retina, and apoptosis quantified by the TUNEL assay. While low basal PKCzeta was observed in the control eyes, TNF induced intense expression of PKCzeta mostly in bipolar cells processes. PKCzeta staining became nuclear when TNF was coinjected with PKCzeta inhibitor. TNF alone did not induce apoptosis in the retina. Coinjection of the PKCzeta-specific inhibitor and TNF, however, induced apoptosis in the inner nuclear and ganglion cell layers. The PKCzeta-specific inhibitor unmasks retinal cells to TNF cytotoxicity showing a link between the proapoptotic effects of TNF and the antiapoptotic PKCzeta signaling pathway.

The role of PKCζ in amikacin-induced apoptosis in the cochlea: Prevention by aspirin

Aminoglycoside antibiotics are ototoxic, inducing irreversible sensorineural hearing loss mediated by oxidative and excitotoxic stresses. The NF-κB pathway is involved in the response to aminoglycoside damage in the cochlea. However, the molecular mechanisms of this ototoxicity remain unclear. We investigated the expression of PKCζ, a key regulator of NF-κB activation, in response to aminoglycoside treatment. Amikacin induced PKCζ cleavage and nuclear translocation. These events were concomitant with chromatin condensation and paralleled the decrease in NF-κB (p65) levels in the nucleus. Amikacin also induced the nuclear translocation of apoptotic inducing factor (AIF). Prior treatment with aspirin prevented PKCζ cleavage and nuclear translocation. Thus, aspirin counteracts the early effects of amikacin, thereby protecting hair cells and spiral ganglion neurons. These results demonstrate that PKCζ acts as sentinel connecting specific survival pathways to mediate cellular responses to amikacin ototoxicity.

The role of PKCζ in NMDA-induced retinal ganglion cell death: Prevention by aspirin

Intravitreal NMDA injection has been shown to induce the excitotoxic loss of retinal cells. The retinal ganglion cell apoptosis induced by NMDA is thought to play an important role in retinal ischemia injury and NMDA-injected rat has been used as a model of neuronal loss in diseases such as glaucoma. In this experimental model, we studied the early effects of NMDA leading to the degeneration of retinal ganglion cells. PKCζ regulates the NF-κB pathway in cellular responses to various stresses and we have shown that aspirin inhibits purified human PKCζ. We therefore investigated the molecular mechanism by which retinal cells limit ocular injury following NMDA treatment. We found that the NMDA-induced apoptosis of ganglion cells was mediated, at least partly, by PKCζ. This enzyme was activated early in the cellular response to NMDA. Prolonged activation was followed by PKCζ cleavage, and nuclear translocation of the C-terminal region of this protein—a critical event for the survival of retinal cells. We also found that pretreatment with aspirin or the coinjection of NMDA with a specific PKCζ inhibitor counteracted the effects of NMDA. These findings provide new insight into the role played by PKCζ in neuronal loss in glaucoma.

A novel cDNA corresponding to transcripts expressed in retina post-mitotic neurons

The long term objective of this study is to isolate genes specifically expressed at the onset of neuronal cell cycle withdrawal. As an experimental paradigm we have used a quail neuroretinal cell clone (clone K2) immortalized by a thermosensitive mutant of Rous Sarcoma Virus. K2 cells proliferate at 36 degrees C but stop synthesizing DNA after a shift to 41.5 degrees C. We have constructed a cDNA library from K2 cells transferred to 41.5 degrees C and autosubtracted with RNAs from K2 cells maintained at 36 degrees C. This strategy has led to the isolation of cDNAs which recognize mRNAs expressed in quail neuroretina (NR) during development. We report here one of these cDNAs, cDNA QN1, that hybridizes with transcripts expressed in retina neurons, in parallel with their withdrawal from the cell cycle. QN1 ORF codes for a 138 kDa polypeptide corresponding to the protein observed in Western blot analysis. A role of QN1 product(s) on neuronal quiescence is suggested by the positive effect of an antisense oligonucleotide on DNA synthesis of K2 cells.

Role of QN1 protein in cell proliferation arrest and differentiation during the neuroretina development

In this report, we describe the involvement of the quail neuroretina 1 (QN1) protein in retinal development. The Qn1 cDNA was isolated as a gene specifically expressed at the onset of neuronal cell cycle withdrawal (Bidou et al., Mech. Dev. 43 (1993) 159). Qn1 is located in the cytoplasm in proliferating cells during the early stages of the development. Its distribution changes, becoming predominantly nuclear, in neurons during establishment of the quiescent state upon the differentiation. We decreased the amount of QN1 protein by an antisense strategy in vitro or in vivo. This decrease of the amount of QN1 protein results in additional mitosis and in severe abnormalities such as retinal dysplasia. Our results suggest that QN1 plays a key role at the onset of neuronal cell cycle withdrawal.