A dynein mutation attenuates motor neuron degeneration in SOD1(G93A) mice.

Cu/Zn SOD1(G93A) transgenic mice develop phenotypical hallmarks of ALS and serve therefore as an established model to study the molecular mechanisms underlying this disease. Recent reports demonstrate that mutations in the motor protein dynein in Legs at odd angles (Loa) and Cramping (Cra1) mice lead to similar but milder phenotypes. Surprisingly, double transgenic mice (Loa/SOD1(G93A)) have been recently shown to attenuate rather than to accelerate the phenotypical expression of motor neuron degeneration. These results raise the question whether other functional relevant mutations in dynein cause a similar effect. To address this question, we have cross-bred SOD1(G93A) with Cra1/+ mice. These double transgenic mice show an attenuated decline of both motor activity and body weight and an increase of survival time compared to SOD1(G93A) mice. Thus, this study confirms that mechanisms associated with dynein such as retrograde axonal transport may play an important role in SOD1(G93A-) toxicity on motor neurons.

ProSAP/Shank postsynaptic density proteins interact with insulin receptor tyrosine kinase substrate IRSp53.

The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA-type and metabotropic glutamate receptors and the actin-based cytoskeleton. In a yeast two hybrid screen utilizing a proline-rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co-immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C-terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline-rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.

The neuron-specific Ca2+-binding protein caldendrin: gene structure, splice isoforms, and expression in the rat central nervous system.

Caldendrin is the founder member of a recently discovered family of calmodulin-like proteins, which are highly abundant in brain. In this study we examined the organization of the murine and human caldendrin gene as well as the expression pattern of transcripts for caldendrin and two novel splice variants. In addition the distribution of caldendrin in rat brain has been assessed by immunohistochemistry. Caldendrin is localized to the somatodendritic compartment of a subpopulation of mainly principal neurons in brain regions with a laminar organization and is present only at a subset of mature excitatory synapses. Caldendrin immunoreactivity (IR) is tightly associated with the cortical cytoskeleton, enriched in the postsynaptic density (PSD) fraction, and associates late during development with the synaptic cytomatrix. The expression is highly heterogenous within cortex, with highest levels of caldendrin IR in layer III of the piriform and layer II/III of the somatosensory cortex. The segregated cortical distribution to areas, which represent the most important primary sensory systems of the rodent brain, may reflect different requirements for dendritic Ca2+-signaling in these neurons. The presence of caldendrin in the PSD of distinct synapses may have important implications for Ca2+-modulated processes of synaptic plasticity.

Synaptic scaffolding proteins in rat brain. Ankyrin repeats of the multidomain Shank protein family interact with the cytoskeletal protein alpha-fodrin.

The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein alpha-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of alpha-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.

The Pars tuberalis of the monkey (Macaca fascicularis) hypophysis: cell types and hormone expression.

The pars tuberalis (Pt) of most mammalian species contains specific cells which are structurally and functionally different from the pars distalis (Pd) cells. Pt-specific cells possess melatonin receptors and reveal morphological changes dependent on the duration of the photoperiod. Furthermore, in hamsters the transmission of photoperiodic stimuli to the endocrine system is influenced by melatonin, an effect which is likely to be mediated by Pt-specific cells. In monkeys, however, only little is known about this cell type. Therefore, we studied the ultrastructural differentiation of Pt-specific cells and describe the expression of different hormones and their mRNA by immunohistochemistry and in situ hybridization. Apparently the Pt consists of (1) cells similar to gonadotropic cells of the Pd, (2) folliculostellate cells and (3) a cell population which is morphologically and functionally clearly distinct from all other cell types found in the Pd. Morphologically they resemble the Pt-specific cells found in other species. Regarding the expression of secretory products there is evidence that they transcribe and translate the beta-TSH subunit. Although there is a strong signal for the mRNA of the common alpha-chain, protein staining is much weaker. POMC mRNA is expressed in the Pt while there is no evidence for PRL mRNA. The present results lead to the conclusion that the Pt of the monkey contains Pt-specific cells which express different hormonal subunits as was already shown for other species. In context with previous findings of melatonin receptors in the monkey Pt further investigations are necessary to establish the possible role of Pt-specific cells in the photoperiod-dependent generation of endocrine rhythms.

The calcium-independent receptor for alpha-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins.

Subtypes of the calcium-independent receptors for alpha-latrotoxin (CIRL1-3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors. The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified. Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain. In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1. The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and ProSAP1 antibodies; only CIRL1, but not CIRL2, was co-immunoprecipitated with ProSAP1. In situ hybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum. Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain. Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.

The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses.

Neuroplastin-65 and -55 (previously known as gp65 and gp55) are glycoproteins of the Ig superfamily that are enriched in rat forebrain synaptic membrane preparations. Whereas the two-Ig domain isoform neuroplastin-55 is expressed in many tissues, the three-Ig domain isoform neuroplastin-65 is brain-specific and enriched in postsynaptic density (PSD) protein preparations. Here, we have assessed the function of neuroplastin in long-term synaptic plasticity. Immunocytochemical studies with neuroplastin-65-specific antibodies differentially stain distinct synaptic neuropil regions of the rat hippocampus with most prominent immunoreactivity in the CA1 region and the proximal molecular layer of the dentate gyrus. Kainate-induced seizures cause a significant enhancement of neuroplastin-65 association with PSDs. Similarly, long-term potentiation (LTP) of CA1 synapses in hippocampal slices enhanced the association of neuroplastin-65 with a detergent-insoluble PSD-enriched protein fraction. Several antibodies against the neuroplastins, including one specific for neuroplastin-65, inhibited the maintenance of LTP. A similar effect was observed when recombinant fusion protein containing the three extracellular Ig domains of neuroplastin-65 was applied to hippocampal slices before LTP induction. Microsphere binding experiments using neuroplastin-F(c) chimeric proteins show that constructs containing Ig1-3 or Ig1 domains, but not Ig2-3 domains mediate homophilic adhesion. These data suggest that neuroplastin plays an essential role in implementing long-term changes in synaptic activity, possibly by means of a homophilic adhesion mechanism.

Susceptibility of retinal ganglion cells to excitotoxicity depends on soma size and retinal eccentricity.

PURPOSE: This study was undertaken to determine if retinal ganglion cell sensitivity to intraocular N-methyl-D-aspartate or kainate injections varied as a function of retinal location (eccentricity) or cell soma size. METHODS: Rat retinal ganglion cells surviving intraocular N-methyl-D-aspartate or intraocular kainate induced lesions were retrogradely labeled with horseradish peroxidase and analyzed using an image analysis system. Control animals were retrogradely labeled after vehicle injection only. Cell counting was performed at 48 sampling points over the entire retina and represented a total area of 1.92 mm2 per retina. RESULTS: Larger cells were more sensitive to kainate than to N-methyl-D-aspartate excitotoxicity; smaller cells more vulnerable to N-methyl-D-aspartate excitotoxicity. Further from the optic nerve, more smaller cells survived kainate administration. After N-methyl-D-aspartate administration, larger cells survived most, noticeably in the central retina. CONCLUSIONS: Our results suggest that loss of retinal ganglion cells after N-methyl-D-aspartate or kainate administration affects distinct populations of retinal ganglion cells, dependent upon soma size and retinal location. The mechanism by which certain classes of cells survive or succumb to such insults has yet to be determined.

Co-expression of c-Jun and ATF-2 characterizes the surviving retinal ganglion cells which maintain axonal connections after partial optic nerve injury.

The expression of c-fos, c-jun, jun-b, jun-d, srf and pc4 mRNA was examined after partial optic nerve crush in the adult rat retina by in situ hybridization. Optic nerve injury led exclusively to the upregulation of c-jun, with cellular label indicative for c-jun mRNA in the retinal ganglion cell layer after two days, three days and one week post-injury. This expression pattern was in accordance with the appearance of c-Jun immunoreactivity in retinal flat mounts. Injection of an antisense but not a missense oligonucleotide against c-jun after partial crush resulted in a reduced number of connected retinal ganglion cells (RGCs) as shown by retrograde labeling. Prelabeling of RGCs with fluorogold before optic nerve section and subsequent antisense targeting against c-jun, however, led to a slightly higher number of surviving but axotomized RGCs. C-Jun antibody staining of retinal whole mounts pre- or postlabeled after crush by intracollicular administration of fluorogold showed strong c-Jun immunoreactivity in connected RGCs and also in a population of disconnected RGCs. Double labeling with an antibody directed against the transcription factor ATF-2 revealed strong co-expression of c-Jun and ATF-2 in connected RGCs but not in axotomized cells. Taken together these data indicate that both RGCs in continuity and those in discontinuity with the superior colliculus respond both equally to the noxious stimulus with c-Jun expression. Moreover, the co-expression of c-Jun with high levels of ATF-2 appears to be essential for either the continuity or survival of RGCs which remain connected with their target. In disconnected RGCs, however, low levels of ATF-2 and the co-expression of c-Jun may be related to cell death.

Apoptotic versus necrotic characteristics of retinal ganglion cell death after partial optic nerve injury.

We have investigated time course and characteristics of retinal ganglion cell (RGC) death after partial optic nerve injury. In situ end labeling of DNA fragments with the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine (dUTP)-biotin nick end labeling (TUNEL) method revealed the presence of apoptotic cells on as early as 5 days postcrush with a very high number of TUNEL-positive cells 1 week postinjury. At the ultrastructural level, features of apoptosis were clearly present in the ganglion cell layer at this time point. Moreover, TUNEL-positive cells could be identified as retinal ganglion cells by retrograde labeling with fluorogold. In addition, DNA laddering characteristic for apoptosis was found 1 week postinjury. A considerable number of TUNEL-labeled cells was still found after 2 weeks postinjury. Retinal whole mounts prepared at postlesion days 2-5, however, revealed that many cell bodies with ruptured membranes as evidenced by nucleosomal Sytox staining were present. These cells were also identified as retinal ganglion cells by retrograde labeling with fluorogold. Moreover, at this early stages of RGC degeneration necrotic cellular profiles could be detected by electron microscopic analysis. Thus, evidence is provided that necrosis and apoptosis follow a distinctly different time course after partial optic nerve injury.

Cell and molecular biology of the pars tuberalis of the pituitary.

The pars tuberalis of the adenohypophysis is mainly composed of a special type of endocrine cells, pars tuberalis-specific cells, lining the primary capillary plexus of the hypophysial portal system. Dense expression of melatonin receptors and marked changes in morphological appearance, production pattern, and secretory activity during annual cycle show that these cells are highly sensitive to changes in photoperiod. This leads to the hypothesis that the pars tuberalis is involved in the transmission of photoperiodic stimuli to endocrine targets. Several investigations support the theory that pars tuberalis-specific cells are multipotential cells exerting a modulatory influence on the secretory activity of the pars distalis. Specifically, there is accumulating evidence that seasonal modulation of prolactin secretion, independent of hypothalamic input, is due to melatonin-regulated activity of pars tuberalis-specific cells. The exact nature of secretory products and their effects within neuroendocrine regulation, however, remain rather enigmatic. Accordingly, molecular mechanisms regulating gene expression under the influence of photoperiod, respectively, circulating melatonin levels are still incomplete. Recent cloning of melatonin receptor genes and new data on intracellular signal transduction will probably lead to new insights on melatonin action and pars tuberalis-specific cell physiology.

The mouse ADP-ribosylation factor-like 4 gene: two separate promoters direct specific transcription in tissues and testicular germ cell.

ADP-ribosylation factor-like protein 4 (ARL4) is a Ras-related GTPase that has been cloned from the 3T3-L1 preadipocyte cell line as an adipocyte-specific cDNA [Schürmann, Breiner, Becker, Huppertz, Kainulainen, Kentrup and Joost (1994) J. Biol. Chem. 269, 15683-15688]. The Arl4 gene maps to the proximal region of mouse chromosome 12 linked to Lamb1-1, Hfhbf1 and Sos2. Compared with all other known genes of Ras-related GTPases, the genomic organization of Arl4 is unusual in that its entire coding region, the 3' untranslated region (UTR) and most of the 5' UTR are located on a single exon. This structure suggests that Arl4 has evolved by retroposition of an Arf (ADP-ribosylation factor) or Arf-like gene. Isolation of the 5' UTR by rapid amplification of cDNA ends (RACE)-PCR revealed heterogeneous transcription initiation sites in alternative exons 1. Both 5'-flanking regions exhibited promoter activity when expressed in COS-7 cells, indicating that the expression of Arl4 is directed by two separate promoters. mRNA transcribed under the control of the downstream promoter was isolated by RACE-PCR from all investigated tissues. In contrast, the upstream promoter seems to drive specifically the expression of Arl4 in adult testis. Hybridization of rat testis in situ indicated that Arl4 is expressed in germ cells of puberal and adult testis, but not in prepuberal testis, suggesting that Arl4 is involved in sperm production.

Axonal injury alters alternative splicing of the retinal NR1 receptor: the preferential expression of the NR1b isoforms is crucial for retinal ganglion cell survival.

Cellular-specific splicing of the retinal NMDAR1 receptor (NR1) and expression of NMDAR2 receptor (NR2) subunits in response to optic nerve injury was investigated by in situ hybridization in adult rats. A controlled optic nerve crush led to a clear alteration in the expression of alternatively spliced NR1 variants in the retinal ganglion cell layer (GCL). The NR1-2b and NR1-4b isoforms were preferentially expressed between 2 d and 1 week after injury, whereas expression for all other isoforms remained either unchanged or decreased to barely detectable levels within 4 weeks. Cellular silver grain density for NR2 subunits also declined in the GCL after trauma. To directly test the hypothesis that NR1b expression is crucial for cell survival after axonal trauma, we administered intraocularly an antisense oligonucleotide against the NR1b isoform 2 and 3 d after injury. This led to a drastic loss of retrogradely labeled retinal ganglion cells (RGCs). Antisense targeting clearly reduced retinal NR1 protein levels, as judged by Western blot analysis, but had no effect on the cell number in control retinas. These findings point toward injury-specific changes in alternative splicing of the NR1 receptor, which are crucial for the survival of RGCs after partial axonal trauma. We therefore propose that this reflects an adaptive, rather than a pathogenic, cellular response to neurotrauma.

Transcripts for secreted and GPI-anchored brevican are differentially distributed in rat brain.

Brevican is a member of the aggrecan/versican family of proteoglycans. In contrast to the other family members, brevican occurs both as soluble isoforms secreted into the extracellular space and membrane-bound isoforms which are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety. Expression of both variants, which are encoded by two differentially processed transcripts from the same gene, is confined to the nervous system. In the current study, we have used in situ hybridization to examine the cellular sites of synthesis for both mRNAs during postnatal development of the rat brain. Whereas the 3.6-kb transcript encoding secreted brevican displays a widespread distribution in grey matter structures, including cerebellar and cerebral cortex, hippocampus and thalamic nuclei with silver grains accumulating over neuronal cell bodies, the smaller transcript (3.3 kb) encoding GPI-anchored isoforms appears to be largely confined to white matter tracts and diffusely distributed glial cells. This expression pattern is further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments with RNA from different glial cell cultures, and by biochemical data demonstrating that the crude membrane fraction from isolated optic nerve contains high amounts of phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive brevican immunoreactivity. During ontogenetic development, both brevican transcripts are generally up-regulated. However, the expression of glypiated brevican is delayed by about 1 week, compared with the expression of the secreted isoform. This late appearance of GPI-linked brevican, its predominant expression in glial cells and its tight association with brain myelin fractions suggest a functional role in neuroglia.

Initial expression of the common alpha-chain in hypophyseal pars tuberalis-specific cells in spontaneous recrudescent hamsters.

When exposed to short-day conditions, hamsters and other long-day breeders undergo gonadal regression. With chronic exposure to short days, however, the animals become photorefractory and gonadal recrudescence occurs. The underlying mechanism for this insensitivity is still unknown. There is growing evidence, however, that specific cells of the pituitary pars tuberalis (PT) mediate these photoperiod/nonphotoperiod-dependent changes as a direct or indirect "Zeitgeber" for the endocrine system. We investigated messenger RNA (mRNA)/protein formation for several hypophyseal hormones (beta-TSH, beta-LH, PRL, common alpha-chain) in the pars distalis (PD) and PT of female Djungarian hamsters in long photoperiod (LP) and after 18, 28, and 38 weeks of short photoperiod (SP). As indicated by gonadal and body weight, the hamsters displayed gonadal regression after 18 and 28 weeks of SP; after 38 weeks of SP, all animals showed recrudescence. At 18 and 28 weeks of SP, only PRL mRNA and protein levels were significantly reduced in the PD and returned to LP values after 38 weeks of SP. The expression of hypothalamic tyrosine hydroxylase in the arcuate nucleus that was determined by immunocytochemistry and by in situ hybridization was also down-regulated in SP18 and SP28 with increasing levels at SP38. Urinary 6-sulfatoxymelatonin levels were elevated in SP with highest levels in the SP18 group. In the PT, beta-TSH mRNA and protein were not detectable in all SP groups compared with the moderate signal intensity in LP. The common alpha-chain mRNA and protein, however, which were also reduced in the animals of the SP18 group, were already elevated after 28 weeks of SP and nearly reached LP-levels after 38 weeks of SP. These results show that, in contrast to LH and TSH, PRL expression in the PD is a sensitive indicator for photoperiod dependent changes of the endocrine system and seems to be tyrosine hydroxylase independent. The increase of common alpha-chain expression in PT-specific cells depending upon duration of SP that precedes the hormonal changes in the PD leads us to speculate that PT-specific cells initiate spontaneous recrudescence via a PT-PD pathway.

Cloning and expression of a brain-derived TSH receptor.

Several hormones not only regulate the activity of endocrine cells and non-endocrine tissues but also serve as neuronal transmitters or modulators of neuronal activity. Accordingly, the expression and physiological significance of hormonal receptors in the central nervous system (CNS) could be demonstrated for a whole set of hormones (e.g. hCG/LH, GH, T3, CRF, TRH). The G-protein coupled TSH receptor is densely expressed in the thyroid gland and mediates the production and secretion of thyroid hormones. Not all TSH effects, especially in neurological and psychiatric disease states, can readily be explained by the action of the hormone on the thyroid gland and/or TRH levels. Therefore, it has been suggested that TSH might exert its effects directly in the CNS, although no direct proof for a TSH receptor in the human brain has been provided yet. Here we describe the cloning of a TSH receptor from an ovine hypothalamic cDNA library that is similar to thyroid derived cDNA clones. The comparison of amino acid sequences indicates that several protein domains important for the function and activity of the receptor are highly conserved. RT-PCR and RNA protection assay demonstrated that the TSH receptor mRNA is widely expressed throughout the ovine brain. The expression of a TSH receptor in the CNS indicates that TSH is not only a hormonal messenger for the thyroid gland but can also act directly in the brain. Further studies should focus on the physiological role of TSH in the CNS and the regulation of TSH receptor expression in the mammalian brain.

Localization of a 42-kDa inositol 1,3,4,5-tetrakisphosphate receptor protein in retina and change in expression after optic nerve injury.

The mRNA and protein expression of a 42-kDa inositol 1,3,4,5-tetrakisphosphate receptor (InsP4R) was investigated in cryostat and paraffin sections from rat, porcine and bovine retina. InsP4R mRNA was localized by in situ hybridization in the ganglion cell layer, the inner nuclear cell layer and the outermost part of the outer nuclear cell layer. For immunocytochemistry, we used an antibody raised against a 19-amino-acid peptide (peptide-3) derived from previous microsequencing of proteolytic fragments of the porcine InsP4R (Stricker et al., FEBS Lett., 370 (1995) 236). The distribution of immunoreactivity was similar in all species investigated. Two cell types, most likely wide-field amacrine and retinal ganglion cells, were intensely stained. Prominent immunoreactivity in the on/off sublaminae of the inner plexiform layer and in the optic nerve layer indicates a pre- and/or post-synaptic localization of the protein. Moreover, significant InsP4R protein expression in the inner segment of photoreceptors points to a putative role of the second messenger InsP4 in signaling processes related to phototransduction. However, also the endfeet of Müller glia cells in the optic nerve layer were intensely stained. Optic nerve crush caused only minor changes in retinal InsP4R mRNA levels whereas InsP4R immunoreactivity was attenuated for more than 4 weeks in the photoreceptor inner segments, wide-field amacrine cells, and in retinal ganglion cells. The immunopositive sublaminae of the inner plexiform layer appeared to have shrunken. However, the signal intensity gradually recovered after 10 weeks. Since in parallel sections stained with a monoclonal antibody directed against the vesicular protein synaptophysin no changes were found, the alterations in InsP4R immunoreactivity induced by nerve injury are not due to a general decline in the expression of pre-synaptic proteins. We, therefore, hypothesize that the InsP4R might be linked to altered intracellular Ca2+ signaling after neuronal injury.

Thyrotropin expression in hypophyseal pars tuberalis-specific cells is 3,5,3'-triiodothyronine, thyrotropin-releasing hormone, and pit-1 independent.

The expression of TSH subunit genes (TSH alpha and -beta) in pituitary thyrotropes is primarily regulated via circulating thyroid hormone levels (T3) and the hypothalamic TRH. Hypophyseal pars tuberalis (PT)-specific cells also express both hormonal subunits of TSH, but do not resemble thyrotropes of the pars distalis (PD) with respect to their distinct morphology, secretion, and direct modulation of TSH expression by photoperiodic inputs and melatonin. To investigate whether this distinct regulation of TSH is related to a different molecular structure or different signaling cascades, we analyzed PT-specific TSH and its transcriptional regulation in ovine PT-specific cells. After construction of PT- and PD-specific complementary DNA (cDNA) libraries, the cloning and sequencing of several TSH alpha and -beta subunit clones revealed identical sizes and sequences for the translated and untranslated regions in both hypophyseal compartments. Transcription start site analysis also displayed three identical start sites for the transcription of TSH beta in PT and PD. After cloning of the ovine TRH receptor cDNA and a partial T3 receptor cDNA, in situ hybridization. Northern blot analysis, and PCR experiments showed that TRH and T3 receptors are not expressed in specific cells of the PT. The transcription factor Pit-1 that is involved in TSH expression of thyrotropes could only be detected in the PD. In additional experiments rats were treated with T4 or TRH, and subsequent in situ hybridization studies showed that TSH beta messenger RNA (mRNA) formation was not altered in the PT. In the PD, however, TSH beta mRNA was significantly reduced in the T4-treated group, but was enhanced in the TRH-treated group. We conclude that PT-specific cells of the pituitary are characterized by the transcription of TSH subunits that are identical to TSH expressed in thyrotropes of the PD. The absence of TRH, T3 receptor mRNA, and Pit-1, respectively, as well as the different reactions compared to PD thyrotropes in in vivo experiments lead to the conclusion that the expression of TSH in PT-specific cells of the pituitary is not regulated via the classical thyrotrope receptors and their intracellular pathways, but through a novel, photoperiod-dependent mechanism.

TSH expression in murine hypophyseal pars tuberalis-specific cells.

Several experiments in photoperiod-dependent species suggest that the hypophyseal pars tuberalis (PT) plays a key role in transducing light/dark information to the endocrine system. In rat and hamster it has been shown that both TSH subunits (TSH-alpha and -beta) are expressed in PT-specific cells, a morphologically distinct cell type which does not resemble thyrotropes of the pars distalis (PD). In order to investigate whether TSH expression is a characteristic and common feature of PT-specific cells we studied cellular morphology and TSH subunit expression in mouse pars tuberalis by electron microscopy, immunocytochemistry and in situ hybridization. In contrast to all other species investigated so far the number and size of secretory granules in mouse PT-specific cells is enlarged. As in rat and hamster, however, TSH subunit expression (mRNA and protein) was found in thyrotropes of the PD and throughout the whole extent of the PT cell layer. We conclude that although mouse PT-specific cells display an ultrastructural morphology that is different from other species, they are nevertheless characterized by TSH subunit expression. Further studies are needed to determine the physiological role of 'PT-specific cell TSH' and to elucidate whether TSH is the only PT-specific cell secretory product.