- Email: [email protected]
threonine-kinase 33 (Stk33) – Component of the neuroendocrine network?
Brain Research xx (xxxx) xxxx–xxxx
Contents lists available at ScienceDirect
Brain Research journal homepage: www.elsevier.com/locate/brainres
Research report
Serine/threonine-kinase 33 (Stk33) – Component of the neuroendocrine network? ⁎
Stefan Reussa, , Bastienne Brauksiepeb, Ursula Disque-Kaiserc, Tim Olivierc a b c
Department of Nuclear Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, Mainz, Germany Department of Anatomy and Cell Biology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany
A R T I C L E I N F O
A BS T RAC T
Keywords: Stk33 Hypothalamus Immunofluorescence Vasopressin Oxytocin Nitric oxide-synthase Substance P Tyrosine-hydroxylase Rat Hamster Tree shrew Baboon Human
The present study was conducted to investigate the expression of serine/threonine-kinase 33 (Stk33) in neuronal structures of the central nervous system in rat and hamster as well as the presence of the protein in the brain of higher mammals, using a polyclonal antibody on cryosections of fixed brains. We found a distinct immunostaining pattern that included intense fluorescence of the ependymal lining of cerebral ventricles, and of hypothalamic tanycytes and their processes. We further observed intense staining of magnocellular neurons in the hypothalamic paraventricular, supraoptic and accessory neurosecretory nuclei, in particular the circular nuclei, and less intense stained neurons in other diencephalic regions. Double-immunostaining experiments showed a partial colocalization of Stk33 with arginine-vasopressin, oxytocin or neuronal nitric oxide-synthase in a large number of neurons of the hypothalamic nuclear regions. Colocalization of Stk33 with substance P or the catecholamine-synthesizing enzyme tyrosine-hydroxylase was not observed. Immunofluorescence was not found in autonomic regions of the lateral horn, suggesting that Stk33 does not contribute to hypothalamo-spinal connections. However, large Stk33-immunoreactive axonal projections from magnocellular hypothalamus to the neurohypophysis were evident. These functionally important connections provide the bridge from neuronal to humoral regulation of the endocrine system. Additionally, Western blots from mouse brain showed two distinct bands representing two Stk33 isoforms. We also present first evidence for the presence of Stk33/STK33 in neuronal structures, ependymal cells and tanycytes in tree shrew, baboon, and human brain.
1. Introduction Serine/threonine kinase 33 is a member of the calcium/calmodulindependent kinases (CaMK) (Manning et al., 2002; Mujica et al., 2001, 2005). Co-immunoprecipitation experiments revealed that Stk33 and the intermediate filament protein vimentin are, in vivo, associated proteins and that Stk33 phosphorylates vimentin in its head domain. Recombinant Stk33 undergoes obligatory autophosphorylation, which might be a requirement for its kinase function, suggesting that Stk33 is involved in intermediate filament assembly/disassembly through the specific and regulated phosphorylation of vimentin (Brauksiepe et al., 2008). This clue to Stk33-function in the regulation of cytoskeleton dynamics by phosphorylation fits to the differential expression pattern of Stk33 (Mujica et al., 2005) that resembles those of some related members of CaMK.
In a recent study (Brauksiepe et al., 2014), we observed the expression of Stk33 and its colocalization to vimentin in ventricular ependymal cells and in tanycytes of rat and hamster as well as its regulation by photoperiod in the Djungarian hamster Phodopus sungorus. The protein was also present in circumventricular organs such as area postrema, subfornical organ, pineal gland and anterior and posterior lobes of the pituitary gland, as well as in magnocellular neurons of the neuroendocrine hypothalamus. As vimentin is present in neurons only in early development (Yabe et al., 2003), other functions of neuronal Stk33 have to be assumed. Since these are unknown as yet, and detailed knowledge of Stk33-expression is an essential prerequisite for the understanding of its function, the present study aimed at characterizing Stk33-neurons with regard to location and co-expression of selected neuroactive substances in rodents. For this purpose, we chose neuroactive substances found in many regions
Abbreviations: ANS, accessory neurosecretory nuclei; AVP, arginine-vasopressin; CiN, circular nucleus; CSF, cerebrospinal fluid; IR, immunoreactive; MCN, magnocellular neurons; nNOS, neuronal nitric oxide-synthase; OT, oxytocin; PVN, paraventricular nucleus; SON, supraoptic nucleus; Stk33, serine/threonine-kinase 33; SP, substance P; TH, tyrosinehydroxylase ⁎ Correspondence to: Department of Nuclear Medicine, University Medical Center, Johannes Gutenberg-University, Langenbeckstraße 1, Bld. 210, 55101 Mainz, Germany. E-mail address: [email protected] (S. Reuss). http://dx.doi.org/10.1016/j.brainres.2016.11.006 Received 9 August 2016; Received in revised form 9 October 2016; Accepted 7 November 2016 Available online xxxx 0006-8993/ © 2016 Published by Elsevier B.V.
Please cite this article as: Reuss, S., Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.11.006
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Fig. 1. Stk33-immunoreactivity in frontal sections of the rat brain. Strong staining was found in ependymal cells of the third ventricle (3 V in A–C). Less intense staining was observed in neurons and their processes. At the level of the optic chiasm (oc in A; approximately interaural +7.9 mm corresponding to Fig. 42 of the rat brain atlas), and in the PVN region at an intermediate level (approximately interaural +7.6 mm corresponding to Fig. 45 of the rat brain atlas), further IR structures are tanycyte processes wrapped around prospective blood capillaries (arrowheads, dorsal periventricular region in A,C; insert in A) and, more ventrally, IR neurons (arrows) of the PVN (A–D). Some of these were located close to the ventricular ependyma and apparently sent processes (arrowheads) into ventricular space (B). Others were located in the lateral PVN aspects (D) and provided beaded fibers advancing to median eminence and neurohypophysis (F). A group of IR neurons (arrows) of the accessory neurosecretory nuclei (ANS) is seen in E. No immunofluorescent neuronal somata were observed in the suprachiasmatic nucleus (SCN in A). All images were taken from the same animal.
2. Results
of the rodent hypothalamus, i.e., arginine-vasopressin, oxytocin, neuronal nitric oxide-synthase, substance P, and tyrosine-hydroxylase (cf. Armstrong, 2015; Harding et al., 2004; van den Pol et al., 1984; Woodside and Amir, 2000). We also studied, more cursory, the presence of Stk33-protein in brain of higher mammals including man. By means of immunofluorescence, we now provide the first overview of the expression of neuronal Stk33-protein in the rodent brain, and demonstrate that the protein is present in the CNS of higher mammals.
2.1. Neuronal expression of Stk33 in rat and hamster Immunostaining of brain sections from adult rats showed a distinct and cell type-specific distribution of Stk33-immunoreactivity. Strong immunostaining was seen in ependymal cells of the ventricular system (Figs. 1A–C, 2E and 3AB) and in tanycytes of the basolateral walls of the third ventricle. The latter cell type exhibited basal, unbeaded processes that were often seen to extend over a long distance and appeared to be wrapped around blood vessels (insert in Fig. 1A and 2
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Fig. 2. Stk33-immunoreactivity in frontal sections of the rat brain (same animal as in Fig. 1). Immunofluorescent neurons (arrows) were found in the supraoptic nucleus (SON in A) and in the circular nucleus (CiN in B) where they exhibit beaded processes. Neurons (arrows) in the epithalamic medial habenular nucleus (MHab in C) were stronger labeled than those in the lateral habenular nucleus (LHab in C). At a posterior level of the hypothalamus (E, approximately interaural +7.2 mm corresponding to Fig. 48 of the rat brain atlas), large neurons with processes (arrows) of the lateral magnocellular part of the paraventricular nucleus (PVN lm) were clearly labeled (D). In the ventral hypothalamus in the same section, strongly stained tanycyte processes extending laterally (arrowheads), and moderately labeled neurons (arrows) of the anterior hypothalamus are seen (F). 3 V third ventricle, sox supraoptic decussation.
nuclei (CiN; Fig. 2B), i.e. in hypothalamic regions containing the neuroendocrine cell groups. Neurons in these nuclei exhibited beaded processes that probably represented axons with varicosities (Fig. 1DF). Additional Stk33-IR MCN were found in the dispersed group of accessory neurosecretory nuclei (ANS; Fig. 1E). Further hypothalamic Sk33-positive neurons of different soma size and less intense immunofluorescence were visible in the anterior hypothalamus (AH in Fig. 2EF), as well as in posterior hypothalamus and premamillary nucleus. Stk33-IR neuronal somata were not observed in the hypothalamic suprachiasmatic nucleus. In addition to hypothalamic neurons, we observed Stk33-immunolabeled neurons in other diencephalic regions of the rat brain. These
2EF). In the sections, it appeared that processes end in the neuropil or terminate close to neuronal somata (Fig. 1B). This pattern of immunoreactivity matches previous data (Brauksiepe et al., 2014). In addition, we observed Stk33-IR neurons in thalamic and hypothalamic nuclei. Their somata exhibited lower immunoreactivity compared to tanycytes in the same section of rat and hamster brain. A prominent neuronal group was observed in the hypothalamus. As judged from size and location, they belonged to the group of neurosecretory magnocellular neurons (MCN; Figs. 1 and 2). Their soma diameters measured approximately 20–35 µm in the paraformaldehyde-fixed rat tissue. They were located predominantly in the paraventricular (PVN; Fig. 1A), supraoptic (SON; Fig. 2A) and circular 3
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Fig. 3. Characterization of Stk33-IR neurons in the rat hypothalamus by double-immunofluorescence. Exemplarily shown is colocalization of Stk33 either with arginine-vasopressin (AVP) in the paraventricular nucleus (PVN; A–C), with oxytocin (OT) in the circular nucleus (CiN; D–F), with neuronal nitric oxide-synthase (nNOS) in the accessory neurosecretory nuclei (ANS in G–I), or with OT in the supraoptic nucleus (SON in K–M). See also Table 1 for percentages of single- and double-labeled neurons.
neuronal nitric oxide-synthase, common marker of magnocellular hypothalamic neurons. Our double-incubation studies in rats showed that Stk33 is colocalized with AVP, OT or nNOS in subpopulations of neuronal perikarya of the hypothalamic neuroendocrine nuclei. As seen in Table 1, the majority of IR neurons in PVN, SON and CiN were Stk33-IR. In the three double-incubation series, 80–86% (sum of columns 1 and 2) of PVN-neurons, 65–84% of SON-neurons and 67– 93% of CiN-neurons exhibited Stk33-immunofluorescence. The portion of double-labeled neurons showed a relative wide range depending on hypothalamic nucleus and colocalized substance (22% with nNOS in the CiN up to 70% with OT in the PVN). Examples of single- and double-labeled neurons are given for AVP in the PVN (Fig. 3A–C), for OT in the CiN (Fig. 3D–F), for nNOS in the ANS (Fig. 3G–I), and for OT in the SON (Fig. 3K–M).
cells were stained with less intensity compared to MCN. The medial habenular nuclei (MHab in Fig. 2C) exhibited neurons with relatively strong immunofluorescence, while neurons of the lateral habenular nuclei (LHab) were only faintly stained. In the thalamus, the posterior nuclei group exhibited IR neurons, while this was barely seen in the neighboring ventral posteromedial nucleus or in the parafascicular nucleus. Fibers tracts such as the fasciculus retroflexus or the superior thalamic radiation were devoid of immunofluorescence. 2.2. Colocalization of Stk33 with AVP, OT or nNOS in the rat hypothalamus To further characterize hypothalamic Stk33-IR neurons, we conducted colocalization studies of Stk33 with vasopressin, oxytocin or 4
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
expression pattern of this enzyme in mostly non-neuronal tissues of mice and men was presented earlier (Mujica et al., 2005). Our biochemical results gained by analysis of Stk33/vimentin interaction in vitro (Brauksiepe et al., 2008) revealed a function of this protein to be related to vimentin phosphorylation/polymerization processes. We recently demonstrated, by means of double-immunofluorescence, colocalization and photoperiodic regulation of Stk33 and vimentin in tanycytes of the hamster brain (Brauksiepe et al., 2014). In recent and in present data, the most prominent staining with the Stk33-antibody was found in the ependymal cells lining cerebral ventricles and in tanycytes, a specialized ependymal cell type located in the walls of the third ventricle. The previous observation of Stk33-positive, vimentin-negative neurons in the hypothalamus of rat and hamster prompted the present study to further describe and characterize this neuronal population. We will therefore concentrate on the contemplation of Stk33-IR neurons rather than ependymal lining cells which nonetheless represent a fascinating aspect of neuroendocrine regulation (cf. Bolborea and Dale, 2013; Elizondo-Vega et al., 2015; Goodman and Hajihosseini, 2015).
Table 1 Stk33-immunoreactive neurons in the rat hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and circular nucleus (CiN). Given are percent of stained magnocellular neurons (mean ± standard deviation from four rats), i.e., double-labeled (column 1), labeled for Stk33 only (column 2) or labeled for either AVP/OT/nNOS only (column 3). Hypothalamic regions
(1)
(2)
PVN SON CiN
Arginine-Vasopressin (AVP) Stk33/AVP Stk33 35.3 ± 5.7 44.7 ± 5.0 45.8 ± 2.6 18.7 ± 3.2 23.9 ± 3.2 42.8 ± 8.9
AVP 20.0 ± 3.3 35.5 ± 2.4 33.3 ± 2.8
PVN SON CiN
Oxytocin (OT) Stk33/OT 70.3 ± 3.4 67.9 ± 3.4 40.7 ± 3.6
OT 14.2 ± 0.7 26.6 ± 2.7 11.8 ± 2.4
Stk33/nNOS PVN SON CiN
Neuronal nitric oxide synthase (nNOS) Stk33/nNOS Stk33 nNOS 56.9 ± 6.1 24.3 ± 3.6 18.8 ± 2.9 73.2 ± 7.9 10.7 ± 1.5 16.1 ± 2.3 22.8 ± 4.2 70.3 ± 31.3 6.9 ± 1.3
Stk33 15.5 ± 5.0 5.5 ± 0.5 47.5 ± 13.9
(3)
3.1. Stk33 in rat and hamster diencephalic neurons In the diencephalon, two populations of Stk33-IR neurons were observed. One was composed of intensely immunofluorescent hypothalamic neurons (as seen in Fig. 1B) in the regions associated with neuroendocrine functions. These magnocellular neurons are part of the paraventricular, supraoptic and circular nuclei as well as of the accessory neurosecretory nuclei. Many of these neurons, which made up 64–93% of the labeled cells (see Table 1), exhibited beaded fibers that probably represented axons with varicosities, while tanycyte processes were unbeaded. The second diencephalic Stk33-positive neuronal population consists of somata exhibiting less intense fluorescence. These were located in the anterior and posterior hypothalamus and in the premamillary nucleus, and were also seen in the epithalamic habenular nuclei and in the posterior thalamic group.
Colocalization of Stk33 and AVP/OT were also observed in beaded fibers that apparently represented ventrally oriented axons located in the vicinity of neuroendocrine cells. In most cases, Stk33-fluorescence of these axons was less intense than the nonapeptide staining. 2.3. No colocalization of Stk33 with TH or SP in the rat hypothalamus We did not observe colocalization of either TH or SP with Stk33 in hypothalamic neurons. However in the same sections, we found TH-IR as well as SP-IR neuronal somata near the third ventricle, in the suprachiasmatic nucleus and in the paraventricular nucleus, sparing its magnocellular part (data not shown). 2.4. Existence of Stk33-immunoreactivity in non-rodent mammalian species
3.2. Stk33 is colocalized in hypothalamic magnocellular neurons
Furthermore, we conducted a more punctual study on the occurrence of Stk33 in the brain of higher mammals by incubating sections from Tupaia, baboon and man. We localized Stk33 in brain sections from all species. In the tree shrew Tupaia belangeri, predominantly immunostained hypothalamic tanycyte processes were seen that extended hundreds of micrometers into the neuropil at the floor and at more dorsally located aspects of the third ventricle (Fig. 4AB). Strong immunofluorescent ependymal cells and processes were striking at the roof of the lateral ventricles (Fig. 4C). In the brain of the baboon Papio hamadryas, we detected Stk33-immunoreactivity in ependymal cells, in processes of ventricular tanycytes and in faintly labeled periventricular neurons (Fig. 4D). In sections of the human brain, ventricular ependymal cells and their processes (Fig. 4E) as well as neuronal somata and axons were labeled by the Stk33-antibody (Fig. 4FG).
Most magnocellular neurons of the PVN, SON and accessory neurosecretory nuclei produce either AVP, OT, or the enzyme responsible for nitric oxide synthesis in neurons, nNOS (cf. Armstrong, 2015). This was also observed in the present study, in which we quantified immunolabeled neurons without considering the subdivisions of PVN and SON. We also did not include all scattered neurons belonging to the ANS system such as the periventricular magnocellular group and the anterior commissural nucleus (cf. Armstrong, 2015), but rather confined their registration to the well-segregated group of MCN making up the circular nucleus. The tightly packed cells in the CiN were frequently associated with vasculature (see Figs. 2B and 3D), as described by Peterson (1966) and Duan and Ju (1998). The double-labeling studies clearly showed that the Sk33-IR MCN do not represent an additional population of hypothalamic neurons but rather are, at least in part, identical to the MCN characterized by expression of AVP, OT or nNOS, but not of SP or TH. Whether the group that here stained only for Stk33 belongs to one of the various other neurochemically defined subpopulations remains to be elucidated. However, our study suggests that Stk33 is a valuable marker substance for the majority of hypothalamic MCN.
2.5. Western blots of Stk33 in mouse brain protein extracts In Western blots using protein extracts from whole mouse brain, we detected two bands corresponding to molecular masses of about 40 and 55 kDa (Fig. 5). They were not seen in blots using an Stk33-antibody preabsorbed with recombinant Stk33-protein.
3.3. Projections of Stk33-neurons 3. Discussion In the present study, we observed beaded axons that originated in the paraventricular region and extended ventrally. It is conceivable that these fibers are axons of Stk33-IR magnocellular neurons, and that
The present study was conducted to analyze the distribution of Stk33-protein in neuronal structures of the mammalian brain. The 5
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Fig. 4. Stk33-immunoreactivity in the primate brain. A–C: Strongly stained tanycyte processes in periventricular hypothalamic regions of Tupaia (A–C). In the baboon, short tanycyte processes and faintly labeled neurons were stained by the antibody (D). In the human brain (floor of the forth ventricle), periventricular processes (E), putative neuronal axons (F) and neuronal somata with processes (G) were present. In F and G, unmarked fluorescent structures represent unspecific autofluorescence. B–D are shown in the extended focal imagingmode.
they provide function as part of the hypothalamo-hypophyseal system. Axons originating in the medial and lateral magnocellular portions of the PVN, and also those that stem from SON and CiN, course in a wide arc to turn medially, then join the tract of Greving and reach the pituitary neural lobe (Vanhatalo and Soinila, 1995), as well as a variety of other intra- and extra-hypothalamic targets (cf. Armstrong, 2015). These axons exhibited local swellings, i.e. clusters of neurosecretory granules that consist of AVP/OT transported along with their associated neurophysins. This finding matches the recent observation of Stk33-IR fibers exhibiting Herring bodies in the mouse pituitary neural lobe (Brauksiepe et al., 2014). The nonapeptides AVP and OT are released by PVN and SON into the hypophyseal-portal blood and the CSF (cf. Post et al., 1983), and this may be the case as well for coexpressed Stk33. There is indeed evidence from Western blots that STK33 is present in the human CSF and blood (unpublished data, Prof. Dr. M. Otto, Dr. P. Steinacker, Dept. of Neurology, University of Ulm, Germany). This may provide broader regions of the CNS with Stk33, however, the exact purpose of Stk33-secretion is unknown as yet. We did not observe Stk33-IR structures in preganglionic autonomic regions of the spinal cord (Brauksiepe et al., 2014). This is of particular interest since nonapeptidergic projections to preganglionic sympathetic and parasympathetic neurons in the spinal intermediolateral nucleus originating from parvocellular subnuclei of the dorsal, ventral, and lateral PVN were described (Hallbeck and Blomqvist, 1999; Motawei et al., 1999; cf. Saper and Stornetta, 2015). Taken together, these
Fig. 5. Western blot of protein extract from mouse brain. Lane 1, molecular weight marker. Lane 2, Coomassie blue staining of the protein extract. Lane 3, signals gained after incubation of the blot with Stk33-antibody are seen in lane 2. Two bands of about 55 kDa (arrow) and of about 40 kDa (arrowhead) representing Stk33-isoforms were visible. The larger isoform is quantitatively more represented in mouse protein extract compared to the smaller isoform.
6
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
findings suggest that paraventricular Stk33 is involved in neuroendocrine regulatory mechanisms, but not in neuroendocrine-autonomic pathways.
4. Experimental procedures 4.1. Animals All procedures concerning animals were performed in accordance with the published European Health Guidelines under a protocol approved by the local Administration District Official Committee. Moreover, all efforts were made to minimize the number of animals and their suffering. Twelve adult male Sprague-Dawley rats and five Balb/c mice were held under constant conditions (12:12 hs light/dark-cycle, 21 °C roomtemperature, food and water ad libitum) at the animal facilities of the Department of Anatomy and Cell Biology and the Institute of Physiological Chemistry (University of Mainz).
3.4. Stk33 in non-rodent species To find out whether Stk33 is expressed in higher mammals, we used the antibody which recognizes an epitope of the Stk33-protein that is conserved throughout mammalian species, in specimens from tree shrew, baboon and man. In all three species studied, we found specific staining in corresponding regions and cell types as in the rodent material. The observed differences in staining intensity and extent may be attributed to technical factors such as perfusion vs. immersion fixation, use of PLP vs. paraformaldehyde. In sections from the tree shrew Tupaia, a species phylogenetically situated between insectivores and primates, we found faintly labeled hypothalamic neurons and dense, intensely labeled ependymal cells or tanycytes with processes extending several hundreds of micrometers into the neuropil neighboring the third ventricle. In the baboon Papio, we observed faintly labeled hypothalamic neurons and stronger stained processes extending from ventricular tanycytes. In human brain, rather short tanycyte processes near the ventricle as well as some neurons and presumptive axons were visible. Taken together, these immunohistochemical data reveal for the first time that Stk33-protein is expressed in the brain of higher mammals and suggest that the protein is a component of the neuroendocrine system also in humans.
4.2. Animal tissue preparation For immunohistochemistry, rats were killed by carbon dioxide at the middle of the light period and perfused transcardially with phosphate-buffered 0.9% saline (PBS), to which 15,000 IU heparin/l were added, at room temperature (RT), followed by an ice-cold fixative (4% paraformaldehyde, 1.37% L-lysine, 0.21% sodium-periodate in phosphate buffer (PLP; according to (McLean and Nakane, 1974). The right atrium was opened to enable venous outflow. Brains were removed, postfixed in PLP for 1 h, cryoprotected in a graded series of sucrose in 10 mM PBS and stored in PBS-30% sucrose. The Tupaia material consisted of brain sections of adult male Tupaia belangeri (the tree shrew) held under long-day conditions (light: dark 16:8 h) in the breeding colony of the German Primate Center (GPC, Göttingen, Germany, see Reuss and Fuchs, 2000), fixed by transcardial perfusion with PLP (see below). The baboon material, fixed by immersion in phosphate-buffered 4% paraformaldehyde, stems from a four-year old male Papio hamadryas from the GPC breeding colony, kindly provided by Prof. Dr. E. Fuchs, GPC. For Western blots, mice were killed by cervical dislocation. Immediately thereafter, the brains were removed, the hypothalamus lateral to the third ventricle was dissected, snap-frozen in liquid nitrogen and stored at −80 °C.
3.5. Stk33-protein in mouse brain (Western blot) Our present application of SDS-PAGE to separate mouse brain proteins according to molecular size, followed by Western blots, yielded two bands with molecular masses of about 55 and 40 kDa. In our previous study, we confirmed the existence of Stk33- transcripts in hypothalamus and spinal cord by reverse-transcriptase-PCR (RT-PCR) followed by sequencing. We extracted RNA from these CNS regions and transcribed it into cDNA, followed by amplification using Stk33specific primers (Brauksiepe et al., 2014). The translation of the DNA sequence into protein with the help of a computer algorithm revealed that the transcripts may correspond to the mouse Stk33-protein sizes.
4.3. Human brainstem preparation These studies were performed on sections from the human brainstem. Material was kindly provided by Dr. Thorsten Fink, Department of Pathology, HSK Wiesbaden, Germany. It stems from a woman who died at the age of 73 years from peritonitis and septic shock, and a man died at the age of 74 years from pneumonia. Neither one suffered from any known neurological disease. The brainstem was taken postmortem, fixed in formalin at 4 °C for two weeks and stored in PBS, then divided in the medial plane. A section of 15 mm thickness was taken from the region bordering the fourth ventricle and cryoprotected as described above.
3.6. Possible functions of neuronal Stk33 A number of clinical aspects underline the importance of this kinase for human developmental and physiological functions. For example, STK33 is one of the genes most markedly down-regulated in the tetralogy of Fallot, a congenital defect of the heart, an organ which, notably, exhibited a strong Stk33-signal in the mouse embryo (Bittel et al., 2011; Mujica et al., 2005). There is also clinical evidence that STK33 takes part in the control of signaling pathways that are involved in tumor genesis (Piccaluga et al., 2016; Wang et al., 2015). It is, however, open what the functions of neuronal Stk33 may be. Other serine/threonine-kinases such as CaMK IV and VI, expressed in defined neuronal populations, are thought to play roles in neuronal plasticity and information transduction (Engels et al., 1999; Sakagami et al., 1992). CaMK, in general, participate in phosphorylation, activation of transcription factors and in gene expression mechanisms (Cohen et al., 2016; Curtis and Finkbeiner, 1999). It is conceivable that neuronal Stk33 plays similar roles, and/or that it is involved in neurofilament assembly. Of particular interest in the present context is the function of Stk33 in hypothalamic magnocellular neurons and their beaded axons. Do they transport Stk33 to the neurohypophysis, and do they release it? The presumable presence of Stk33 in cerebrospinal fluid and blood may shed additional light on the function of this kinase.
4.4. Immunohistochemistry Tissues were cryosectioned at 40 µm thickness on a freezing microtome in the frontal plane. Sections were washed three times in 10 mM PBS pH 7.4 for 15 min before they were incubated free-floating overnight at room-temperature in primary antibodies diluted in PBS /0.1% Triton X-100/1% normal swine serum. Antibodies were a polyclonal rabbit-raised antibody against synthetic Stk33 (1:100 in PBS, see Mujica et al., (2005) for further details), a polyclonal guinea pig antibody raised against synthetic arginine-vasopressin (AVP; 1:200, Peninsula Labs, San Carlos, CA, USA), a polyclonal guinea pig antibody raised against synthetic oxytocin (OT, 1:200, Peninsula), a polyclonal sheep antibody raised against neuronal nitric oxide-synthase (nNOS, 1:50, Abcam, Cambridge, UK), a rat monoclonal antibody raised 7
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Acknowledgments
against substance P (SP, 1:100, Abcam), and a mouse monoclonal antibody raised against tyrosine hydroxylase (TH, 1:200, Chemicon, Temecula, CA, USA). They were used and characterized in our laboratory previously (Brauksiepe et al., 2014; Mujica et al., 2005; Reuss et al., 2009a, 2009b, 2016a, 2016b). Control incubations were carried out on material from either species by (1) using antibodies preabsorbed with the respective antigen, (2) using rabbit preimmuneserum instead of the antibodies, and (3) by omitting secondary antibodies. The results of controls all demonstrated specificity of the antibodies. Double-immunostaining experiments were conducted on five series of rat brain frontal sections (antibodies against Stk33 combined with anti-AVP, anti-OT, anti-nNOS, anti-SP or anti-TH). Both primary antibodies of a given series were applied to the sections simultaneously. After three rinses in PBS, the Stk33-immunoreactions were visualized using Cy3-conjugated F(ab´)2 fragment donkey anti-rabbit IgG (Dianova, Hamburg) at 1:100 dilution for 90 min at room-temperature. The additional antibody was visualized using a Cy2-conjugated F(ab´)2 fragment donkey anti-guinea pig IgG (Dianova) at 1:100 dilution. After three rinses in PBS, sections were mounted on gelatinized glass slides, dried, cleared in xylene and covered with Merckoglas (Merck, Darmstadt, Germany). All sections were analyzed using an Olympus BX51 research microscope equipped with an epifluorescence unit, highly specific single and dual band filter sets allowing the single or simultaneous excitation and observation of dyes without overlapping-artefacts (Olympus fluorescence dichromatic mirror cubes, maximal excitation/maximal emission, Cy2: 489 nm/506 nm, Cy3: 552 nm/ 565 nm). Photomicrographs were taken using a digital color camera, processed by the AnalySIS software (Soft Imaging System, Münster, Germany) and, if indicated in legends, arranged using the options “multiple image alignment” (MIA) and “extended focal imaging” (EFI). The Adobe Photoshop program was then used to adjust image contrast and brightness and to add labels. From each section of the double immunostaining experiments, labeled cells were counted with regard to their location within the hypothalamus. Neurons labeled by the antibodies tested were counted when immunoreactivity was clearly over the background level. Singleand double-labeled neurons were quantified separately and the respective percentages were calculated for each nucleus. Brain regions were identified according to the stereotaxic brain atlas of the rat (Paxinos and Watson, 2014).
This work was conducted while SR was Head of the Functional Neuroanatomy Group at the Department of Anatomy and Cell Biology, University Medical Center, Mainz. We thank Dr. Lisa Baumgarten, Department of Anatomy and Cell Biology, for technical help, Dr. Thorsten Fink, Department of Pathology, HSK Wiesbaden, Germany, for providing the postmortem human brainstem specimens, Prof. Dr. E. Fuchs, German Primate Center, Göttingen, Germany, for the baboon brain material, and Prof. Dr. E. Schmidt, Institute of Molecular Genetics, for providing the Stk33-antibody. BB thanks the Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, for making her contribution to this study possible. The study is part of project focusing on the functional analysis of Stk33, initiated by the Institute of Molecular Genetics. SR was supported by the RöttgerStiftung and Hoffmann-Klose-Stiftung. The authors have no conflicts of interest to declare. References Armstrong, W.E., 2015. Hypothalamic supraoptic and paraventricular nuclei. (4th editions)In: Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, Amsterdam, 295–314. Bittel, D.C., Butler, M.G., Kibiryeva, N., Marshall, J.A., Chen, J., Lofland, G.K., O'Brien, J.E., Jr., 2011. Gene expression in cardiac tissues from infants with idiopathic conotruncal defects. BMC Med. Genom. 4, 1. Bolborea, M., Dale, N., 2013. Hypothalamic tanycytes: potential roles in the control of feeding and energy balance. Trends Neurosci. 36, 91–100. Brauksiepe, B., Mujica, A.O., Herrmann, H., Schmidt, E.R., 2008. The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin. BMC Biochem. 9, 25. Brauksiepe, B., Baumgarten, L., Reuss, S., Schmidt, E.R., 2014. Co-localization of serine/ threonine kinase 33 (Stk33) and vimentin in the hypothalamus. Cell Tissue Res. 355, 189–199. Cohen, S.M., Ma, H., Kuchibhotla, K.V., Watson, B.O., Buzsaki, G., Froemke, R.C., Tsien, R.W., 2016. Excitation-transcription coupling in parvalbumin-positive interneurons employs a novel CaM kinase-dependent pathway distinct from excitatory neurons. Neuron 90, 292–307. Curtis, J., Finkbeiner, S., 1999. Sending signals from the synapse to the nucleus: possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth. J. Neurosci. Res. 58, 88–95. Duan, X., Ju, G., 1998. The organization of chemically characterized afferents to the perivascular neuronal groups of the hypothalamic magnocellular neurosecretory system in the rat. Brain Res. Bull. 46, 409–415. Elizondo-Vega, R., Cortes-Campos, C., Barahona, M.J., Oyarce, K.A., Carril, C.A., GarciaRobles, M.A., 2015. The role of tanycytes in hypothalamic glucosensing. J. Cell. Mol. Med. 19, 1471–1482. Engels, B.M., Lucassen, P.J., de Kloet, E.R., Vreugdenhil, E., 1999. Regional distribution of a novel calcium/calmodulin-dependent protein kinase mRNA in the rat brain. Brain Res. 835, 365–368. Goodman, T., Hajihosseini, M.K., 2015. Hypothalamic tanycytes - masters and servants of metabolic, neuroendocrine, and neurogenic functions. Front. Neurosci. 9, 387.. Hallbeck, M., Blomqvist, A., 1999. Spinal cord-projecting vasopressinergic neurons in the rat paraventricular hypothalamus. J. Comp. Neurol. 411, 201–211. Harding, A., Paxinos, G., Halliday, G., 2004. The serotonin and tachykinin systems. In: Paxinos, G. (Ed.), The Rat Nervous System3rd edition. Elsevier Academic Press, Amsterdam, 1203–1256. Manning, G., Whyte, D.B., Martinez, R., Hunter, T., Sudarsanam, S., 2002. The protein kinase complement of the human genome. Science 298, 1912–1934. McLean, I.W., Nakane, P.K., 1974. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083. Motawei, K., Pyner, S., Ranson, R.N., Kamel, M., Coote, J.H., 1999. Terminals of paraventricular spinal neurones are closely associated with adrenal medullary sympathetic preganglionic neurones: immunocytochemical evidence for vasopressin as a possible neurotransmitter in this pathway. Exp. Brain Res. 126, 68–76. Mujica, A.O., Hankeln, T., Schmidt, E.R., 2001. A novel serine/threonine kinase gene, STK33, on human chromosome 11p15.3. Gene 280, 175–181. Mujica, A.O., Brauksiepe, B., Saaler-Reinhardt, S., Reuss, S., Schmidt, E.R., 2005. Differential expression pattern of the novel serine/threonine kinase, STK33, in mice and men. FEBS J. 272, 4884–4898. Paxinos, G., Watson, C., 2014. The Rat Brain in Stereotaxic Coordinates 7th edition. Academic Press, San Diego. Peterson, R.P., 1966. Magnocellular neurosecretory centers in the rat hypothalamus. J. Comp. Neurol. 128, 181–190. Piccaluga, P.P., Navari, M., De Falco, G., Ambrosio, M.R., Lazzi, S., Fuligni, F., Bellan, C., Rossi, M., Sapienza, M.R., Laginestra, M.A., Etebari, M., Rogena, E.A., Tumwine, L., Tripodo, C., Gibellini, D., Consiglio, J., Croce, C.M., Pileri, S.A., Leoncini, L., 2016. Virus-encoded microRNA contributes to the molecular profile of EBV-positive Burkitt lymphomas. Oncotarget 7, 224–240. Post, R.M., Gold, P.W., Rubinow, D.R., Bunney, W.E., Ballenger, J.C., Goodwin, F.K.,
4.5. Western blots of mouse brain proteins Proteins were extracted by homogenizing the brain material in preheated RIPA-buffer (95 °C) (10 mM Tris, 1 mM CaCl2, 0.5% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 150 mM NaCl, 10 mM NaF, 25 mM β-glycerophosphate pH 7.4), followed by sonification and centrifugation for 45 min at 4 °C to remove cellular debris. Proteins were dissolved in Laemmli sample buffer and boiled for 5 min with occasional vortexing, then loaded on a 12% polyacrylamide/SDS gel and electrophoresed. The separated proteins were electrophoretically transferred to PVDF-membrane (Roth). Membranes were incubated in the polyclonal rabbit-raised antibody directed against synthetic Stk33, mentioned in paragraph 4.4., in a dilution of 1:100, and visualized using a monoclonal anti-rabbit IgG peroxidase conjugate (1:320.000, Sigma). The Immobilion Western-HRP chemiluminescence substrate (Millipore) was applied to the membrane according to the manufacturer´s instructions. Signals were detected by exposure to Fuji Medical X-ray film Super RX for several minutes. A duplicate of the SDS/PAGE was stained by Coomassie brilliant blue to visualize the total protein content. The film and the stained gel were scanned with a transmission-scanner. 8
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al. 1983. Cerebrospinal fluid as neuroregulatory pathway: peptides in neuropsychiatric illness. In: Wood, J.H. (Ed.), Neurobiology of Cerebrospinal Fluid2nd edition. Plenum Press, New York, 107–142. Reuss, S., Fuchs, E., 2000. Anterograde tracing of retinal afferents to the tree shrew hypothalamus and raphe. Brain Res. 874, 66–74.. Reuss, S., Disque-Kaiser, U., Antoniou-Lipfert, P., Najaf Gholi, M., Riemann, E., Riemann, R., 2009a. Neurochemistry of olivocochlear neurons in the hamster. Anat. Rec. 292, 461–471. Reuss, S., Kühn, I., Windoffer, R., Riemann, R., 2009b. Neurochemistry of identified motoneurons of the tensor tympani muscle in rat middle ear. Hear. Res. 248, 69–79. Reuss, S., Banica, O., Elgurt, M., Mitz, S., Disque-Kaiser, U., Riemann, R., Hill, M., Jaquish, D.V., Koehrn, F.J., Burmester, T., Hankeln, T., Woolf, N.K., 2016a. Neuroglobin expression in the mammalian auditory system. Mol. Neurobiol. 53, 1461–1477. Reuss, S., Wystub, S., Disque-Kaiser, U., Hankeln, T., Burmester, T., 2016b. Distribution of cytoglobin in the mouse brain. Front. Neuroanat. 10, 47. Sakagami, H., Watanabe, M., Kondo, H., 1992. Gene expression of Ca2+/calmodulindependent protein kinase of the cerebellar granule cell type or type IV in the mature
and developing rat brain. Mol. Brain Res. 16, 20–28. Saper, C.P., Stornetta, R.L., 2015. Central autonomic system. (ed.)In: Paxinos, G. (Ed.), The Rat Nervous System. Vol.4th edition. Academic Press, Amsterdam, 629–673. van den Pol, A.N., Herbst, R.S., Powell, J.F., 1984. Tyrosine hydroxylase-immunoreactive neurons of the hypothalamus: a light and electron microscopic study. Neuroscience 13, 1117–1156. Vanhatalo, S., Soinila, S., 1995. Nitric oxide synthase in the hypothalamo-pituitary pathways. J. Chem. Neuroanat. 8, 165–173. Wang, P., Cheng, H., Wu, J., Yan, A., Zhang, L., 2015. STK33 plays an important positive role in the development of human large cell lung cancers with variable metastatic potential. Acta Biochim Biophys. Sin. (Shanghai). 47, 214–223. Woodside, B., Amir, S., 2000. Nitric oxide signaling in the hypothalamus. In: Steinbusch, H.W.M., De Vente, J., Vincent, S.R. (Eds.), Functional Neuroanatomy of the Nitric Oxide System. Handbook of Chemical Neuroanatomy Vol. 17. Elsevier, Amsterdam, 147–176. Yabe, J.T., Chan, W.K., Wang, F.S., Pimenta, A., Ortiz, D.D., Shea, T.B., 2003. Regulation of the transition from vimentin to neurofilaments during neuronal differentiation. Cell. Motil. Cytoskelet. 56, 193–205.
9
Contents lists available at ScienceDirect
Brain Research journal homepage: www.elsevier.com/locate/brainres
Research report
Serine/threonine-kinase 33 (Stk33) – Component of the neuroendocrine network? ⁎
Stefan Reussa, , Bastienne Brauksiepeb, Ursula Disque-Kaiserc, Tim Olivierc a b c
Department of Nuclear Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, Mainz, Germany Department of Anatomy and Cell Biology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany
A R T I C L E I N F O
A BS T RAC T
Keywords: Stk33 Hypothalamus Immunofluorescence Vasopressin Oxytocin Nitric oxide-synthase Substance P Tyrosine-hydroxylase Rat Hamster Tree shrew Baboon Human
The present study was conducted to investigate the expression of serine/threonine-kinase 33 (Stk33) in neuronal structures of the central nervous system in rat and hamster as well as the presence of the protein in the brain of higher mammals, using a polyclonal antibody on cryosections of fixed brains. We found a distinct immunostaining pattern that included intense fluorescence of the ependymal lining of cerebral ventricles, and of hypothalamic tanycytes and their processes. We further observed intense staining of magnocellular neurons in the hypothalamic paraventricular, supraoptic and accessory neurosecretory nuclei, in particular the circular nuclei, and less intense stained neurons in other diencephalic regions. Double-immunostaining experiments showed a partial colocalization of Stk33 with arginine-vasopressin, oxytocin or neuronal nitric oxide-synthase in a large number of neurons of the hypothalamic nuclear regions. Colocalization of Stk33 with substance P or the catecholamine-synthesizing enzyme tyrosine-hydroxylase was not observed. Immunofluorescence was not found in autonomic regions of the lateral horn, suggesting that Stk33 does not contribute to hypothalamo-spinal connections. However, large Stk33-immunoreactive axonal projections from magnocellular hypothalamus to the neurohypophysis were evident. These functionally important connections provide the bridge from neuronal to humoral regulation of the endocrine system. Additionally, Western blots from mouse brain showed two distinct bands representing two Stk33 isoforms. We also present first evidence for the presence of Stk33/STK33 in neuronal structures, ependymal cells and tanycytes in tree shrew, baboon, and human brain.
1. Introduction Serine/threonine kinase 33 is a member of the calcium/calmodulindependent kinases (CaMK) (Manning et al., 2002; Mujica et al., 2001, 2005). Co-immunoprecipitation experiments revealed that Stk33 and the intermediate filament protein vimentin are, in vivo, associated proteins and that Stk33 phosphorylates vimentin in its head domain. Recombinant Stk33 undergoes obligatory autophosphorylation, which might be a requirement for its kinase function, suggesting that Stk33 is involved in intermediate filament assembly/disassembly through the specific and regulated phosphorylation of vimentin (Brauksiepe et al., 2008). This clue to Stk33-function in the regulation of cytoskeleton dynamics by phosphorylation fits to the differential expression pattern of Stk33 (Mujica et al., 2005) that resembles those of some related members of CaMK.
In a recent study (Brauksiepe et al., 2014), we observed the expression of Stk33 and its colocalization to vimentin in ventricular ependymal cells and in tanycytes of rat and hamster as well as its regulation by photoperiod in the Djungarian hamster Phodopus sungorus. The protein was also present in circumventricular organs such as area postrema, subfornical organ, pineal gland and anterior and posterior lobes of the pituitary gland, as well as in magnocellular neurons of the neuroendocrine hypothalamus. As vimentin is present in neurons only in early development (Yabe et al., 2003), other functions of neuronal Stk33 have to be assumed. Since these are unknown as yet, and detailed knowledge of Stk33-expression is an essential prerequisite for the understanding of its function, the present study aimed at characterizing Stk33-neurons with regard to location and co-expression of selected neuroactive substances in rodents. For this purpose, we chose neuroactive substances found in many regions
Abbreviations: ANS, accessory neurosecretory nuclei; AVP, arginine-vasopressin; CiN, circular nucleus; CSF, cerebrospinal fluid; IR, immunoreactive; MCN, magnocellular neurons; nNOS, neuronal nitric oxide-synthase; OT, oxytocin; PVN, paraventricular nucleus; SON, supraoptic nucleus; Stk33, serine/threonine-kinase 33; SP, substance P; TH, tyrosinehydroxylase ⁎ Correspondence to: Department of Nuclear Medicine, University Medical Center, Johannes Gutenberg-University, Langenbeckstraße 1, Bld. 210, 55101 Mainz, Germany. E-mail address: [email protected] (S. Reuss). http://dx.doi.org/10.1016/j.brainres.2016.11.006 Received 9 August 2016; Received in revised form 9 October 2016; Accepted 7 November 2016 Available online xxxx 0006-8993/ © 2016 Published by Elsevier B.V.
Please cite this article as: Reuss, S., Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.11.006
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Fig. 1. Stk33-immunoreactivity in frontal sections of the rat brain. Strong staining was found in ependymal cells of the third ventricle (3 V in A–C). Less intense staining was observed in neurons and their processes. At the level of the optic chiasm (oc in A; approximately interaural +7.9 mm corresponding to Fig. 42 of the rat brain atlas), and in the PVN region at an intermediate level (approximately interaural +7.6 mm corresponding to Fig. 45 of the rat brain atlas), further IR structures are tanycyte processes wrapped around prospective blood capillaries (arrowheads, dorsal periventricular region in A,C; insert in A) and, more ventrally, IR neurons (arrows) of the PVN (A–D). Some of these were located close to the ventricular ependyma and apparently sent processes (arrowheads) into ventricular space (B). Others were located in the lateral PVN aspects (D) and provided beaded fibers advancing to median eminence and neurohypophysis (F). A group of IR neurons (arrows) of the accessory neurosecretory nuclei (ANS) is seen in E. No immunofluorescent neuronal somata were observed in the suprachiasmatic nucleus (SCN in A). All images were taken from the same animal.
2. Results
of the rodent hypothalamus, i.e., arginine-vasopressin, oxytocin, neuronal nitric oxide-synthase, substance P, and tyrosine-hydroxylase (cf. Armstrong, 2015; Harding et al., 2004; van den Pol et al., 1984; Woodside and Amir, 2000). We also studied, more cursory, the presence of Stk33-protein in brain of higher mammals including man. By means of immunofluorescence, we now provide the first overview of the expression of neuronal Stk33-protein in the rodent brain, and demonstrate that the protein is present in the CNS of higher mammals.
2.1. Neuronal expression of Stk33 in rat and hamster Immunostaining of brain sections from adult rats showed a distinct and cell type-specific distribution of Stk33-immunoreactivity. Strong immunostaining was seen in ependymal cells of the ventricular system (Figs. 1A–C, 2E and 3AB) and in tanycytes of the basolateral walls of the third ventricle. The latter cell type exhibited basal, unbeaded processes that were often seen to extend over a long distance and appeared to be wrapped around blood vessels (insert in Fig. 1A and 2
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Fig. 2. Stk33-immunoreactivity in frontal sections of the rat brain (same animal as in Fig. 1). Immunofluorescent neurons (arrows) were found in the supraoptic nucleus (SON in A) and in the circular nucleus (CiN in B) where they exhibit beaded processes. Neurons (arrows) in the epithalamic medial habenular nucleus (MHab in C) were stronger labeled than those in the lateral habenular nucleus (LHab in C). At a posterior level of the hypothalamus (E, approximately interaural +7.2 mm corresponding to Fig. 48 of the rat brain atlas), large neurons with processes (arrows) of the lateral magnocellular part of the paraventricular nucleus (PVN lm) were clearly labeled (D). In the ventral hypothalamus in the same section, strongly stained tanycyte processes extending laterally (arrowheads), and moderately labeled neurons (arrows) of the anterior hypothalamus are seen (F). 3 V third ventricle, sox supraoptic decussation.
nuclei (CiN; Fig. 2B), i.e. in hypothalamic regions containing the neuroendocrine cell groups. Neurons in these nuclei exhibited beaded processes that probably represented axons with varicosities (Fig. 1DF). Additional Stk33-IR MCN were found in the dispersed group of accessory neurosecretory nuclei (ANS; Fig. 1E). Further hypothalamic Sk33-positive neurons of different soma size and less intense immunofluorescence were visible in the anterior hypothalamus (AH in Fig. 2EF), as well as in posterior hypothalamus and premamillary nucleus. Stk33-IR neuronal somata were not observed in the hypothalamic suprachiasmatic nucleus. In addition to hypothalamic neurons, we observed Stk33-immunolabeled neurons in other diencephalic regions of the rat brain. These
2EF). In the sections, it appeared that processes end in the neuropil or terminate close to neuronal somata (Fig. 1B). This pattern of immunoreactivity matches previous data (Brauksiepe et al., 2014). In addition, we observed Stk33-IR neurons in thalamic and hypothalamic nuclei. Their somata exhibited lower immunoreactivity compared to tanycytes in the same section of rat and hamster brain. A prominent neuronal group was observed in the hypothalamus. As judged from size and location, they belonged to the group of neurosecretory magnocellular neurons (MCN; Figs. 1 and 2). Their soma diameters measured approximately 20–35 µm in the paraformaldehyde-fixed rat tissue. They were located predominantly in the paraventricular (PVN; Fig. 1A), supraoptic (SON; Fig. 2A) and circular 3
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Fig. 3. Characterization of Stk33-IR neurons in the rat hypothalamus by double-immunofluorescence. Exemplarily shown is colocalization of Stk33 either with arginine-vasopressin (AVP) in the paraventricular nucleus (PVN; A–C), with oxytocin (OT) in the circular nucleus (CiN; D–F), with neuronal nitric oxide-synthase (nNOS) in the accessory neurosecretory nuclei (ANS in G–I), or with OT in the supraoptic nucleus (SON in K–M). See also Table 1 for percentages of single- and double-labeled neurons.
neuronal nitric oxide-synthase, common marker of magnocellular hypothalamic neurons. Our double-incubation studies in rats showed that Stk33 is colocalized with AVP, OT or nNOS in subpopulations of neuronal perikarya of the hypothalamic neuroendocrine nuclei. As seen in Table 1, the majority of IR neurons in PVN, SON and CiN were Stk33-IR. In the three double-incubation series, 80–86% (sum of columns 1 and 2) of PVN-neurons, 65–84% of SON-neurons and 67– 93% of CiN-neurons exhibited Stk33-immunofluorescence. The portion of double-labeled neurons showed a relative wide range depending on hypothalamic nucleus and colocalized substance (22% with nNOS in the CiN up to 70% with OT in the PVN). Examples of single- and double-labeled neurons are given for AVP in the PVN (Fig. 3A–C), for OT in the CiN (Fig. 3D–F), for nNOS in the ANS (Fig. 3G–I), and for OT in the SON (Fig. 3K–M).
cells were stained with less intensity compared to MCN. The medial habenular nuclei (MHab in Fig. 2C) exhibited neurons with relatively strong immunofluorescence, while neurons of the lateral habenular nuclei (LHab) were only faintly stained. In the thalamus, the posterior nuclei group exhibited IR neurons, while this was barely seen in the neighboring ventral posteromedial nucleus or in the parafascicular nucleus. Fibers tracts such as the fasciculus retroflexus or the superior thalamic radiation were devoid of immunofluorescence. 2.2. Colocalization of Stk33 with AVP, OT or nNOS in the rat hypothalamus To further characterize hypothalamic Stk33-IR neurons, we conducted colocalization studies of Stk33 with vasopressin, oxytocin or 4
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
expression pattern of this enzyme in mostly non-neuronal tissues of mice and men was presented earlier (Mujica et al., 2005). Our biochemical results gained by analysis of Stk33/vimentin interaction in vitro (Brauksiepe et al., 2008) revealed a function of this protein to be related to vimentin phosphorylation/polymerization processes. We recently demonstrated, by means of double-immunofluorescence, colocalization and photoperiodic regulation of Stk33 and vimentin in tanycytes of the hamster brain (Brauksiepe et al., 2014). In recent and in present data, the most prominent staining with the Stk33-antibody was found in the ependymal cells lining cerebral ventricles and in tanycytes, a specialized ependymal cell type located in the walls of the third ventricle. The previous observation of Stk33-positive, vimentin-negative neurons in the hypothalamus of rat and hamster prompted the present study to further describe and characterize this neuronal population. We will therefore concentrate on the contemplation of Stk33-IR neurons rather than ependymal lining cells which nonetheless represent a fascinating aspect of neuroendocrine regulation (cf. Bolborea and Dale, 2013; Elizondo-Vega et al., 2015; Goodman and Hajihosseini, 2015).
Table 1 Stk33-immunoreactive neurons in the rat hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and circular nucleus (CiN). Given are percent of stained magnocellular neurons (mean ± standard deviation from four rats), i.e., double-labeled (column 1), labeled for Stk33 only (column 2) or labeled for either AVP/OT/nNOS only (column 3). Hypothalamic regions
(1)
(2)
PVN SON CiN
Arginine-Vasopressin (AVP) Stk33/AVP Stk33 35.3 ± 5.7 44.7 ± 5.0 45.8 ± 2.6 18.7 ± 3.2 23.9 ± 3.2 42.8 ± 8.9
AVP 20.0 ± 3.3 35.5 ± 2.4 33.3 ± 2.8
PVN SON CiN
Oxytocin (OT) Stk33/OT 70.3 ± 3.4 67.9 ± 3.4 40.7 ± 3.6
OT 14.2 ± 0.7 26.6 ± 2.7 11.8 ± 2.4
Stk33/nNOS PVN SON CiN
Neuronal nitric oxide synthase (nNOS) Stk33/nNOS Stk33 nNOS 56.9 ± 6.1 24.3 ± 3.6 18.8 ± 2.9 73.2 ± 7.9 10.7 ± 1.5 16.1 ± 2.3 22.8 ± 4.2 70.3 ± 31.3 6.9 ± 1.3
Stk33 15.5 ± 5.0 5.5 ± 0.5 47.5 ± 13.9
(3)
3.1. Stk33 in rat and hamster diencephalic neurons In the diencephalon, two populations of Stk33-IR neurons were observed. One was composed of intensely immunofluorescent hypothalamic neurons (as seen in Fig. 1B) in the regions associated with neuroendocrine functions. These magnocellular neurons are part of the paraventricular, supraoptic and circular nuclei as well as of the accessory neurosecretory nuclei. Many of these neurons, which made up 64–93% of the labeled cells (see Table 1), exhibited beaded fibers that probably represented axons with varicosities, while tanycyte processes were unbeaded. The second diencephalic Stk33-positive neuronal population consists of somata exhibiting less intense fluorescence. These were located in the anterior and posterior hypothalamus and in the premamillary nucleus, and were also seen in the epithalamic habenular nuclei and in the posterior thalamic group.
Colocalization of Stk33 and AVP/OT were also observed in beaded fibers that apparently represented ventrally oriented axons located in the vicinity of neuroendocrine cells. In most cases, Stk33-fluorescence of these axons was less intense than the nonapeptide staining. 2.3. No colocalization of Stk33 with TH or SP in the rat hypothalamus We did not observe colocalization of either TH or SP with Stk33 in hypothalamic neurons. However in the same sections, we found TH-IR as well as SP-IR neuronal somata near the third ventricle, in the suprachiasmatic nucleus and in the paraventricular nucleus, sparing its magnocellular part (data not shown). 2.4. Existence of Stk33-immunoreactivity in non-rodent mammalian species
3.2. Stk33 is colocalized in hypothalamic magnocellular neurons
Furthermore, we conducted a more punctual study on the occurrence of Stk33 in the brain of higher mammals by incubating sections from Tupaia, baboon and man. We localized Stk33 in brain sections from all species. In the tree shrew Tupaia belangeri, predominantly immunostained hypothalamic tanycyte processes were seen that extended hundreds of micrometers into the neuropil at the floor and at more dorsally located aspects of the third ventricle (Fig. 4AB). Strong immunofluorescent ependymal cells and processes were striking at the roof of the lateral ventricles (Fig. 4C). In the brain of the baboon Papio hamadryas, we detected Stk33-immunoreactivity in ependymal cells, in processes of ventricular tanycytes and in faintly labeled periventricular neurons (Fig. 4D). In sections of the human brain, ventricular ependymal cells and their processes (Fig. 4E) as well as neuronal somata and axons were labeled by the Stk33-antibody (Fig. 4FG).
Most magnocellular neurons of the PVN, SON and accessory neurosecretory nuclei produce either AVP, OT, or the enzyme responsible for nitric oxide synthesis in neurons, nNOS (cf. Armstrong, 2015). This was also observed in the present study, in which we quantified immunolabeled neurons without considering the subdivisions of PVN and SON. We also did not include all scattered neurons belonging to the ANS system such as the periventricular magnocellular group and the anterior commissural nucleus (cf. Armstrong, 2015), but rather confined their registration to the well-segregated group of MCN making up the circular nucleus. The tightly packed cells in the CiN were frequently associated with vasculature (see Figs. 2B and 3D), as described by Peterson (1966) and Duan and Ju (1998). The double-labeling studies clearly showed that the Sk33-IR MCN do not represent an additional population of hypothalamic neurons but rather are, at least in part, identical to the MCN characterized by expression of AVP, OT or nNOS, but not of SP or TH. Whether the group that here stained only for Stk33 belongs to one of the various other neurochemically defined subpopulations remains to be elucidated. However, our study suggests that Stk33 is a valuable marker substance for the majority of hypothalamic MCN.
2.5. Western blots of Stk33 in mouse brain protein extracts In Western blots using protein extracts from whole mouse brain, we detected two bands corresponding to molecular masses of about 40 and 55 kDa (Fig. 5). They were not seen in blots using an Stk33-antibody preabsorbed with recombinant Stk33-protein.
3.3. Projections of Stk33-neurons 3. Discussion In the present study, we observed beaded axons that originated in the paraventricular region and extended ventrally. It is conceivable that these fibers are axons of Stk33-IR magnocellular neurons, and that
The present study was conducted to analyze the distribution of Stk33-protein in neuronal structures of the mammalian brain. The 5
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Fig. 4. Stk33-immunoreactivity in the primate brain. A–C: Strongly stained tanycyte processes in periventricular hypothalamic regions of Tupaia (A–C). In the baboon, short tanycyte processes and faintly labeled neurons were stained by the antibody (D). In the human brain (floor of the forth ventricle), periventricular processes (E), putative neuronal axons (F) and neuronal somata with processes (G) were present. In F and G, unmarked fluorescent structures represent unspecific autofluorescence. B–D are shown in the extended focal imagingmode.
they provide function as part of the hypothalamo-hypophyseal system. Axons originating in the medial and lateral magnocellular portions of the PVN, and also those that stem from SON and CiN, course in a wide arc to turn medially, then join the tract of Greving and reach the pituitary neural lobe (Vanhatalo and Soinila, 1995), as well as a variety of other intra- and extra-hypothalamic targets (cf. Armstrong, 2015). These axons exhibited local swellings, i.e. clusters of neurosecretory granules that consist of AVP/OT transported along with their associated neurophysins. This finding matches the recent observation of Stk33-IR fibers exhibiting Herring bodies in the mouse pituitary neural lobe (Brauksiepe et al., 2014). The nonapeptides AVP and OT are released by PVN and SON into the hypophyseal-portal blood and the CSF (cf. Post et al., 1983), and this may be the case as well for coexpressed Stk33. There is indeed evidence from Western blots that STK33 is present in the human CSF and blood (unpublished data, Prof. Dr. M. Otto, Dr. P. Steinacker, Dept. of Neurology, University of Ulm, Germany). This may provide broader regions of the CNS with Stk33, however, the exact purpose of Stk33-secretion is unknown as yet. We did not observe Stk33-IR structures in preganglionic autonomic regions of the spinal cord (Brauksiepe et al., 2014). This is of particular interest since nonapeptidergic projections to preganglionic sympathetic and parasympathetic neurons in the spinal intermediolateral nucleus originating from parvocellular subnuclei of the dorsal, ventral, and lateral PVN were described (Hallbeck and Blomqvist, 1999; Motawei et al., 1999; cf. Saper and Stornetta, 2015). Taken together, these
Fig. 5. Western blot of protein extract from mouse brain. Lane 1, molecular weight marker. Lane 2, Coomassie blue staining of the protein extract. Lane 3, signals gained after incubation of the blot with Stk33-antibody are seen in lane 2. Two bands of about 55 kDa (arrow) and of about 40 kDa (arrowhead) representing Stk33-isoforms were visible. The larger isoform is quantitatively more represented in mouse protein extract compared to the smaller isoform.
6
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
findings suggest that paraventricular Stk33 is involved in neuroendocrine regulatory mechanisms, but not in neuroendocrine-autonomic pathways.
4. Experimental procedures 4.1. Animals All procedures concerning animals were performed in accordance with the published European Health Guidelines under a protocol approved by the local Administration District Official Committee. Moreover, all efforts were made to minimize the number of animals and their suffering. Twelve adult male Sprague-Dawley rats and five Balb/c mice were held under constant conditions (12:12 hs light/dark-cycle, 21 °C roomtemperature, food and water ad libitum) at the animal facilities of the Department of Anatomy and Cell Biology and the Institute of Physiological Chemistry (University of Mainz).
3.4. Stk33 in non-rodent species To find out whether Stk33 is expressed in higher mammals, we used the antibody which recognizes an epitope of the Stk33-protein that is conserved throughout mammalian species, in specimens from tree shrew, baboon and man. In all three species studied, we found specific staining in corresponding regions and cell types as in the rodent material. The observed differences in staining intensity and extent may be attributed to technical factors such as perfusion vs. immersion fixation, use of PLP vs. paraformaldehyde. In sections from the tree shrew Tupaia, a species phylogenetically situated between insectivores and primates, we found faintly labeled hypothalamic neurons and dense, intensely labeled ependymal cells or tanycytes with processes extending several hundreds of micrometers into the neuropil neighboring the third ventricle. In the baboon Papio, we observed faintly labeled hypothalamic neurons and stronger stained processes extending from ventricular tanycytes. In human brain, rather short tanycyte processes near the ventricle as well as some neurons and presumptive axons were visible. Taken together, these immunohistochemical data reveal for the first time that Stk33-protein is expressed in the brain of higher mammals and suggest that the protein is a component of the neuroendocrine system also in humans.
4.2. Animal tissue preparation For immunohistochemistry, rats were killed by carbon dioxide at the middle of the light period and perfused transcardially with phosphate-buffered 0.9% saline (PBS), to which 15,000 IU heparin/l were added, at room temperature (RT), followed by an ice-cold fixative (4% paraformaldehyde, 1.37% L-lysine, 0.21% sodium-periodate in phosphate buffer (PLP; according to (McLean and Nakane, 1974). The right atrium was opened to enable venous outflow. Brains were removed, postfixed in PLP for 1 h, cryoprotected in a graded series of sucrose in 10 mM PBS and stored in PBS-30% sucrose. The Tupaia material consisted of brain sections of adult male Tupaia belangeri (the tree shrew) held under long-day conditions (light: dark 16:8 h) in the breeding colony of the German Primate Center (GPC, Göttingen, Germany, see Reuss and Fuchs, 2000), fixed by transcardial perfusion with PLP (see below). The baboon material, fixed by immersion in phosphate-buffered 4% paraformaldehyde, stems from a four-year old male Papio hamadryas from the GPC breeding colony, kindly provided by Prof. Dr. E. Fuchs, GPC. For Western blots, mice were killed by cervical dislocation. Immediately thereafter, the brains were removed, the hypothalamus lateral to the third ventricle was dissected, snap-frozen in liquid nitrogen and stored at −80 °C.
3.5. Stk33-protein in mouse brain (Western blot) Our present application of SDS-PAGE to separate mouse brain proteins according to molecular size, followed by Western blots, yielded two bands with molecular masses of about 55 and 40 kDa. In our previous study, we confirmed the existence of Stk33- transcripts in hypothalamus and spinal cord by reverse-transcriptase-PCR (RT-PCR) followed by sequencing. We extracted RNA from these CNS regions and transcribed it into cDNA, followed by amplification using Stk33specific primers (Brauksiepe et al., 2014). The translation of the DNA sequence into protein with the help of a computer algorithm revealed that the transcripts may correspond to the mouse Stk33-protein sizes.
4.3. Human brainstem preparation These studies were performed on sections from the human brainstem. Material was kindly provided by Dr. Thorsten Fink, Department of Pathology, HSK Wiesbaden, Germany. It stems from a woman who died at the age of 73 years from peritonitis and septic shock, and a man died at the age of 74 years from pneumonia. Neither one suffered from any known neurological disease. The brainstem was taken postmortem, fixed in formalin at 4 °C for two weeks and stored in PBS, then divided in the medial plane. A section of 15 mm thickness was taken from the region bordering the fourth ventricle and cryoprotected as described above.
3.6. Possible functions of neuronal Stk33 A number of clinical aspects underline the importance of this kinase for human developmental and physiological functions. For example, STK33 is one of the genes most markedly down-regulated in the tetralogy of Fallot, a congenital defect of the heart, an organ which, notably, exhibited a strong Stk33-signal in the mouse embryo (Bittel et al., 2011; Mujica et al., 2005). There is also clinical evidence that STK33 takes part in the control of signaling pathways that are involved in tumor genesis (Piccaluga et al., 2016; Wang et al., 2015). It is, however, open what the functions of neuronal Stk33 may be. Other serine/threonine-kinases such as CaMK IV and VI, expressed in defined neuronal populations, are thought to play roles in neuronal plasticity and information transduction (Engels et al., 1999; Sakagami et al., 1992). CaMK, in general, participate in phosphorylation, activation of transcription factors and in gene expression mechanisms (Cohen et al., 2016; Curtis and Finkbeiner, 1999). It is conceivable that neuronal Stk33 plays similar roles, and/or that it is involved in neurofilament assembly. Of particular interest in the present context is the function of Stk33 in hypothalamic magnocellular neurons and their beaded axons. Do they transport Stk33 to the neurohypophysis, and do they release it? The presumable presence of Stk33 in cerebrospinal fluid and blood may shed additional light on the function of this kinase.
4.4. Immunohistochemistry Tissues were cryosectioned at 40 µm thickness on a freezing microtome in the frontal plane. Sections were washed three times in 10 mM PBS pH 7.4 for 15 min before they were incubated free-floating overnight at room-temperature in primary antibodies diluted in PBS /0.1% Triton X-100/1% normal swine serum. Antibodies were a polyclonal rabbit-raised antibody against synthetic Stk33 (1:100 in PBS, see Mujica et al., (2005) for further details), a polyclonal guinea pig antibody raised against synthetic arginine-vasopressin (AVP; 1:200, Peninsula Labs, San Carlos, CA, USA), a polyclonal guinea pig antibody raised against synthetic oxytocin (OT, 1:200, Peninsula), a polyclonal sheep antibody raised against neuronal nitric oxide-synthase (nNOS, 1:50, Abcam, Cambridge, UK), a rat monoclonal antibody raised 7
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al.
Acknowledgments
against substance P (SP, 1:100, Abcam), and a mouse monoclonal antibody raised against tyrosine hydroxylase (TH, 1:200, Chemicon, Temecula, CA, USA). They were used and characterized in our laboratory previously (Brauksiepe et al., 2014; Mujica et al., 2005; Reuss et al., 2009a, 2009b, 2016a, 2016b). Control incubations were carried out on material from either species by (1) using antibodies preabsorbed with the respective antigen, (2) using rabbit preimmuneserum instead of the antibodies, and (3) by omitting secondary antibodies. The results of controls all demonstrated specificity of the antibodies. Double-immunostaining experiments were conducted on five series of rat brain frontal sections (antibodies against Stk33 combined with anti-AVP, anti-OT, anti-nNOS, anti-SP or anti-TH). Both primary antibodies of a given series were applied to the sections simultaneously. After three rinses in PBS, the Stk33-immunoreactions were visualized using Cy3-conjugated F(ab´)2 fragment donkey anti-rabbit IgG (Dianova, Hamburg) at 1:100 dilution for 90 min at room-temperature. The additional antibody was visualized using a Cy2-conjugated F(ab´)2 fragment donkey anti-guinea pig IgG (Dianova) at 1:100 dilution. After three rinses in PBS, sections were mounted on gelatinized glass slides, dried, cleared in xylene and covered with Merckoglas (Merck, Darmstadt, Germany). All sections were analyzed using an Olympus BX51 research microscope equipped with an epifluorescence unit, highly specific single and dual band filter sets allowing the single or simultaneous excitation and observation of dyes without overlapping-artefacts (Olympus fluorescence dichromatic mirror cubes, maximal excitation/maximal emission, Cy2: 489 nm/506 nm, Cy3: 552 nm/ 565 nm). Photomicrographs were taken using a digital color camera, processed by the AnalySIS software (Soft Imaging System, Münster, Germany) and, if indicated in legends, arranged using the options “multiple image alignment” (MIA) and “extended focal imaging” (EFI). The Adobe Photoshop program was then used to adjust image contrast and brightness and to add labels. From each section of the double immunostaining experiments, labeled cells were counted with regard to their location within the hypothalamus. Neurons labeled by the antibodies tested were counted when immunoreactivity was clearly over the background level. Singleand double-labeled neurons were quantified separately and the respective percentages were calculated for each nucleus. Brain regions were identified according to the stereotaxic brain atlas of the rat (Paxinos and Watson, 2014).
This work was conducted while SR was Head of the Functional Neuroanatomy Group at the Department of Anatomy and Cell Biology, University Medical Center, Mainz. We thank Dr. Lisa Baumgarten, Department of Anatomy and Cell Biology, for technical help, Dr. Thorsten Fink, Department of Pathology, HSK Wiesbaden, Germany, for providing the postmortem human brainstem specimens, Prof. Dr. E. Fuchs, German Primate Center, Göttingen, Germany, for the baboon brain material, and Prof. Dr. E. Schmidt, Institute of Molecular Genetics, for providing the Stk33-antibody. BB thanks the Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, for making her contribution to this study possible. The study is part of project focusing on the functional analysis of Stk33, initiated by the Institute of Molecular Genetics. SR was supported by the RöttgerStiftung and Hoffmann-Klose-Stiftung. The authors have no conflicts of interest to declare. References Armstrong, W.E., 2015. Hypothalamic supraoptic and paraventricular nuclei. (4th editions)In: Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, Amsterdam, 295–314. Bittel, D.C., Butler, M.G., Kibiryeva, N., Marshall, J.A., Chen, J., Lofland, G.K., O'Brien, J.E., Jr., 2011. Gene expression in cardiac tissues from infants with idiopathic conotruncal defects. BMC Med. Genom. 4, 1. Bolborea, M., Dale, N., 2013. Hypothalamic tanycytes: potential roles in the control of feeding and energy balance. Trends Neurosci. 36, 91–100. Brauksiepe, B., Mujica, A.O., Herrmann, H., Schmidt, E.R., 2008. The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin. BMC Biochem. 9, 25. Brauksiepe, B., Baumgarten, L., Reuss, S., Schmidt, E.R., 2014. Co-localization of serine/ threonine kinase 33 (Stk33) and vimentin in the hypothalamus. Cell Tissue Res. 355, 189–199. Cohen, S.M., Ma, H., Kuchibhotla, K.V., Watson, B.O., Buzsaki, G., Froemke, R.C., Tsien, R.W., 2016. Excitation-transcription coupling in parvalbumin-positive interneurons employs a novel CaM kinase-dependent pathway distinct from excitatory neurons. Neuron 90, 292–307. Curtis, J., Finkbeiner, S., 1999. Sending signals from the synapse to the nucleus: possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth. J. Neurosci. Res. 58, 88–95. Duan, X., Ju, G., 1998. The organization of chemically characterized afferents to the perivascular neuronal groups of the hypothalamic magnocellular neurosecretory system in the rat. Brain Res. Bull. 46, 409–415. Elizondo-Vega, R., Cortes-Campos, C., Barahona, M.J., Oyarce, K.A., Carril, C.A., GarciaRobles, M.A., 2015. The role of tanycytes in hypothalamic glucosensing. J. Cell. Mol. Med. 19, 1471–1482. Engels, B.M., Lucassen, P.J., de Kloet, E.R., Vreugdenhil, E., 1999. Regional distribution of a novel calcium/calmodulin-dependent protein kinase mRNA in the rat brain. Brain Res. 835, 365–368. Goodman, T., Hajihosseini, M.K., 2015. Hypothalamic tanycytes - masters and servants of metabolic, neuroendocrine, and neurogenic functions. Front. Neurosci. 9, 387.. Hallbeck, M., Blomqvist, A., 1999. Spinal cord-projecting vasopressinergic neurons in the rat paraventricular hypothalamus. J. Comp. Neurol. 411, 201–211. Harding, A., Paxinos, G., Halliday, G., 2004. The serotonin and tachykinin systems. In: Paxinos, G. (Ed.), The Rat Nervous System3rd edition. Elsevier Academic Press, Amsterdam, 1203–1256. Manning, G., Whyte, D.B., Martinez, R., Hunter, T., Sudarsanam, S., 2002. The protein kinase complement of the human genome. Science 298, 1912–1934. McLean, I.W., Nakane, P.K., 1974. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083. Motawei, K., Pyner, S., Ranson, R.N., Kamel, M., Coote, J.H., 1999. Terminals of paraventricular spinal neurones are closely associated with adrenal medullary sympathetic preganglionic neurones: immunocytochemical evidence for vasopressin as a possible neurotransmitter in this pathway. Exp. Brain Res. 126, 68–76. Mujica, A.O., Hankeln, T., Schmidt, E.R., 2001. A novel serine/threonine kinase gene, STK33, on human chromosome 11p15.3. Gene 280, 175–181. Mujica, A.O., Brauksiepe, B., Saaler-Reinhardt, S., Reuss, S., Schmidt, E.R., 2005. Differential expression pattern of the novel serine/threonine kinase, STK33, in mice and men. FEBS J. 272, 4884–4898. Paxinos, G., Watson, C., 2014. The Rat Brain in Stereotaxic Coordinates 7th edition. Academic Press, San Diego. Peterson, R.P., 1966. Magnocellular neurosecretory centers in the rat hypothalamus. J. Comp. Neurol. 128, 181–190. Piccaluga, P.P., Navari, M., De Falco, G., Ambrosio, M.R., Lazzi, S., Fuligni, F., Bellan, C., Rossi, M., Sapienza, M.R., Laginestra, M.A., Etebari, M., Rogena, E.A., Tumwine, L., Tripodo, C., Gibellini, D., Consiglio, J., Croce, C.M., Pileri, S.A., Leoncini, L., 2016. Virus-encoded microRNA contributes to the molecular profile of EBV-positive Burkitt lymphomas. Oncotarget 7, 224–240. Post, R.M., Gold, P.W., Rubinow, D.R., Bunney, W.E., Ballenger, J.C., Goodwin, F.K.,
4.5. Western blots of mouse brain proteins Proteins were extracted by homogenizing the brain material in preheated RIPA-buffer (95 °C) (10 mM Tris, 1 mM CaCl2, 0.5% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 150 mM NaCl, 10 mM NaF, 25 mM β-glycerophosphate pH 7.4), followed by sonification and centrifugation for 45 min at 4 °C to remove cellular debris. Proteins were dissolved in Laemmli sample buffer and boiled for 5 min with occasional vortexing, then loaded on a 12% polyacrylamide/SDS gel and electrophoresed. The separated proteins were electrophoretically transferred to PVDF-membrane (Roth). Membranes were incubated in the polyclonal rabbit-raised antibody directed against synthetic Stk33, mentioned in paragraph 4.4., in a dilution of 1:100, and visualized using a monoclonal anti-rabbit IgG peroxidase conjugate (1:320.000, Sigma). The Immobilion Western-HRP chemiluminescence substrate (Millipore) was applied to the membrane according to the manufacturer´s instructions. Signals were detected by exposure to Fuji Medical X-ray film Super RX for several minutes. A duplicate of the SDS/PAGE was stained by Coomassie brilliant blue to visualize the total protein content. The film and the stained gel were scanned with a transmission-scanner. 8
Brain Research xx (xxxx) xxxx–xxxx
S. Reuss et al. 1983. Cerebrospinal fluid as neuroregulatory pathway: peptides in neuropsychiatric illness. In: Wood, J.H. (Ed.), Neurobiology of Cerebrospinal Fluid2nd edition. Plenum Press, New York, 107–142. Reuss, S., Fuchs, E., 2000. Anterograde tracing of retinal afferents to the tree shrew hypothalamus and raphe. Brain Res. 874, 66–74.. Reuss, S., Disque-Kaiser, U., Antoniou-Lipfert, P., Najaf Gholi, M., Riemann, E., Riemann, R., 2009a. Neurochemistry of olivocochlear neurons in the hamster. Anat. Rec. 292, 461–471. Reuss, S., Kühn, I., Windoffer, R., Riemann, R., 2009b. Neurochemistry of identified motoneurons of the tensor tympani muscle in rat middle ear. Hear. Res. 248, 69–79. Reuss, S., Banica, O., Elgurt, M., Mitz, S., Disque-Kaiser, U., Riemann, R., Hill, M., Jaquish, D.V., Koehrn, F.J., Burmester, T., Hankeln, T., Woolf, N.K., 2016a. Neuroglobin expression in the mammalian auditory system. Mol. Neurobiol. 53, 1461–1477. Reuss, S., Wystub, S., Disque-Kaiser, U., Hankeln, T., Burmester, T., 2016b. Distribution of cytoglobin in the mouse brain. Front. Neuroanat. 10, 47. Sakagami, H., Watanabe, M., Kondo, H., 1992. Gene expression of Ca2+/calmodulindependent protein kinase of the cerebellar granule cell type or type IV in the mature
and developing rat brain. Mol. Brain Res. 16, 20–28. Saper, C.P., Stornetta, R.L., 2015. Central autonomic system. (ed.)In: Paxinos, G. (Ed.), The Rat Nervous System. Vol.4th edition. Academic Press, Amsterdam, 629–673. van den Pol, A.N., Herbst, R.S., Powell, J.F., 1984. Tyrosine hydroxylase-immunoreactive neurons of the hypothalamus: a light and electron microscopic study. Neuroscience 13, 1117–1156. Vanhatalo, S., Soinila, S., 1995. Nitric oxide synthase in the hypothalamo-pituitary pathways. J. Chem. Neuroanat. 8, 165–173. Wang, P., Cheng, H., Wu, J., Yan, A., Zhang, L., 2015. STK33 plays an important positive role in the development of human large cell lung cancers with variable metastatic potential. Acta Biochim Biophys. Sin. (Shanghai). 47, 214–223. Woodside, B., Amir, S., 2000. Nitric oxide signaling in the hypothalamus. In: Steinbusch, H.W.M., De Vente, J., Vincent, S.R. (Eds.), Functional Neuroanatomy of the Nitric Oxide System. Handbook of Chemical Neuroanatomy Vol. 17. Elsevier, Amsterdam, 147–176. Yabe, J.T., Chan, W.K., Wang, F.S., Pimenta, A., Ortiz, D.D., Shea, T.B., 2003. Regulation of the transition from vimentin to neurofilaments during neuronal differentiation. Cell. Motil. Cytoskelet. 56, 193–205.
9