Molecular mechanism of kallikrein-related peptidase 8/neuropsin-induced hyperkeratosis in inflamed skin.

BACKGROUND: Hyperkeratosis and acanthosis occur in inflamed skin. Proliferation and differentiation of keratinocytes are important processes during epidermal repair after inflammation. Neuropsin and its human homologue kallikrein-related peptidase 8 (KLK8) have been reported to be involved in epidermal proliferation and differentiation, but the involved molecular mechanisms are obscure. OBJECTIVES: To explore the molecular mechanism of KLK8/neuropsin-induced hyperkeratosis and acanthosis in inflamed skin. METHODS: The molecular mechanism involved in KLK8/neuropsin-induced hyperkeratosis and acanthosis in inflamed skin was investigated both in vivo and in vitro using neuropsin knockout mice and KLK8 knockdown human keratinocytes. Neuropsin-related genes were identified by differential gene display. The localization and functional relationship of the molecules affected downstream of KLK8/neuropsin in normal and inflamed skin were analysed by in situ hybridization and immunohistochemistry. RESULTS: Hyperkeratosis and acanthosis in sodium lauryl sulphate-stimulated skin were markedly inhibited in neuropsin knockout mice. Knockdown of KLK8/neuropsin increased transcription factor activator protein-2α (AP-2α) expression and decreased keratin 10 expression in human keratinocytes and mouse skin, respectively. AP-2α has been reported to inhibit epidermal proliferation and keratin 10 expression. Distributional analysis showed that KLK8/neuropsin was expressed in the stratum spinosum, AP-2α was expressed in the stratum basale and the lower part of the stratum spinosum, and keratin 10 was expressed throughout the stratum spinosum. CONCLUSIONS: The above findings suggest the following mechanism of events underlying KLK8/neuropsin-induced hyperkeratosis: (i) skin inflammation increases KLK8/neuropsin expression in the stratum spinosum; (ii) the released KLK8/neuropsin inhibits AP-2α expression in the cells of the stratum basale and stratum spinosum; (iii) the decrease in AP-2α results in cell proliferation in the stratum basale and cell differentiation in the stratum spinosum, with an increase in keratin 10 expression.

Ablation of neuropsin-neuregulin 1 signaling imbalances ErbB4 inhibitory networks and disrupts hippocampal gamma oscillation.

Parvalbumin-expressing interneurons are pivotal for the processing of information in healthy brain, whereas the coordination of these functions is seriously disrupted in diseased brain. How these interneurons in the hippocampus participate in pathological functions remains unclear. We previously reported that neuregulin 1 (NRG1)-ErbB4 signaling, which is actuated by neuropsin, is important for coordinating brain plasticity. Neuropsin cleaves mature NRG1 (bound to extracellular glycosaminoglycans) in response to long-term potentiation or depression, liberating a soluble ligand that activates its receptor, ErbB4. Here, we show in mice that kainate-induced status epilepticus transiently elevates the proteolytic activity of neuropsin and stimulates cFos expression with a time course suggesting that activation of ErbB4- and parvalbumin-expressing interneurons follows the excitation and subsequent silencing of pyramidal neurons. In neuropsin-deficient mice, kainate administration impaired signaling and disrupted the neuronal excitation-inhibition balance (E/I balance) in hippocampal networks, by decreasing the activity of parvalbumin-positive interneurons while increasing that of pyramidal neurons, resulting in the progression of status epilepticus. Slow, but not fast, gamma oscillations in neuropsin-deficient mice showed reduced power. Intracerebroventricular infusion of the soluble NRG1 ligand moiety restored the E/I balance, status epilepticus and gamma oscillations to normal levels. These results suggest that the neuropsin-NRG1 signaling system has a role in pathological processes underlying temporal lobe epilepsy by regulating the activity of parvalbumin-expressing interneurons, and that neuropsin regulates E/I balance and gamma oscillations through NRG1-ErbB4 signaling toward parvalbumin-expressing interneurons. This neuronal system may be a useful target of pharmacological therapies against cognitive disorders.

Hippocampal CA1 synaptic plasticity as a gamma transfer function.

The capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output conversion efficiency at the synapses. In the adult mouse hippocampus in vivo, we carefully compared the input-output relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e. conforming to the so-called gamma curve. In extreme cases, long-term potentiation and depression coexist in the same synaptic pathway with long-term potentiation dominating over long-term depression at higher levels of presynaptic activity. These findings predict a novel function of synaptic plasticity, i.e. a contrast-enhancing filtering of neural information through a gamma correction-like process.

cDNA cloning and expression of a novel serine protease, TLSP.

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.

Expression of neuropsin in the keratinizing epithelial tissue-immunohistochemical analysis of wild-type and nude mice.

Neuropsin is a trypsin-type serine protease that was first cloned from the mouse brain as a factor related to neural plasticity. Subsequent in situ hybridization histochemical analysis indicated a broad localization of its mRNA throughout the whole body, although the details remain obscure. In this study, we showed that neuropsin immunoreactivity is localized in the keratinized stratified epithelia of the mouse epidermis, hair, tongue, palate, nasal cavity, pharynges, esophagus, and forestomach. In the skin and mucous membranes, neuropsin immunoreactivity was found in the stratum spinosum and the stratum granulosum. The immunoreactivity in the former sublayer was mainly present in the cytoplasm, but that in the latter sublayer was exclusively present in the intercellular space or on the outer surface of the cell membrane and thus exhibited a lamellar-like peripheral distribution. During development, the appearance of neuropsin immunoreactivity in the various epithelia was found at embryonic days 14.5-15.5, prior to formation of the stratum corneum. More extensive expression of neuropsin immunoreactivity was found in the nude mouse skin and mucous membranes than in wild-type mice. Because the nude mouse is characterized by genetic impairment of keratinization, such abnormal neuropsin expression might be caused or affected by this impairment. Therefore, neuropsin, an extracellular serine protease, is suggested to be involved in keratinization in the stratified epithelia.

Immunoreactive glucagon in the smooth muscle cells of the rat cerebral artery: an immunohistochemical analysis.

The presence of immunoreactive glucagon (IRG) in a smooth muscle of the cerebral arteries of rats was demonstrated immunohistochemically using two antisera against pancreatic glucagon, OAL -123 and Unger 's 30K . Based on the results and on our previous radioimmunoassay and gel filtration study, the smooth muscle cells of the blood vessels may be one of the extrapancreatic sources of IRG in the plasma.

Sequence analysis and expression of human neuropsin cDNA and gene.

Neuropsin is a serine protease which is thought to function in a variety of tissues including the brain and skin. This protease has been shown to have important roles in neural plasticity in mice. Here we have cloned a cDNA and analyzed the gene for human neuropsin by polymerase chain reaction-based strategies. The cDNA had 72% identity to mouse neuropsin. The deduced amino acid sequence showed 72% identity to mouse neuropsin. Key amino acid residues for the enzyme activity and all cysteine residues were conserved between human and mouse neuropsin. The gene for human neuropsin had six exons and five introns, and the gene organization is similar to trypsin-type serine proteases. The mRNA was expressed in primary cultures of keratinocytes.

Experimental immunohistochemical studies on the substance P neuron system in the lateral habenular nucleus of the rat: distribution and origins.

We examined the distribution and afferent projections of substance P-like immunoreactive (SPI) fibers in the lateral habenular nucleus of the rats by using the indirect immunofluorescence method. On the basis of the distribution, a dense plexus of SPI fibers in the lateral habenular nucleus (LHb) could be divided into two parts: medial (mLHb) and lateral (lLHb). The present study demonstrates that SPI fibers in the lLHb originate from SPI cells in the rostral entopeduncular nucleus (rEP) and the adjacent area, while those in the mLHb originate from SPI cells in the medial habenular nucleus. Moreover, we showed that the axons from SPI cells in the rEP reached the lLHB via the "pallidohabenular tract."

Gap junction protein in rat hippocampus: correlative light and electron microscope immunohistochemical localization.

Immunohistochemical techniques and an affinity-purified antibody directed against the 27-kD gap-junctional protein (GJP) from rat liver were used to determine the ultrastructural localization of GJP in the rat hippocampus. At the light microscope level, dense GJP immunoreactivity having a stringlike appearance was seen in a very small percentage of medium-sized neuronal somata located in the stratum pyramidale, and diffuse immunostaining was seen in many small cell bodies in the stratum pyramidale, stratum oriens, and the alveus. Abundant GJP-immunoreactive (GJP-IR) varicose fibers were observed in the strata pyramidale, radiatum, and oriens but were less concentrated in the alveus. Numerous punctate GJP-IR elements were observed in all hippocampal layers. Upon EM analysis, GJP-IR neuronal somata in the stratum pyramidale were found to be, without exception, nonpyramidal neurons as judged by such distinguishing features as their fusiform perikarya, indented nucleus, and well-developed rough endoplasmic reticulum (RER). Immunostaining within these cells was largely localized to the Golgi apparatus and associated vesicular components. Small, diffusely GJP-IR cells were identified ultrastructurally as protoplasmic and fibrous astrocytes. Immunostaining within these cells was localized to the Golgi apparatus, RER, and small, ribosomelike bodies 15-25 nm in diameter. Among neuronal processes GJP immunoreactivity was found within dendrites, axons, and axonal terminals. The latter structures contained numerous GJP-IR vesicles having an average diameter of about 40 nm. A frequent observation indicating some degree of specificity of the anti-GJP antibody employed here was immunostaining of typical gap junctions between dendrites and, more commonly, between processes of glial cells. Occasionally, however, GJP-IR dendrodendritic, axodendritic, and axoaxonic contacts were found that could be considered, at best, as being gap-junction-like (gj-L). In these cases, asymmetric immunostaining of adjacent plasma membranes forming gj-L structures was not uncommon. These results confirm the existence of gap junctions between dendrites in the rat hippocampus and demonstrate that GJP immunoreactivity on cytoplasmic membranes is restricted either to typical neuronal and glial gap junctions or to gj-L structures at circumscribed sites of contact between various types of neuronal elements where GJP may contribute to a novel mechanism of neural communication.

Gap junction protein in rat hippocampus: light microscope immunohistochemical localization.

An affinity-purified antibody against a 27-kD rat liver gap-junctional protein (GJP) was used to determine the distribution of GJP immunoreactivity in sections of rat hippocampus. Four heterogeneously distributed GJP-immunostaining patterns were observed. The two most common were punctate immunoreactive elements ranging in size from 0.3 to 0.7 microns and networks of immunoreactive varicose fibers coursing in a variety of directions within the various hippocampal layers and ranging in length from a few microns up to 200 microns. The density of punctate immunostaining was highest within a portion of the stratum pyramidale, at the border between the stratum pyramidale and stratum oriens, and at the border between the molecular and granule cell layers of the dentate gyrus. Moderate to low densities were observed in other hippocampal areas. Immunoreactive fibers were most concentrated within the border portions of the stratum pyramidale and oriens, moderately distributed in the stratum radiatum and the remaining part of the stratum oriens, and sparse in the alveus. In the dentate gyrus, fiber networks were most evident at the border between the granule cell and molecular layers and very unevenly distributed in the molecular layer. The two other patterns observed included intense filamentous immunostaining within a small number of neuronal perikarya located mainly in the stratum pyramidale of areas CA2 and CA3, but rarely in area CA1 or the dentate gyrus, and diffuse immunostaining of small cell bodies dispersed throughout the hippocampus but most numerous in the vicinity of the stratum pyramidale and in the alveus. All of these immunostaining patterns were seen at all rostrocaudal hippocampal levels. These results suggest that if GJP-immunoreactive fibers and neurons observed in the hippocampus have the capacity to form gap junctions, then electrotonic transmission may constitute an important means of information processing within this structure.

Ontogeny of the peptidergic system in the rat spinal cord: immunohistochemical analysis.

The ontogeny of neuropeptides, such as somatostatin (SRIF), substance P (SP), leucine-enkephalin (LE), and neurotensin (NT) in the spinal cord (including the spinal ganglion) of the rat, was examined by means of the indirect immunofluorescence method. SRIF and SP appear in the early fetal period before the establishment of the spinal synaptic transmission system, and their appearance precedes that of LE and NT, thus suggesting that SRIF and SP might have some important role in the development of the spinal cord. Furthermore, a number of SRIF-positive structures are found during the fetal period in the spinal cord; however, SRIF-positive fibers in the ventral horn, lamina V, VI, and X tend to decrease remarkably in number after birth, while those found in the dorsal horn maintain their immunoreactivity even in the adult rats. These facts suggest that SRIF in the latter area might function as a neurotransmitter, whereas in the former areas, SRIF might have another role in the development of the spinal cord. SP-positive structures also made their appearance during the fetal period. SP-positive fibers continue to increase in number after birth, and they can be seen throughout the entire spinal cord even in the adult rats. It becomes difficult to identify SP-positive neurons as the rats grow. Numerous SP-positive cells are demonstrated, however, by colchicine pretreatment, thus suggesting that this system is functioning actively in the adult rats. LE- and NT-positive structures appear at perinatal stages and they continue to increase in number after birth. These facts suggest that these peptides (SP, LE and NT) might act as neurotransmitters.

Origins of substance P-containing fibers in the lateral septal area of young rats: immunohistochemical analysis of experimental manipulations.

The majority of substance P-like immunoreactive (SPLI) fibers in the lateral septal area (LS) are supplied by SPLI cells in the area (BAL) between the anterior hypothalamic nucleus and the lateral hypothalamus, and by those in the nucleus latero-dorsalis tegmenti (TLD). These conclusions are based on following: (1) Unilateral destruction of the BAL resulted in an ipsilateral decrease in the septal SPLI fibers similar to that seen after the destruction of the TLD, and (2) simultaneous destruction of the BAL and TLD caused a marked reduction of SPLI fibers in the LS on the operated side. The possibility that the destruction of the BAL affected the ascending SPLI system from the TLD seems to be excluded, because (1) the destruction of the TLD resulted in a decrease in SPLI fibers in the ipsilateral medial forebrain bundle (MFB), but failed to reduce the number of SPLI fibers in the BAL, and (2) the destruction of the BAL caused a decrease in SPLI fibers in the perifornical area rostral to the lesion, but failed to reduce the number of SPLI fibers in the MFB. These facts further suggest that ascending SPLI fibers from the BAL travel in the perifornical area and those from the TLD pass through the MFB. It should be noted that a few SPLI fibers remained intact following the simultaneous destruction of the BAL and TLD. The present study suggests that these remaining SPLI fibers might be innervated by intrinsic SPLI cells. In support of this, several SPLI cells were detected in the septal area after colchicine pretreatment.

Topographic atlas of somatostatin-containing neurons system in the avian brain in relation to catecholamine-containing neurons system. I. Telencephalon and diencephalon.

The morphological organization of the somatostatin (SRIF)-positive neurons in the forebrain (telencephalon and diencephalon) of the warbling grass parakeet (Melopsittacus undulatus) was studied using the indirect immunohistochemical technique of Coons and co-workers ('58). In the telencephalon, a number of SRIF-positive neurons was detected in the lobus paraolfactorius, hippocampus, and paleostriatum. Furthermore, scattered SRIF-labeled cells were noticed in the area corticoidea dorsolateralis and area temporoparieto-occipitalis. A moderate density of immunoreactive fibers was found in the above areas. In addition, although the septal areas was devoid of SRIF-positive neurons, this area contained a moderate occurred in the following hypothalamic areas: (1) nucleus medialis hypothalami posterior, (2) lateral hypothalamus, and (3) mammillary nucleus. The bird hypothalamus also received a strikingly massive SRIF innervation. The heaviest concentration of SRIF-labeled fibers was detected in the medial eminence. Many SRIF-labeled fibers were also observed in other hypothalamic regions. Their locations roughly corresponded in many cases to the areas in which SRIF-positive neurons were disclosed. The overall distribution of the catecholamine system (CA) of the avian forebrain is also represented by means of histofluorescent technique. A possible interaction between SRIF and CA neurons systems is briefly discussed.

Topographic atlas of somatostatin-containing neuron system in the avian brain in relation to catecholamine-containing neuron system. II. Mesencephalon, rhombencephalon, and spinal cord.

With the indirect immunofluorescence technique of Coon's and collaborators, overall distribution of the somatostatin (SRIF)-positive neurons system in the avian lower brain stem was explored. Numerous cell somata containing SRIF were identified in the interpeduncular nucleus and substantia grisea centralis (GCT) at the level of the nucleus nervi trochlearis. Furthermore, a moderate number of SRIF-positive neurons were seen in the tectum opticum, nucleus tractus solitarii, and spinal cord. Scattered labeled cells were noticed in the rhombencephalon. A dense network of SRIF-positive fibers was distributed widely in the lower brain stem of birds. Their locations corresponded in many cases to the areas where SRIF-positive neurons were found. The present study also presents the distribution of the catecholamine (CA) neuron system in the avian lower brain stem. Possible interactions between SRIF and CA neurons systems are briefly discussed.

Distribution of somatostatin in the frog brain, Rana catesbiana, in relation to location of catecholamine-containing neuron system.

The distribution of somatostatin (SRIF)-immunoreactive structures in the central nervous system of the bull frog (both with and without treatment of colchicine) was studied, using the indirect immunofluorescence technique of Coons and co-workers (Coons, '58). SRIF-containing cells were observed in more than ten areas including the spinal cord. These SRIF-positive cells showed segmental distribution, in that SRIF-positive neurons were identified in various areas at various brain levels. An extensive network of SRIF-positive fibers was found in most parts of the central nervous system. The distribution of a catecholamine (CA)-containing neuron system in the frog brain is also presented in this study. The possible interactions between SRIF and CA neurons systems are briefly discussed.

Ontogeny of somatostatin-containing neuron system of the rat: immunohistochemical observations. I. Lower brainstem.

The ontogeny of the somatostatin (SRIF) neuron system in the lower brainstem of the rat was analyzed using the indirect immunofluorescence technique of Coons. SRIF-positive structures first appeared in the primordium of the ventral nucleus of the lemniscus lateralis of the rat fetus corresponding to gestational day 15 (12-14-mm embryos). On and after gestational day 15, these structures appeared in progressively more diverse areas of the lower brainstem and continued to show an increase in number and intensity up till birth. The maximum SRIF-positive structures were histochemically identified in the lower brainstem at the perinatal stage. However, after birth, the numbers of SRIF-positive structures in the lower brainstem began to decrease as the rat grew and none or only a few SRIF-positive structures were detected in the adult rats. This ontogenetical study of the SRIF neuron system on the lower brainstem of the rat strongly suggests that SRIF might play an important role in the development of the lower brainstem other than in its neurotransmitter or neuromodulator function.

Morphological study of noradrenaline innervation in the caudal raphe nuclei with special reference to fine structure.

Previous histofluorescence studies have demonstrated that the caudal raphe nuclei (CRa) of the rat, particularly the ventral portion (VCRa), contains a very high density of noradrenaline (NA) terminals and in the present study we attempted to elucidate the origins and free structure of the NA terminals in this region. The majority of NA terminals found in the VCRa originated from A1 and A3 NA neurons and disclosed that a small number of very strongly fluorescent fibers located along the blood vessels arise from the superior cervical ganglion (SGC). Electron microscopic analysis after potassium permanganate fixation demonstrated that the NA terminals originated from A1 and A3 NA neurons are connected with neuronal elements, while NA fibers from the SGC were identified among the vascular elements. Axodendritic contacts were found to be predominant among the synapselike contacts of NA terminals in this area.

Fine structure of peptidergic and catecholaminergic nerve fibers in the anterior cerebral artery and their interrelationship: an immunoelectron microscopic study.

This study shows (1) the ultrastructure of vasoactive intestinal polypeptide (VIP)-, substance P (SP)-, and neuropeptide Y (NPY)-containing nerve fibers in the walls of the cerebral arteries and (2) the relationship between these peptidergic (VIP, SP, and NPY) and catecholaminergic (CA) nerve terminals by immunohistochemistry combined with false transmitter (5-hydroxydopamine) histochemistry under the electron microscope. VIP-, SP-, and NPY-like immunoreactivity (VIPI, SPI, and NPYI) were found diffusely in the axoplasm and around the small clear vesicles in the nerve terminals. In a few cases, SPI was found within the large vesicles. Most of the VIPI terminals were ensheathed by the cytoplasm of the Schwann cells together with CA terminals, identified as those with a number of small granulated vesicles. In some cases, they were directly apposed to the smooth muscle cells at a distance of about 100 nm. SPI terminals were frequently solitary but about 30% were located together with CA and other (neither SPI nor CA) terminals ensheathed by Schwann cells, directly apposed to the smooth muscle cells at a distance of about 100 nm. On the other hand, NPYI terminals were also identified as CA terminals, indicating the coexistence of these two substances. These findings suggest a close interrelationship between peptidergic and CA nerve terminals in the neurogenic control of cerebral blood vessel function.

Ontogeny of somatostatin-containing neuron system of the rat: immunohistochemical analysis. II. Forebrain and diencephalon.

The ontogeny of the somatostatin (SRIF) neuron system in the forebrain and diencephalon the rat was investigated by means of the indirect immunofluorescence method. SRIF-positive cells first appear in the primordium of the hypothalamus surrounding the fasciculus mammillothalamicus of the fetus at gestational day 14 (10-12-mm embryo). At gestational days 16-17 (14-17-mm embryo, SRIF-positive cells appear in the developing piriform cortex and entopeduncular nucleus. The fetus at gestational days 18-19 (17-26-mm embryos) showed a remarkable increase in the number of SRIF-positive cells and numerous groups of such cells are detectable in various forebrain and diencephalic areas such as the hypothalamic periventricular zone, zona incerta, area lateral to the commissura posterior (ACP), area between the optic tract and capsula interna, pars retrolenticularis (AOR), n. caudatus putamen, hippocampus, somatic sensory cortex, and n. accumbens, etc. At gestational day 20, SRIF-positive cells newly appear in the septum, olfactory bulb, diagonal band of Broca, claustrum, lateral preoptic area, and lateral habenular nucleus. The majority of SRIF-positive structures found in the forebrain and diencephalon increase in number during the perinatal stage (between gestational day 21 and postnatal day 2) and more or less maintain their immunoreactivity even in adult rats. However, SRIF-positive cells located in the AOR, ACP, and lateral septal area, etc., begin to decrease in number during the perinatal stage and no or only a few SRIF-containing cells are identified in these areas of infant and adult rats.

Ontogeny of the leucine-enkephalin neuron system of the rat: immunohistochemical analysis. I. Lower brainstem.

Ontogeny of the leucine-enkephalin (L-Enk) neuron system in the lower brainstem of the rat was investigated by means of indirect immunofluorescence. L-Enk-containing cells first appear in the primordium of the medullary reticular formation just medial to the n. tractus spinalis nerve trigemini at the level of the the rostral half of the inferior olivary nucleus, in the n. cuneiformis, and in the mesencephalic reticular formation of the fetus at gestational day 16 (14-15-mm embryos). From that time onward, L-Enk-containing cells appear in various areas of the lower brainstem one after another until birth. After birth, although L-Enk-containing cells decrease slightly in number as the rats grow, L-Enk-containing fibers continue to increase in number.