Expression of PINK1 in the brain, eye and ear of mouse during embryonic development.

PINK1 is a 581 amino acid protein with a serine/threonine kinase domain and an N-terminal mitochondrial targeting motif. The enzyme is expressed in the brain as well as in several tissues such as heart, skeletal muscle, liver, kidney, pancreas and testis. In the present study, we have investigated by Western blot analysis and immunohistochemistry the presence and distribution of PINK1 in the brain, eye and inner ear of mouse during embryonic development. In the brain we detected two PINK1 molecular isoforms of 55 kDa and 66 kDa. Immunoreactive perikarya first appeared at stage E15 in the diencephalon within the thalamus, the hypothalamus, the periventricular layers of the third ventricle and in the rhombencephalon at level of the pons. Subsequently, new PINK1-positive neurons were found in the midbrain within the floor and the periventricular layers of the ventral wall of the mesencephalic vesicle (stage E17) as well as in the neopallial cortex, the tegmentum of the midbrain and the periventricular region of the caudal part of the rhombencephalon (stage E19). At P0, PINK1-immunoreactive cells appeared in the striatum, the mantle layer and caudal part of the medulla oblongata and the cerebellum. The spatio-temporal expression of PINK1 and its heterogeneous distribution suggest that the enzyme might be involved in neuroregulatory processes during embryogenesis. In the eye, PINK1-immunoreactivity was found in the lens and in the cornea, whereas in the inner ear the enzyme was expressed in the ependymal and subependymal cells of the saccule and in the semicircular canals indicating that PINK1 plays a role in the development of these sensory organs.

Expression pattern of mUBPy in the brain and sensory organs of mouse during embryonic development.

Mouse UBPy (mUBPy) belongs to the family of ubiquitin-specific processing proteases (UBPs). In this study we have investigated the expression of mUBPy in the brain and sensory organs of mouse at different embryonic stages (E9, E11, E13, E15, E17, E19) and during the postnatal stages P0, P1, P2, P4 and P5 using Western blot and immunohistochemistry. mUBPy-immunoreactive cell bodies first appeared at stage E11 in several brain regions, particularly in the walls surrounding the vesicles and the ventricles. Subsequently, at stage E13, new mUBPy-positive cells appeared in the corpus striatum, the caudate nucleus, the thalamus, the epithalamus, the hypothalamus and the pons. At E15 the mUBPy pattern was very similar to that observed at E13, whereas at stage E17 mUBPy-immunoreactivity significantly decreased and a high number of mUBPy-immunoreactive cells was found only to line the third ventricle and within the mantle layer of the fourth ventricle. At E19 and P0, no mUBPy-immunoreactive element was found in the brain. At the postnatal stages P2 and P5, mUBPy-positive cells were detected in all subdivisions of the brain, with high concentrations in several cortex regions. Double labeling with the mUBPy antiserum and antisera against specific cell markers showed that the enzyme is expressed both in neurons and astrocytes. Outside the brain, mUBPy was detected, from stage E11, in the eye, within the lens and the cornea, in the inner ear, at the level of the cochlear and vestibular systems and in the olfactory epithelium. The spatio-temporal expression of mUBPy suggests that the enzyme may be involved in neuroregulatory processes during embryogenesis.

Expression of the deubiquitinating enzyme mUBPy in the mouse brain.

Mouse UBPy (mUBPy) is an ubiquitin-specific protease which belongs to a family of deubiquitinating enzymes (DUBs) implicated in several cellular processes related to both cell growth and differentiation. Previously, Northern blot analysis revealed an important expression of mUBPy in the testis and brain. However, a more comprehensive map of mUBPy localization in the central nervous system (CNS) is still lacking. In this study, we mapped the distribution of mUBPy in the mouse brain using nonradioactive in situ hybridization and immunohistochemical techniques. In general, transcript and protein showed a similar and widespread distribution. In particular, mUBPy was strongly expressed in the hippocampal formation, septal region, ventral pallidum, preoptic nucleus, periventricular nucleus of hypothalamus, compact part of the substantia nigra, ventral tegmental area, cochlear nucleus and granular cell layer of cerebellum. A moderate expression of mUBPy was found in the amygdaloid complex, supraoptic nucleus, arcuate and ventromedial nuclei of hypothalamus, lateral hypothalamic area and lateral and reticular part of the substantia nigra. Double labelling with the mUBPy antiserum and antisera against specific cell markers showed that the enzyme is generally expressed in neurons and, in specific regions, also in oligodendrocytes. Moreover, by using antisera to TH and mUBPy we found that mUBPy is localized in dopaminergic neurons. The different distribution of mUBPy in the distinct regions of the brain suggests that it could be related to different deubiquitinating processes; in particular, in the areas where it is expressed at high levels, mUBPy could exert a specialized function through its interaction with specific protein substrates.

Ontogeny of PAC1-R and VPAC1-R in the frog, Rana esculenta.

The distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptors in the brain of amphibians has been previously described. In the present study, we have investigated the ontogeny of the selective PACAP receptor, PAC1-R, and the PACAP-vasoactive intestinal polypeptide (VIP) mutual receptor, VPAC1-R, in frog embryos by whole-mount in situ hybridization histochemistry. At stage 20, expression of PAC1-R and/or VPAC1-R mRNAs was detected in the brain, the auditory vesicles, the external gills, the buds of the lateral lines and the coelomatic cavity. At stage 25, PAC1-R and/or VPAC1-R mRNAs were observed in the buds of the orbital lateral line, the pancreas and heart. At stage 30, PAC1-R and VPAC1-R mRNAs were widely distributed in the telencephalon and diencephalon as well as in the bud of the lateral line, the heart and the pancreas. The anatomical distribution of PAC1-R and VPAC1-R mRNAs, although similar, did not totally overlap, indicating that PACAP and VIP may exert differential effects in frog during development.

Marine sponge-derived polymeric alkylpyridinium salts as a novel tumor chemotherapeutic targeting the cholinergic system in lung tumors.

Previous studies have shown that the cholinergic system plays a pivotal rule in small cell lung cancer (SCLC) cell growth through an autocrine loop that activates the nicotinic cholinergic receptor, which together with the activation of this receptor by nicotine links SCLC evolution with tobacco use. Non-small cell lung cancer (NSCLC) is the most common form of lung cancer and is also linked to tobacco use. Here we describe the presence of molecules of the cholinergic system in NSCLC samples and cell lines and investigate the implications of the cholinergic system in cell growth regulation. Cholino-acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE) were observed in NSCLC tumor biopsies and in NSCLC cell lines. Polymeric alkylpyridinium salts (poly-APS) are AChE inhibitors isolated from the crude extract of the marine sponge, Reniera sarai. These metabolites were characterized as a mixture of two polymers of 3-octylpyridinium, including 29 and 99 monomeric units. Exposure of normal lung fibroblast and NSCLC cell lines to poly-APS revealed a selective cytotoxicity for cancer cells as compared to the normal fibroblast cell lines. FACS analysis indicated poly-APS induced apoptosis in NSCLC cells but not in normal lymphocytes. Non-toxic doses of poly-APS also potently reduced NSCLC cell-cell adhesion in suspension cultures. The limited toxicity of poly-APS on normal cells was confirmed by injection in the caudal vein of mice. No overt effects on health parameters, such as weight gain and physical behavior, were observed, and histological analysis of major organs did not reveal differences between the treated animals as compared to controls. These data demonstrate that NSCLC cells express cholinergic molecules that may be involved in cell growth regulation and that the cholinesterase inhibitor, poly-APS, shows selective toxicity toward NSCLC cells while having no apparent toxicity towards normal cells and tissue in vitro and in vivo.

Expression of PACAP receptors in the frog brain during development.

This study describes the expression of PAC1 and VPAC1 receptor (PAC1-R and VPAC1-R) mRNAs in the brain of frog (Rana esculenta) during development. PAC1-R mRNA was detected in the periventricular and subependymal layers of the thalamus and epithalamus and in the ependymal layer of the mesencephalon and rhombencephalon (stage 20), in the amygdala, in the habenular complex, in the periventricular nucleus of the hypothalamus, and in the ventral cerebellum (stage 30). VPAC1-R mRNA expression was observed only at stage 20, in the floor of the hypothalamus. These results suggest that, in amphibians, pituitary adenylate cyclase-activating polypeptide (PACAP) may play a role in brain development.