Tamás Kozicz

Distribution of urocortin-like immunoreactivity in the central nervous system of the rat.

Urocortin was recently cloned from the rat midbrain. Urocortin is a member of the corticotropin releasing factor (CRF) peptide family and shows 45% sequence identity to CRF and 63% sequence identity to urotensin. It binds with a high affinity to CRF1 and CRF2 receptors, resulting in the stimulation of their adenylate cyclase activity. We used a polyclonal antibody against rat urocortin to define the distribution of urocortin‐like immunoreactivity in the rat central nervous system. Several immunostained cell bodies were found in the supraoptic, paraventricular, and ventromedial hypothalamic nuclei. A large number of neurons with urocortin‐like immunoreactivity were seen in the dorsolateral tegmental nucleus, in the linear and dorsal raphe nuclei, and in the substantia nigra. The most abundant immunoreactive (ir) perikarya were found in the Edinger‐Westphal nucleus. Some neurons showed immunoreactivity in the interstitial nucleus of Cajal, the nucleus of Darkeschewitsch, and the periaqueductal gray. A dense immunoreactive fiber network was found in the lateral septal area. Some faintly stained axon terminals were observed among urocortin‐ir perikarya in the supraoptic and paraventricular nuclei, in the central and periaqueductal gray, and in the Edinger‐Westphal nucleus. No fibers with urocortin‐ir were seen in the median eminence or the posterior pituitary. The distribution of urocortin‐ir overlapped with the expression of the mRNA for the CRF2 receptor in several brain areas. These data support the hypothesis that this peptide is the endogenous ligand for the CRF2 receptor. Urocortin has been implicated in various endocrine responses, such as blood pressure regulation, as well as in higher cognitive functions. J. Comp. Neurol. 391:1–10, 1998.

Delayed Systemic Administration of PACAP38 Is Neuroprotective in Transient Middle Cerebral Artery Occlusion in the Rat

BACKGROUND AND PURPOSE Many substances have been shown to reduce brain damage in models of stroke, but mainly when given either before or shortly after the onset of ischemia. Delayed systemic administration of pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to attenuate the neuronal damage in the hippocampus in a model of global ischemia in rats. The present study examined the neuroprotective action of delayed systemic administration of PACAP38 in a model of transient focal ischemia produced by middle cerebral artery occlusion (MCAO) in rats. METHODS We administered PACAP38 as an intravenous bolus (20 nmol/kg body wt) followed by an intravenous infusion for 48 hours using a micro-osmotic pump at a rate of 160 pmol/microL per hour, beginning 4, 8, or 12 hours after a 2-hour transient MCAO using a filament model. The size of the infarct was determined by examining 2-mm-thick brain sections stained with triphenyltetrazolium chloride, followed by image analysis. Control animals received intravenously 0.1% bovine serum albumin in 0.9% saline as a bolus and infusion at the same time intervals. RESULTS The administration of PACAP38 beginning 4 hours after MCAO significantly reduced the infarct size by 50.88%. Treatment with PACAP38 starting 8 or 12 hours after the onset of ischemia did not result in a significant reduction of the infarct size, although infarct volumes tended to be smaller than in the control groups. CONCLUSIONS Systemic administration of PACAP38 should be clinically useful for reducing brain damage resulting from stroke even when administration is delayed for several hours.

Ghrelin Regulates the Hypothalamic-Pituitary-Adrenal Axis and Restricts Anxiety After Acute Stress

BACKGROUND Ghrelin plays important roles in glucose metabolism, appetite, and body weight regulation, and recent evidence suggests ghrelin prevents excessive anxiety under conditions of chronic stress. METHODS We used ghrelin knockout (ghr-/-) mice to examine the role of endogenous ghrelin in anxious behavior and hypothalamic-pituitary-adrenal axis (HPA) responses to acute stress. RESULTS Ghr-/- mice are more anxious after acute restraint stress, compared with wild-type (WT) mice, with three independent behavioral tests. Acute restraint stress exacerbated neuronal activation in the hypothalamic paraventricular nucleus and medial nucleus of the amygdala in ghr-/- mice compared with WT, and exogenous ghrelin reversed this effect. Acute stress increased neuronal activation in the centrally projecting Edinger-Westphal nucleus in WT but not ghr-/- mice. Ghr-/- mice exhibited a lower corticosterone response after stress, suggesting dysfunctional glucocorticoid negative feedback in the absence of ghrelin. We found no differences in dexamethasone-induced Fos expression between ghr-/- and WT mice, suggesting central feedback was not impaired. Adrenocorticotropic hormone replacement elevated plasma corticosterone in ghr-/-, compared with WT mice, indicating increased adrenal sensitivity. The adrenocorticotropic hormone response to acute stress was significantly reduced in ghr-/- mice, compared with control subjects. Pro-opiomelanocortin anterior pituitary cells express significant growth hormone secretagogue receptor. CONCLUSIONS Ghrelin reduces anxiety after acute stress by stimulating the HPA axis at the level of the anterior pituitary. A novel neuronal growth hormone secretagogue receptor circuit involving urocortin 1 neurons in the centrally projecting Edinger-Westphal nucleus promotes an appropriate stress response. Thus, ghrelin regulates acute stress and offers potential therapeutic efficacy in human mood and stress disorders.

Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness.

Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking.

THE EDINGER-WESTPHAL NUCLEUS: A HISTORICAL, STRUCTURAL AND FUNCTIONAL PERSPECTIVE ON A DICHOTOMOUS TERMINOLOGY

The eponymous term nucleus of Edinger‐Westphal (EW) has come to be used to describe two juxtaposed and somewhat intermingled cell groups of the midbrain that differ dramatically in their connectivity and neurochemistry. On one hand, the classically defined EW is the part of the oculomotor complex that is the source of the parasympathetic preganglionic motoneuron input to the ciliary ganglion (CG), through which it controls pupil constriction and lens accommodation. On the other hand, EW is applied to a population of centrally projecting neurons involved in sympathetic, consumptive, and stress‐related functions. This terminology problem arose because the name EW has historically been applied to the most prominent cell collection above or between the somatic oculomotor nuclei (III), an assumption based on the known location of the preganglionic motoneurons in monkeys. However, in many mammals, the nucleus designated as EW is not made up of cholinergic, preganglionic motoneurons supplying the CG and instead contains neurons using peptides, such as urocortin 1, with diverse central projections. As a result, the literature has become increasingly confusing. To resolve this problem, we suggest that the term EW be supplemented with terminology based on connectivity. Specifically, we recommend that 1) the cholinergic, preganglionic neurons supplying the CG be termed the Edinger‐Westphal preganglionic (EWpg) population and 2) the centrally projecting, peptidergic neurons be termed the Edinger‐Westphal centrally projecting (EWcp) population. The history of this nomenclature problem and the rationale for our solutions are discussed in this review. J. Comp. Neurol. 519:1413–1434, 2011.

Urocortinergic neurons respond in a differentiated manner to various acute stressors in the Edinger-Westphal nucleus in the rat

Corticotropin‐releasing factor (CRF) was implicated as being a major contributor to the neurochemically mediated central regulation of stress response; however, an increasing body of evidence suggests that, besides CRF, other members of this neuropeptide family, such as urocortin (Ucn), may also play a role in modifying the efferent components of immune, endocrine, and behavioral responses to stress. Ucns distribution in the rat brain has been demonstrated, with the most abundant Ucn‐immunoreactive perikarya present in the Edinger‐Westphal nucleus (E‐WN). Acute pain and immobilization stresses recruit E‐WN neurons, however, the activation pattern of E‐WN Ucn neurons in response to various acute systemic and neurogenic challenges has not been compared in a single study. We therefore combined quantitative Fos imaging as a marker for neuronal activation with urocortin immunohistochemistry to visualize neurons induced by intravenous lipopolysaccharide (LPS; 100 μg/kg), ether inhalation, restraint, hyperosmotic (1.5 M NaCl i.p.), and hypotensive hemorrhage challenges. Neurons in the E‐WN responded with the strongest Fos induction to LPS, but ether and restraint stress also resulted in massive Fos immunoreactivity 2 hours after stress. Unexpectedly, hyperosmotic and hypotensive hemorrhage stresses did not induce urocortinergic perikarya in this brain area 2 hours poststress. This challenge‐specific recruitment of E‐WN neurons was independent of stress‐induced adrenal response. The biological significance and the stress‐specific activation of E‐WN urocortinergic neurons will be discussed. J. Comp. Neurol. 480:170–179, 2004.

Chronic stress induces sex-specific alterations in methylation and expression of corticotropin-releasing factor gene in the rat

Background Although the higher prevalence of depression in women than in men is well known, the neuronal basis of this sex difference is largely elusive. Methods Male and female rats were exposed to chronic variable mild stress (CVMS) after which immediate early gene products, corticotropin-releasing factor (CRF) mRNA and peptide, various epigenetic-associated enzymes and DNA methylation of the Crf gene were determined in the hypothalamic paraventricular nucleus (PVN), oval (BSTov) and fusiform (BSTfu) parts of the bed nucleus of the stria terminalis, and central amygdala (CeA). Results CVMS induced site-specific changes in Crf gene methylation in all brain centers studied in female rats and in the male BST and CeA, whereas the histone acetyltransferase, CREB-binding protein was increased in the female BST and the histone-deacetylase-5 decreased in the male CeA. These changes were accompanied by an increased amount of c-Fos in the PVN, BSTfu and CeA in males, and of FosB in the PVN of both sexes and in the male BSTov and BSTfu. In the PVN, CVMS increased CRF mRNA in males and CRF peptide decreased in females. Conclusions The data confirm our hypothesis that chronic stress affects gene expression and CRF transcriptional, translational and secretory activities in the PVN, BSTov, BSTfu and CeA, in a brain center-specific and sex-specific manner. Brain region-specific and sex-specific changes in epigenetic activity and neuronal activation may play, too, an important role in the sex specificity of the stress response and the susceptibility to depression.

Neurons colocalizing urocortin and cocaine and amphetamine-regulated transcript immunoreactivities are induced by acute lipopolysaccharide stress in the Edinger-Westphal nucleus in the rat.

Besides corticotropin releasing factor, central stress regulatory pathways utilize various neurotransmitters/neuropeptides, such as urocortin and cocaine and amphetamine-regulated transcript, which play an important role in modifying the efferent components of endocrine, immune and behavioral responses to stress. Urocortins distribution in the rats brain has been demonstrated, with the most abundant urocortin-ir perikarya present in Edinger-Westphal nucleus. Cocaine and amphetamine-regulated transcript is widely expressed in the rat brain, with a dominant seat of cellular expression also in the Edinger-Westphal nucleus. Since immediate early gene expressions were seen in several midbrain regions, such as in the Edinger-Westphal nucleus, following various acute stresses, the Edinger-Westphal nucleus has been postulated to exert a regulatory/modulatory control over stress responses. Based on these data we decided to investigate the possible colocalization of urocortin and cocaine and amphetamine-regulated transcript-ir in the Edinger-Westphal nucleus using semithin double-label immunofluorescence technique. Furthermore, we also studied whether urocortinergic neurons colocalizing with cocaine and amphetamine-regulated transcript are recruited by lipopolysaccharide stress. Our experiments revealed that urocortin and cocaine and amphetamine-regulated transcript immunoreactivities colocalize in the Edinger-Westphal nucleus. In addition, our studies using the inducible immediate early gene c-fos as a marker of activated neurons demonstrated a significant stress-induced activation in perikarya colocalizing urocortin- and cocaine and amphetamine-regulated transcript-ir in the Edinger-Westphal nucleus. In view of these data it can be postulated that neurons colocalizing cocaine and amphetamine-regulated transcript and urocortin immunoreactivities respond to acute stress, and may play a role in modulating various physiological functions, such as feeding behaviors.

Congenital disorders of glycosylation: new defects and still counting

Almost 50 inborn errors of metabolism have been described due to congenital defects in N-linked glycosylation. These phenotypically diverse disorders typically present as clinical syndromes, affecting multiple systems including the central nervous system, muscle function, transport, regulation, immunity, endocrine system, and coagulation. An increasing number of disorders have been discovered using novel techniques that combine glycobiology with next-generation sequencing or use tandem mass spectrometry in combination with molecular gene-hunting techniques. The number of “classic” congenital disorders of glycosylation (CDGs) due to N-linked glycosylation defects is still rising. Eight novel CDGs affecting N-linked glycans were discovered in 2013 alone. Newly discovered genes teach us about the significance of glycosylation in cell–cell interaction, signaling, organ development, cell survival, and mosaicism, in addition to the consequences of abnormal glycosylation for muscle function. We have learned how important glycosylation is in posttranslational modification and how glycosylation defects can imitate recognizable, previously described phenotypes. In many CDG subtypes, patients unexpectedly presented with long-term survival, whereas some others presented with nonsyndromic intellectual disability. In this review, recently discovered N-linked CDGs are described, with a focus on clinical presentations and therapeutic ideas. A diagnostic approach in unsolved N-linked CDG cases with abnormal transferrin screening results is also suggested.

Leptin and the hypothalamo-pituitary-adrenal stress axis

Leptin is a 16-kDa protein mainly produced and secreted by white adipose tissue and informing various brain centers via leptin receptor long and short forms about the amount of fat stored in the body. In this way leptin exerts a plethora of regulatory functions especially related to energy intake and metabolism, one of which is controlling the activity of the hypothalamo-pituitary-adrenal (HPA) stress axis. First, this review deals with the basic properties of leptins structure and signaling at the organ, cell and molecule level, from lower vertebrates to humans but with emphasis on rodents because these have been investigated in most detail. Then, attention is given to the various interactions of adipose leptin with the HPA-axis, at the levels of the hypothalamus (especially the paraventricular nucleus), the anterior lobe of the pituitary gland (action on corticotropes) and the adrenal gland, where it releases corticosteroids needed for adequate stress adaptation. Also, possible local production and autocrine and paracrine actions of leptin at the hypothalamic and pituitary levels of the HPA-axis are being considered. Finally, a schematic model is presented showing the ways peripherally and centrally produced leptin may modulate, via the HPA-axis, stress adaptation in conjunction with the control of energy homeostasis.