Pronounced and sustained central hypernoradrenergic function in major depression with melancholic features: Relation to hypercortisolism and corticotropin-releasing hormone

Both stress-system activation and melancholic depression are characterized by fear, constricted affect, stereotyped thinking, and similar changes in autonomic and neuroendocrine function. Because norepinephrine (NE) and corticotropin-releasing hormone (CRH) can produce these physiological and behavioral changes, we measured the cerebrospinal fluid (CSF) levels each hour for 30 consecutive hours in controls and in patients with melancholic depression. Plasma adrenocorticotropic hormone (ACTH) and cortisol levels were obtained every 30 min. Depressed patients had significantly higher CSF NE and plasma cortisol levels that were increased around the clock. Diurnal variations in CSF NE and plasma cortisol levels were virtually superimposable and positively correlated with each other in both patients and controls. Despite their hypercortisolism, depressed patients had normal levels of plasma ACTH and CSF CRH. However, plasma ACTH and CSF CRH levels in depressed patients were inappropriately high, considering the degree of their hypercortisolism. In contrast to the significant negative correlation between plasma cortisol and CSF CRH levels seen in controls, patients with depression showed no statistical relationship between these parameters. These data indicate that persistent stress-system dysfunction in melancholic depression is independent of the conscious stress of the disorder. These data also suggest mutually reinforcing bidirectional links between a central hypernoradrenergic state and the hyperfunctioning of specific central CRH pathways that each are driven and sustained by hypercortisolism. We postulate that α-noradrenergic blockade, CRH antagonists, and treatment with antiglucocorticoids may act at different loci, alone or in combination, in the treatment of major depression with melancholic features.

Modulation of N-methyl-d-aspartate receptor function by glycine transport

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-d-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.

Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide

The effects of oleamide, an amidated lipid isolated from the cerebrospinal fluid of sleep-deprived cats, on serotonin receptor-mediated responses were investigated in cultured mammalian cells. In rat P11 cells, which endogenously express the 5-hydroxytryptamine2A (5HT2A) receptor, oleamide significantly potentiated 5HT-induced phosphoinositide hydrolysis. In HeLa cells expressing the 5HT7 receptor subtype, oleamide caused a concentration-dependent increase in cAMP accumulation but with lower efficacy than that observed by 5HT. This effect was not observed in untransfected HeLa cells. Clozapine did not prevent the increase in cAMP elicited by oleamide, and ketanserin caused an ≈65% decrease. In the presence of 5HT, oleamide had the opposite effect on cAMP, causing insurmountable antagonism of the concentration-effect curve to 5HT, but had no effect on cAMP levels elicited by isoproterenol or forskolin. These results indicate that oleamide can modulate 5HT-mediated signal transduction at different subtypes of mammalian 5HT receptors. Additionally, our data indicate that oleamide acts at an apparent allosteric site on the 5HT7 receptor and elicits functional responses via activation of this site. This represents a unique mechanism of activation for 5HT G protein-coupled receptors and suggests that G protein-coupled neurotransmitter receptors may act like their iontropic counterparts (i.e., γ-aminobutyric acid type A receptors) in that there may be several binding sites on the receptor that regulate functional activity with varying efficacies.

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Simvastatin strongly reduces levels of Alzheimer's disease β-amyloid peptides Aβ42 and Aβ40 in vitro and in vivo

Recent epidemiological studies show a strong reduction in the incidence of Alzheimer's disease in patients treated with cholesterol-lowering statins. Moreover, elevated Aβ42 levels and the ɛ4 allele of the lipid-carrier apolipoprotein E are regarded as risk factors for sporadic and familial Alzheimer's disease. Here we demonstrate that the widely used cholesterol-lowering drugs simvastatin and lovastatin reduce intracellular and extracellular levels of Aβ42 and Aβ40 peptides in primary cultures of hippocampal neurons and mixed cortical neurons. Likewise, guinea pigs treated with high doses of simvastatin showed a strong and reversible reduction of cerebral Aβ42 and Aβ40 levels in the cerebrospinal fluid and brain homogenate. These results suggest that lipids are playing an important role in the development of Alzheimer's disease. Lowered levels of Aβ42 may provide the mechanism for the observed reduced incidence of dementia in statin-treated patients and may open up avenues for therapeutic interventions.

Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.

We have investigated whether side chain-hydroxylated cholesterol species are important for elimination of cholesterol from the brain. Plasma concentrations of 24-hydroxycholesterol (24-OH-Chol) in the internal jugular vein and the brachial artery in healthy volunteers were consistent with a net flux of this steroid from the brain into the circulation, corresponding to elimination of approximately 4 mg cholesterol during a 24-h period in adults. Results of experiments with rats exposed to 18O2 were also consistent with a flux of 24-OH-Chol from the brain into the circulation. No other oxysterol measured showed a similar behavior as 24-OH-Chol. These results and the finding that the concentration of 24-OH-Chol was 30- to 1500-fold higher in the brain than in any other organ except the adrenals indicate that the major part of 24-OH-Chol present in the circulation originates from the brain. Both the 24-OH-Chol present in the brain and in the circulation were the 24S-stereoisomer. In contrast to other oxysterols, levels of plasma 24-OH-Chol were found to be markedly dependent upon age. The ratio between 24-OH-Chol and cholesterol in plasma was approximately 5 times higher during the first decade of life than during the sixth decade. There was a high correlation between levels of 24-OH-Chol in plasma and cerebrospinal fluid. It is suggested that the flux of 24-OH-Chol from the brain is important for cholesterol homeostasis in this organ.

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Brain lipids that induce sleep are novel modulators of 5-hydroxytrypamine receptors.

Amide derivatives of fatty acids were recently isolated from cerebrospinal fluid of sleep-deprived animals and found to induce sleep in rats. To determine which brain receptors might be sensitive to these novel neuromodulators, we tested them on a range of receptors expressed in Xenopus oocytes. cis-9,10-Octadecenamide (ODA) markedly potentiated the action of 5-hydroxytryptamine (5-HT) on 5-HT2A and 5-HT2C receptors, but this action was not shared by related compounds such as oleic acid and trans-9,10-octacenamide. ODA was active at concentrations as low as 1 nM. The saturated analog, octadecanamide, inhibited rather than potentiated 5-HT2C responses. ODA had either no effect or only weak effects on other receptors, including muscarinic cholinergic, metabotropic glutamate, GABA(A), N-methyl-D-asparate, or alpha-amino-3-hydroxy-5-methyl-4-isoxozolepropionic acid receptors. Modulation of 5-HT2 receptors by ODA and related lipids may represent a novel mechanism for regulation of receptors that activate G proteins and thereby play a role in alertness, sleep, and mood as well as disturbances of these states.

OB protein binds specifically to the choroid plexus of mice and rats.

Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor.

Serine-1321-independent regulation of the mu 1 adult skeletal muscle Na+ channel by protein kinase C.

The adult skeletal muscle Na+ channel mu1 possesses a highly conserved segment between subunit domains III and IV containing a consensus protein kinase C (PKC) phosphorylation site that, in the neuronal isoform, acts as a master control for "convergent" regulation by PKC and cAMP-dependent protein kinase. It lacks an approximately 200-aa segment between domains I and II though to modulate channel gating. We here demonstrate that mu1 is regulated by PKC (but not cAMP-dependent protein kinase) in a manner distinct from that observed for the neuronal isoforms, suggesting that under the same conditions muscle excitation could be uncoupled from motor neuron input. Maximal phosphorylation by PKC, in the presence of phosphatase inhibitors, reduced peak Na+ currents by approximately 90% by decreasing the maximal conductance, caused a -15 mV shift in the midpoint of steady-state inactivation, and caused a slight speeding of inactivation. Surprisingly, these effects were not affected by mutation of the conserved serine (serine-1321) in the interdomain III-IV loop. the pattern of current suppression and gating modification by PKC resembles the response of muscle Na+ channels to inhibitory factors present in the serum and cerebrospinal fluid of patients with Guillain-Barré syndrome, multiple sclerosis, and idiopathic demyelinating polyradiculoneuritis.

Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes.

Two water channel homologs were cloned recently from rat kidney, mercurial-insensitive water channel (MIWC) and glycerol intrinsic protein (GLIP). Polyclonal antibodies were raised against synthetic C-terminal peptides and purified by affinity chromatography. MIWC and GLIP antibodies recognized proteins in rat kidney with an apparent molecular mass of 30 and 27 kDa, respectively, and did not cross-react. By immunofluorescence, MIWC and GLIP were expressed together on the basolateral plasma membrane of collecting duct principal cells in kidney. By immunohistochemistry, MIWC and GLIP were expressed on tracheal epithelial cells with greater expression of GLIP on the basal plasma membrane and MIWC on the lateral membrane; only MIWC was expressed in bronchial epithelia. In eye, GLIP was expressed in conjunctival epithelium, whereas MIWC was found in iris, ciliary body, and neural cell layers in retina. MIWC and GLIP colocalized on the basolateral membrane of villus epithelial cells in colon and brain ependymal cells. Expression of MIWC and GLIP was not detected in small intestine, liver, spleen, endothelia, and cells that express water channels CHIP28 or WCH-CD. These studies suggest water/solute transporting roles for MIWC and GLIP in the urinary concentrating mechanism, cerebrospinal fluid absorption, ocular fluid balance, fecal dehydration, and airway humidification. The unexpected membrane colocalization of MIWC and GLIP in several tissues suggests an interaction at the molecular and/or functional levels.

Nitric oxide mediates the increase in local cerebral blood flow during focal seizures.

The role of nitric oxide (NO) in the increase in local cerebral blood flow (LCBF) elicited by focal cortical epileptic seizures was investigated in anesthetized adult rats. Seizures were induced by topical bicuculline methiodide applied through two cranial windows drilled over homotopic sites of the frontal cortex, and LCBF was measured by quantitative autoradiography by using 4-iodo[N-methyl-14C]antipyrine. Superfusion of an inhibitor of NO synthase, N omega-nitro-L-arginine (NA; 1 mM), for 45 min abolished the increase of LCBF induced by topical bicuculline methiodide (10 mM) [164 +/- 18 ml/100 g per min in the artificial cerebrospinal fluid (aCSF)-superfused side and 104 +/- 12 ml/100 g per ml in the NA-superfused side; P < 0.005]. This effect was reversed by coapplication of an excess of L-arginine substrate (10 mM) (218 +/- 22 ml/100 g per min in the aCSF-superfused side and 183 +/- 31 ml/100 g per min in the NA + L-Arg-superfused side) but not by 10 mM D-arginine, a stereoisomer with poor affinity for NO synthase (193 +/- 17 ml/100 g per min in the aCSF-superfused side and 139 +/- 21 ml/100 g per min in the NA + D-Arg-superfused side; P < 0.005). Superfusion of the guanylyl cyclase inhibitor methylene blue attenuated the LCBF increase elicited by topical bicuculline methiodide by 25% +/- 16% (P < 0.05). The present findings suggest that NO is the mediator of the vasodilation in response to focal epileptic seizures.

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)adenine on visna virus infection in lambs: a model for in vivo testing of candidate anti-human immunodeficiency virus drugs.

The acyclic nucleoside phosphonate analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was recently found to be effective as an inhibitor of visna virus replication and cytopathic effect in sheep choroid plexus cultures. To study whether PMEA also affects visna virus infection in sheep, two groups of four lambs each were inoculated intracerebrally with 10(6.3) TCID50 of visna virus strain KV1772 and treated subcutaneously three times a week with PMEA at 10 and 25 mg/kg, respectively. The treatment was begun on the day of virus inoculation and continued for 6 weeks. A group of four lambs were infected in the same way but were not treated. The lambs were bled weekly or biweekly and the leukocytes were tested for virus. At 7 weeks after infection, the animals were sacrificed, and cerebrospinal fluid (CSF) and samples of tissue from various areas of the brain and from lungs, spleen, and lymph nodes were collected for isolation of virus and for histopathologic examination. The PMEA treatment had a striking effect on visna virus infection, which was similar for both doses of the drug. Thus, the frequency of virus isolations was much lower in PMEA-treated than in untreated lambs. The difference was particularly pronounced in the blood, CSF, and brain tissue. Furthermore, CSF cell counts were much lower and inflammatory lesions in the brain were much less severe in the treated lambs than in the untreated controls. The results indicate that PMEA inhibits the propagation and spread of visna virus in infected lambs and prevents brain lesions, at least during early infection. The drug caused no noticeable side effects during the 6 weeks of treatment.

Prevention of autoimmune demyelination in non-human primates by a cAMP-specific phosphodiesterase inhibitor.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that serves as a model for the human disease multiple sclerosis. We evaluated rolipram, a type IV phosphodiesterase inhibitor, for its efficacy in preventing EAE in the common marmoset Callithrix jacchus. In a blinded experimental design, clinical signs of EAE developed within 17 days of immunization with human white matter in two placebo-treated animals but in none of three monkeys that received rolipram (10 mg/kg s.c. every other day) beginning 1 week after immunization. In controls, signs of EAE were associated with development of cerebrospinal fluid pleocytosis and cerebral MRI abnormalities. In the treatment group, there was sustained protection from clinical EAE, transient cerebrospinal fluid pleocytosis in only one of three animals, no MRI abnormality, and marked reduction in histopathologic findings. Rolipram-treated and control animals equally developed circulating antibodies to myelin basic protein. Thus, inhibition of type IV phosphodiesterase, initiated after sensitization to central nervous system antigens, protected against autoimmune demyelinating disease.