The increased density of p38 mitogen-activated protein kinase-immunoreactive microglia in the sensorimotor cortex of aged TgCRND8 mice is associated predominantly with smaller dense-core amyloid plaques.

The role for phosphorylated p38 mitogen-activated protein kinase [p-p38(MAPK)] in ß-amyloid plaque deposition [a hallmark of Alzheimer's disease (AD) pathology] remains ambiguous. We combined immunohistochemistry and stereological sampling to quantify the distribution of plaques and p-p38(MAPK)-immunoreactive (IR) cells in the sensorimotor cortex of 3-, 6- and 10-month-old TgCRND8 mice. The aggressive nature of the AD-related human amyloid-ß protein precursor expressed in these mice was confirmed by the appearance of both dense-core (thioflavin-S-positive) and diffuse plaques, even in the youngest mice. p-p38(MAPK)-IR cells of the sensorimotor cortex were predominantly co-immunoreactive for the Macrophage-1 (CD11b/CD18) microglial marker. These p-p38(MAPK)-IR microglia were associated with both dense-core and diffuse plaques, but the expected age-dependent increase in the density of plaque-associated p-p38(MAPK)-IR microglia was restricted to dense-core plaques. Furthermore, the density of dense-core plaque-associated p-p38(MAPK)-IR microglia was inversely correlated with the size of the core within the given plaque, which supports a role for these microglia in restricting core growth. p-p38(MAPK)-IR microglia were also observed throughout wildtype and TgCRND8 mouse cortical parenchyma, but the density of these non-plaque-associated microglia remained constant, regardless of age or genotype. We conclude that the constitutive presence of p-p38(MAPK)-IR microglia in aging mouse brain is indicative of a longitudinal role for this kinase in normal brain physiology. We suggest that this fact, as well as the fact that a pool of p-p38(MAPK)-IR microglia appears to restrict ß-amyloid plaque core development, needs to be duly considered when ascribing functions for p38(MAPK) signalling in the AD brain.

Dephosphorylation of Akt in C6 cells grown in serum-free conditions corresponds with redistribution of p85/PI3K to the nucleus.

Withdrawal of serum from cell cultures constitutes a useful model for the study of mechanisms involved in the regulation of Akt function in vitro. However, there have been several reports of changes in Akt activity that are not fully explained by the current model of phosphatidylinositol 3'-kinase (PI3K)/Akt signaling. We demonstrate the expected loss of Akt phosphorylation in C6 glioma cells cultured in serum-free conditions, yet we also observed a paradoxical increase in PI3K-lipid kinase activity in the same cultures. These events corresponded with relocalization of p85, the regulatory subunit of PI3K, to the perinuclear region and a local increase in PI3K-lipid kinase products. Treatment with platelet-derived growth factor (PDGF) maintained the association between p85 and the PDGF receptor during serum withdrawal and restored PI3K-lipid production at the plasma membrane. Although this protected Akt from dephosphorylation, it only slightly reversed cell-cycle arrest. These effects were not sensitive to treatment with epidermal growth factor, thus precluding a generalized role for growth factors. Our data suggest that loss of growth factor signaling, including PDGF signaling, may disrupt recruitment and/or anchoring of an active p85(PI3K) complex at the plasma membrane during serum withdrawal, which could account for the concurrent loss of Akt function.

Lacunar-canalicular network in femoral cortical bone is reduced in aged women and is predominantly due to a loss of canalicular porosity.

The lacunar-canalicular network (LCN) of bone contains osteocytes and their dendritic extensions, which allow for intercellular communication, and are believed to serve as the mechanosensors that coordinate the processes of bone modeling and remodeling. Imbalances in remodeling, for example, are linked to bone disease, including fragility associated with aging. We have reported that there is a reduction in scale for one component of the LCN, osteocyte lacunar volume, across the human lifespan in females. In the present study, we explore the hypothesis that canalicular porosity also declines with age. To visualize the LCN and to determine how its components are altered with aging, we examined samples from young (age: 20-23 y; n = 5) and aged (age: 70-86 y; n = 6) healthy women donors utilizing a fluorescent labelling technique in combination with confocal laser scanning microscopy. A large cross-sectional area of cortical bone spanning the endosteal to periosteal surfaces from the anterior proximal femoral shaft was examined in order to account for potential trans-cortical variation in the LCN. Overall, we found that LCN areal fraction was reduced by 40.6% in the samples from aged women. This reduction was due, in part, to a reduction in lacunar density (21.4% decline in lacunae number per given area of bone), but much more so due to a 44.6% decline in canalicular areal fraction. While the areal fraction of larger vascular canals was higher in endosteal vs. periosteal regions for both age groups, no regional differences were observed in the areal fractions of the LCN and its components for either age group. Our data indicate that the LCN is diminished in aged women, and is largely due to a decline in the canalicular areal fraction, and that, unlike vascular canal porosity, this diminished LCN is uniform across the cortex.

4-Methoxytranylcypromine, a monoamine oxidase inhibitor: effects on biogenic amines in rat brain following chronic administration.

4-Methoxytranylcypromine (MeOTCP), a ring-substituted analogue of the monoamine oxidase (MAO)-inhibiting antidepressant tranylcypromine (TCP), was investigated in the rat after chronic (28-day) administration and the results compared with those observed with TCP using equimolar doses of both drugs. At the dose tested, MeOTCP produced a greater inhibition of type A MAO in brain, liver, and heart than did TCP. Both drugs caused a reduction in the specific binding to beta-adrenergic and tryptamine receptors in cortex from brain. MeOTCP produced a marked increase in 5-hydroxytryptamine levels in pons-medulla, hypothalamus, and hippocampus relative to values in vehicle-treated rats and also produced a significant increase in these levels over those observed in the TCP-treated rats.

Beta-adrenoceptors and antidepressants: possible 2-phenylethylamine mediation of chronic phenelzine effects.

Effects of chronic administration of antidepressant drugs on beta-adrenoceptor function were assessed. Tricyclics (imipramine 30 mg/kg/day, desipramine 5 and 10 mg/kg/day) and monoamine oxidase inhibitors [(+/-)-tranylcypromine 1 mg/kg/day, phenelzine 5 and 10 mg/kg/day] were administered to Male Sprague-Dawley rats (n = 8), via Alzet 2ML2 osmotic minipumps for 28 days. Pumps were implanted subcutaneously in the interscapular region and replaced after 14 days. On days 21 and 22 motor-suppressant actions of the beta-adrenoceptor agonist salbutamol (3 mg/kg intraperitoneally [IP]) were assessed as a measure of beta-adrenergic receptor sensitivity. On day 28 the animals were killed and their brains used for measurement of drug levels and monoamine oxidase activity. Liver tissue was used to measure the trace amine 2-phenylethylamine. Each drug induced a decrease in the response to salbutamol. With phenelzine the decreased response to salbutamol was not observed at the lower dose. Differences in monoamine oxidase inhibition following phenelzine did not correspond to differential effects on functional beta-adrenergic sensitivity. Levels of 2-phenylethylamine, an endogenous amine that is also a metabolite of phenelzine, were significantly higher in the 10-mg/kg/day phenelzine group. These data suggest that 2-phenylethylamine may be one mediator of the chronic actions of phenelzine on beta-adrenoceptors.

A gas chromatographic procedure for separation and quantitation of the enantiomers of the antidepressant tranylcypromine.

A novel assay procedure has been developed that allows for the separation and quantification of the enantiomers of the monoamine oxidase inhibitor tranylcypromine (TCP) in brain and liver of rats. The analytical method involves extraction of the drug from rat tissue with an organic solvent. TCP is then derivatized with S-(-)-N-(trifluoroacetyl)-prolyl chloride to allow gas chromatographic analysis of the resulting diastereoisomers. Conditions for analysis by a gas chromatograph equipped with a nitrogen-phosphorus detector and a capillary column are described. The method has been applied to the separation and quantification of the enantiomers of TCP in samples of brain and liver of rats that had been injected with this drug alone and after pretreatment with iprindole, a drug known to block aromatic ring hydroxylation.

Trace amines in hepatic encephalopathy.

In 1971 Fischer and Baldessarini proposed the hypothesis that hepatic encephalopathy (HE), a neuropsychiatric syndrome associated with hepatic dysfunction, could result from the direct decarboxylation of amino acids leading to trace amines such as tyramine and octopamine which could then act as false neurotransmitters. This was supported by the observation that the clinical symptoms of HE appeared to improve following treatment with L-Dopa, which cannot be metabolized to either of these trace amines. In addition to serum and urine levels of octopamine correlating roughly with the grade of clinical HE, levels of octopamine were also significantly increased in rat brain following coma induced by hepatic devascularization and in portacaval-shunted rats fed high aromatic amino acid content diets. This hypothesis was questioned, however, given the lack of observable adverse behavioural effects following treatments with octopamine. Finally, the equivocal results of a limited number of clinical trials (using L-Dopa) argued against a direct intervention by catecholamine-like trace amines in HE. An alternative hypothesis was advanced by Sourkes in 1978 implicating increased tryptophan metabolism as a factor in the etiology of HE. Hepatic dysfunction in humans alters CNS concentrations of tryptophan which correlate well with levels of the tryptamine metabolite indoleacetic acid (IAA). Furthermore, regional densities of [3H]tryptamine receptors in HE patient brain tissue are significantly decreased. These data support a pathophysiologic role for tryptophan and its neuroactive trace amine metabolite tryptamine in HE.

A high-affinity [3H]tryptamine binding site in human brain.

In vitro filtration binding revealed high-affinity specific [3H]tryptamine binding sites in human brain. These binding sites are heterogeneously distributed throughout brain, ranging from 280 fmol/mg protein in hippocampus and thalamus to approximately 90 fmol/mg protein in medulla oblongata and cerebellum. Preliminary autoradiographic studies indicate a heterogeneous distribution within layers of the frontal cortex. The observed stereoselectivity of the site, the interaction of the site with a G protein and the observed region-selective downregulation of the site in a human pathological condition, i.e. hepatic encephalopathy (Mousseau et al., 1994), suggests that this binding site is a functional [3H]tryptamine receptor. A similarity in kinetics and distribution of the [3H]tryptamine receptor in human and rat brain indicates that these two entities represent homologous structures, although the difference in pharmacological profiles suggests species variants. One cannot exclude the possibility that the rat and human [3H]tryptamine receptors do represent distinct subtypes. Finally, the suggested role for tryptamine in neuropsychiatric disorders as originally suggested by Dewhurst (1968) is supported by the present series of experiments.

Identification of a novel receptor mediating substance P-induced behavior in the mouse.

To determine whether opioid receptors or the more recently characterized naloxone-sensitive substance P (SP) N-terminal binding sites play a role in desensitization to the behavioral effects of SP, we assessed the effects of selective antagonists at mu-(naloxonazine and beta-funaltrexamine), delta- (naltrindole) and kappa- (nor-binaltorphimine) opioid receptors, as well as the effect of [D-Pro2,D-Leu7]SP-(1-7) D-SP-(1-7) (D-SP (1-7)), an inhibitor of [3H]SP-(1-7) binding, on behaviors induced by intrathecally administered SP in mice. Whereas naloxone, a non-selective opioid antagonist, inhibited the development of behavioral desensitization to SP, the response to repeated SP administration remained unaffected by pretreatment with selective opioid antagonists. Like naloxone, however, the SP-(1-7) antagonist inhibited SP-induced desensitization. The protection against desensitization to SP by D-SP-(1-7), but not by selective antagonists of mu, delta or kappa receptors, suggests that desensitization to the behavioral effects of SP does not appear to be mediated by an action at an opioid receptor but by an action at the SP-(1-7) binding site.

Vesicular dysfunction during experimental thiamine deficiency is indicated by alterations in dopamine metabolism.

Experimental and clinical studies indicate that catecholamines play an important role in the neurobehavioural symptomatology of thiamine deficiency. Given the cerebral region-selective vulnerability and the behavioural impairment commonly encountered in thiamine deficiency, we undertook to investigate regional catecholamine metabolism in the brains of pyrithiamine-induced thiamine-deficient rats. Dopamine metabolism was unaffected in the striatum. In contrast, other regions also known to be involved in sensory processing and intellectual function (e.g., frontal cortex, hypothalamus, thalamus), but having a greater noradrenergic input, had increased levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and decreased levels of other dopaminergic metabolites including noradrenaline. In these regions levels of the vesicular amine transporter, defined by tetrabenazine-sensitive [3H]ketanserin binding, were also decreased. Our data suggest a region-selective vesicular dysfunction resulting in intraneuronal release, and subsequent degradation, of dopamine. These disruptions of dopamine and consequently noradrenaline metabolism may account for certain neurobehavioural deficits commonly encountered in thiamine deficiency.

Na+,K(+)-ATPase activity is selectively increased in thalamus in thiamine deficiency prior to the appearance of neurological symptoms.

The relationship between progression of neurological status and the activities of both Na+,K(+)- and Mg(2+)-dependent-ATPase (adenosine 5'-triphosphate phosphohydrolase) was investigated in brain regions of pyrithiamine-induced thiamine deficient rats. Thalamic Na+,K(+)-ATPase activity was selectively increased by 200% (P < 0.01) prior to the appearance of symptoms of thiamine deficiency and normalized in symptomatic rats. This selective transitory activation precludes a mediation by brain soluble fraction Na+,K(+)-ATPase modifiers as does the unaltered distribution in regional high-affinity [3H]ouabain binding densities observed throughout the time-course used in these experiments. Na+,K(+)-ATPase maintains cellular ionic gradients and has been implicated in neurotransmitter uptake and release mechanisms. The fact that the increased thalamic Na+,K(+)-ATPase activity coincides with the early alterations in serotonin metabolism observed in similarly treated animals and the concomitantly early increase in glucose utilization previously observed in the thalamus of thiamine-deficient rats is discussed.

Selective loss of pallidal dopamine D2 receptor density in hepatic encephalopathy.

The binding parameters of [3H]SCH 23390 and [3H]spiperone (radioligands for dopamine D1 and D2 receptors, respectively) were investigated in autopsied frontal cortex, caudate nucleus and globus pallidus/putamen of cirrhotic patients who died in hepatic coma as well as in age- and sex-matched controls. Specific [3H]SCH 23390 binding site densities were unchanged in all regions; in contrast, specific [3H]spiperone binding site density was decreased (by 44%, P < 0.001) in the globus pallidus/putamen of patients with HE. Decreased densities of pallidal D2 binding sites could relate to the motor dysfunctions commonly encountered in human HE.

Nitric oxide synthase activities are selectively decreased in vulnerable brain regions in thiamine deficiency.

Pyrithiamine-induced thiamine deficiency in the rat exhibits many neuropathological and biochemical similarities to Wernicke's Encephalopathy in human. Activities of constitutive nitric oxide synthase (NOS) were measured in vulnerable (thalamus and cerebellum) and non-vulnerable (hippocampus and striatum) brain regions of pyrithiamine-induced thiamine-deficient rats. NOS activities were significantly decreased in the thalamus (by 26%, P < 0.05) of presymptomatic thiamine-deficient rats compared to pair-fed controls. Following onset of symptoms, in addition to thalamus (-38%, P < 0.01), cerebellum (-50%, P < 0.01) also manifested significantly decreased activities of NOS. Hippocampal and striatal activities of NOS were unchanged at both presymptomatic and symptomatic stages of thiamine deficiency. Selectively decreased activities of neuronal NOS in the thalamus and the cerebellum extends the previous observations of region-selective metabolic changes and, ultimately, neuronal cell loss observed in thiamine deficiency.

Ring-substituted analogues of tranylcypromine as monoamine oxidase inhibitors.

4-Fluorotranylcypromine and 4-methoxytranylcypromine, in which the 4-position of the phenyl ring is protected from metabolic ring hydroxylation, were tested for their ability to inhibit, relative to tranylcypromine, monoamine oxidase (MAO) -A and -B in rat brain after administration of low doses (1.2 and 3.7 mumol/kg) of the drugs. One hour after intraperitoneal injection of the lower dose, tranylcypromine was weaker than 4-fluorotranylcypromine and 4-methoxytranylcypromine at inhibiting MAO-A. After long-term (28 day) administration of a dose of 3.7 mumol/kg/day (administered via osmotic minipumps), 4-fluorotranylcypromine had a slightly stronger inhibitory effect on MAO-B than did the other two drugs. At the same time and dose both 4-substituted analogues were slightly more potent inhibitors of MAO-A than was tranylcypromine. After 28 days of administration at a daily dose of 1.2 mumol/kg/day, both analogues produced greater inhibition of MAO-A and -B than did tranylcypromine. 4-Methoxytranylcypromine and 4-fluorotranylcypromine were similar in their extent of inhibition of MAO-B but the former was more potent than the latter at inhibition MAO-A.

Monoamine oxidase inhibitors: effects on tryptophan concentrations in rat brain.

It has been suggested that inhibition of tryptophan (Trp) pyrrolase and a subsequent elevation of brain Trp may contribute to the actions of antidepressant drugs. In our laboratories, we have conducted a series of experiments measuring brain Trp levels in the rat after both acute and chronic administration of several monoamine oxidase (MAO) inhibitors. The drugs studied during the course of the long-term (28 day) experiments were phenelzine, N2-acetylphenelzine, tranylcypromine, 4-fluorotranylcypromine, 4-methoxytranylcypromine and (-)-deprenyl. High-pressure liquid chromatography with electrochemical detection was employed to measure Trp levels in brains of both MAO inhibitor- and vehicle-treated animals. No significant increases in brain Trp levels were observed as a consequence of MAO inhibitor treatment. Acute time-response (up to 24 h) and dose-response studies were conducted following the administration of phenelzine and tranylcypromine. Only after administration of high doses of these drugs was an elevation in brain Trp observed and the increase was relatively short-lived. These results suggest that elevation of brain Trp may be an important factor in the actions of MAO inhibitors only at high doses of these drugs.

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1.

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.

Haloperidol induces apoptosis via the sigma2 receptor system and Bcl-XS.

Toxicity of the typical antipsychotic haloperidol (HAL) comprises an apoptotic component that we link to pro-apoptotic Bcl-XS in PC12 preneuronal and N2a neuroblastoma cells. The mitochondrial translocation of Bcl-XS and its interaction with the pore-forming voltage-dependent anion channel (VDAC) correlates with the redistribution of cytochrome c and the cleavage of Poly(ADP-ribose) polymerase. Haloperidol-induced apoptosis is mediated by the sigma2 (sigma2) receptor system and does not involve the expected antagonism of the dopamine D(2) receptor, nor is it influenced by Vitamin E- or p53/Bax-mediated events. Pathological relevance is demonstrated by the cytotoxic synergism between HAL and the Alzheimer disease-related peptide beta-amyloid(1-40), which correlates with Bcl-XS expression and its interaction with VDAC, and with cytosolic cytochrome c translocation. These data provide for a unique apoptotic mechanism that could underscore the clinical risks associated with HAL, particularly following chronic regimens or in the elderly.

Tryptamine: a metabolite of tryptophan implicated in various neuropsychiatric disorders.

Although early interest in the biomedical relevance of tryptamine has waned in recent years, it is clear from the above discussion that the study of tryptamine is worthy of serious consideration as a factor in neuropsychiatric disorders. The study of [3H]-tryptamine binding sites indicates an adaptive responsiveness characteristic of functional receptors. The question raised by Jones (1982d) on whether tryptamine is acting centrally as a neurotransmitter or a neuromodulator still remains mostly unanswered, although the evidence cited within this review strongly suggests a modulatory role for this neuroactive amine (see also Juorio and Paterson, 1990). The synthesis and degradative pathways of tryptamine, as well as the intricate neurochemical and behavioral consequences of altering these pathways, are now more fully understood. It is not yet clear what the role of tryptamine is under normal physiological [homeostatic] conditions, however, its role during pathological conditions such as mental and physical stress, hepatic dysfunction and other disorders of metabolism (i.e. electrolyte imbalance, increased precursor availability, enzyme induction or alterations in enzyme co-factor availability) may be quite subtle, perhaps accounting for various sequelae hitherto considered idiopathic. The evidence for a primary role for tryptamine in the etiology of mental or neurological diseases is still relatively poor, although the observations that endogenous concentrations of tryptamine are particularly susceptible to pharmacological as well as physiological manipulations serve to reinforce the proposition that this indoleamine is not simply a metabolic accident but rather a neuroactive compound in its own right. Finally, one might wonder what proportion of the data attributed to modifications of 5-HT metabolism might, in fact, involve unrecognized changes in the concentrations of other neuroactive metabolites of tryptophan such as tryptamine.

The [3H]tryptamine receptor in human brain: kinetics, distribution, and pharmacologic profile.

The kinetics and distribution of [3H]tryptamine binding sites in human brain were investigated. Specific [3H]tryptamine binding in frontal cortex was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. A heterogeneous distribution for this binding site was demonstrated, with the highest density observed in hippocampus, thalamus >> caudate nucleus, frontal cortex, pons, temporal cortex > globus pallidus/putamen, cerebellum. The similarities in kinetics and distribution of the [3H]tryptamine binding site in human and rat brain indicate that these two binding sites represent homologous structures. However, the present displacement studies using various ligands (indoleamines and other tryptophan metabolites, phenylethylamines, and miscellaneous drugs) and salts (Na+, K+, Ca2+, Mg2+, Cu2+) indicate stereospecific displacement as well as a rank-order potency profile that is different from that reported for the rat [3H]tryptamine binding site. This suggests the presence of distinct species-dependent [3H]tryptamine binding site subtypes. Taken together with the documented electrophysiological and behavioral evidence of tryptamine-mediated effects in the rat and the recent report of a significant loss of these binding sites in human portal systemic encephalopathy, as well as the present demonstration of an effect of guanine nucleotides on [3H]-tryptamine binding affinity, these findings suggest that these binding sites might be functional receptors. The implied role of tryptamine in neuropsychiatric disorders is supported by this demonstration of a receptor for [3H]-tryptamine in human brain.