Extra-nuclear estrogen receptor GPR30 regulates serotonin function in rat hypothalamus.

Selective serotonin reuptake inhibitors (SSRIs), such as Prozac, are used to treat mood disorders. SSRIs attenuate (i.e. desensitize) serotonin 1A (5-HT(1A)) receptor signaling, as demonstrated in rats through decreased release of oxytocin and adrenocorticotropin hormone (ACTH) following 5-HT(1A) receptor stimulation. Maximal therapeutic effects of SSRIs for treatment of mood disorders, as well as effects on hypothalamic 5-HT(1A) receptor signaling in animals, take 1 to 2 weeks to develop. Estradiol also attenuates 5-HT(1A) receptor signaling, but, in rats, these effects occur within 2 days; thus, estrogens or selective estrogen receptor modulators may serve as useful short-term tools to accelerate desensitization of 5-HT(1A) receptors in response to SSRIs if candidate estrogen receptor targets in the hypothalamus are identified. We found high levels of GPR30, which has been identified recently as a pertussis-toxin (PTX) sensitive G-protein-coupled estrogen receptor, in the hypothalamic paraventricular nucleus (PVN) of rats. Double-label immunohistochemistry revealed that GPR30 co-localizes with 5-HT(1A) receptors, corticotrophin releasing factor (CRF) and oxytocin in neurons in the PVN. Pretreatment with PTX to the PVN before peripheral injections of 17-beta-estradiol 3-benzoate completely prevented the reduction of the oxytocin response to the 5-HT(1A) receptor agonist, (+)-8-hydroxy-2-dipropylaminotetralin (DPAT). Treatment with the selective GRP30 agonist, G-1, attenuated 5-HT(1A) receptor signaling in the PVN as measured by an attenuated oxytocin (by 29%) and ACTH (by 31%) response to DPAT. This study indicates that a putative extra-nuclear estrogen receptor, GPR30, may play a role in estradiol-mediated attenuation of 5-HT(1A) receptor signaling, and potentially in accelerating the effects of SSRIs in treatment of mood disorders.

Transglutaminase-catalyzed transamidation: a novel mechanism for Rac1 activation by 5-hydroxytryptamine2A receptor stimulation.

Transglutaminase (TGase)-induced activation of small G proteins via 5-hydroxytryptamine (HT)(2A) receptor signaling leads to platelet aggregation (Cell 115:851-862, 2003). We hypothesize that stimulation of 5-HT(2A) receptors in neurons activates TGase, resulting in transamidation of serotonin to a small G protein, Rac1, thereby constitutively activating Rac1. Using immunoprecipitation and immunoblotting, we show that, in rat cortical cell line A1A1v, serotonin increases TGase-catalyzed transamidation of Rac1. This transamidation occurs in both undifferentiated and differentiated cells. Treatment with a 5-HT(2A/2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine, but not the 5-HT(1A) receptor agonist 5-hydroxy-2-dipropylamino tetralin, increases transamidation of Rac1 by TGase. In A1A1v cells, 5-HT(2A) receptors mediate the transamidation reaction because expression of 5-HT(2C) receptors was not detectable and the selective 5-HT(2A) receptor antagonist blocked transamidation. Time course studies demonstrate that transamidation of Rac1 is significantly elevated after 5 and 15 min of serotonin treatment, but returns it to control levels after 30 min. The activity of Rac1 is also transiently increased following serotonin stimulation. Inhibition of TGase by cystamine or small interfering RNA reduces TGase modification of Rac1, and cystamine also prevents Rac1 activation. Serotonin itself is bound to Rac1 by TGase following 5-HT(2A) receptor stimulation as demonstrated by coimmunoprecipitation experiments and a dose-dependent decrease of serotonin-associated Rac1 by cystamine. These data support the hypothesis that Rac1 activity is transiently increased due to TGase-catalyzed transamidation of serotonin to Rac1 via stimulation of 5-HT(2A) receptors. Activation of Rac1 via TGase is a novel effector and second messenger of the 5-HT(2A) receptor-signaling cascade in neurons.

Chronic olanzapine activates the Stat3 signal transduction pathway and alters expression of components of the 5-HT2A receptor signaling system in rat frontal cortex.

The mechanisms underlying desensitization of serotonin 2A (5-HT(2A)) receptor signaling by antagonists are unclear but may involve changes in gene expression mediated via signal transduction pathways. In cells in culture, olanzapine causes desensitization of 5-HT(2A) receptor signaling and increases the levels of regulators of G protein signaling (RGS) 7 protein dependent on phosphorylation/activation of the Janus kinase 2 (Jak2)/signal transducers and activators of transcription 3 (Stat3) signaling pathway. In the current study, the 5-HT(2A) receptor signaling system in rat frontal cortex was examined following 7 days of daily treatment with 0.5, 2.0 or 10.0 mg/kg i.p. olanzapine. Olanzapine increased phosphorylation of Stat3 in rats treated daily with 10 mg/kg olanzapine and caused a dose-dependent desensitization of 5-HT(2A) receptor-mediated phospholipase C activity. There were dose-dependent increases in the levels of membrane-associated 5-HT(2A) receptor, G(alpha11) and G(alphaq) protein levels but no changes in the G(beta) protein levels. With olanzapine treatment, RGS4 protein levels increase in the membrane-fraction and decrease in the cytosolic fraction by similar amounts suggesting a redistribution of RGS4 protein within neurons. RGS7 protein levels increase in both the membrane and cytosolic fractions in rats treated daily with 10mg/kg olanzapine. The olanzapine-induced increase in Stat3 activity could underlie the increase in RGS7 protein expression in vivo as previously demonstrated in cultured cells. Furthermore, the increases in membrane-associated RGS proteins could play a role in desensitization of signaling by terminating the activated G(alphaq/11) proteins more rapidly.

Cocaine-mediated supersensitivity of 5-HT2A receptors in hypothalamic paraventricular nucleus is a withdrawal-induced phenomenon.

We previously reported that treatment and withdrawal from cocaine increases: (1) 5-HT2A receptor-mediated neuroendocrine responses, and (2) Galphaq and Galpha11 G-protein levels in the hypothalamic paraventricular nucleus (PVN) at 48 h post-treatment. This study investigates changes in the initial 24 h of withdrawal to discern whether 5-HT2A receptor supersensitivity is due to cocaine treatment or is induced during the withdrawal period. We report here increases in 5-HT2A receptor-mediated neuroendocrine responses only 12 or 24 h post-treatment, but not during the initial 4 h withdrawal period. Levels of membrane- or cytosol-associated Galphaq or Galpha11 proteins in PVN are not altered during the first 24 h of withdrawal. However, the density of 125I-(-)-1-(2,5 dimethoxy-4-iodophenyl)-2-amino-propane HCl (DOI)-labeled high-affinity 5-HT2A receptors in PVN increased 35% in rats withdrawn from cocaine for 24 h. These findings demonstrate that cocaine-induced increases in 5-HT2A receptor function in PVN represents a withdrawal-induced phenomena that: (1) is likely attributed to increased G-protein coupled/high-affinity conformational state of the 5-HT2A receptor, and (2) occurs in the absence of changes in the levels of associated G proteins during the first 24 h.

Evidence that G(z)-proteins couple to hypothalamic 5-HT(1A) receptors in vivo.

Using in situ hybridization and immunoblot analysis, the present studies identified G(z) mRNA and G(z)-protein in the hypothalamic paraventricular nucleus. The role of G(z)-proteins in hypothalamic 5-HT(1A) receptor signaling was examined in vivo. Activation of 5-HT(1A) receptors increases the secretion of oxytocin and ACTH, but not prolactin. Intracerebroventricular infusion (3-4 d) of G(z) antisense oligodeoxynucleotides, with different sequences and different phosphorothioate modification patterns, reduced the levels of G(z)-protein in the hypothalamic paraventricular nucleus, whereas missense oligodeoxynucleotides had no effect. Neither antisense nor missense oligodeoxynucleotide treatment altered basal plasma levels of ACTH, oxytocin, or prolactin, when compared with untreated controls. An antisense-induced decrease in hypothalamic G(z)-protein levels was paralleled by a significant decrease in the oxytocin and ACTH responses to the 5-HT(1A) agonist 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT). In contrast, the prolactin response to 8-OH-DPAT (which cannot be blocked by 5-HT(1A) antagonists) was not inhibited by G(z) antisense oligodeoxynucleotides. G(z)-proteins are the only members of the G(i)/G(o)-protein family that are not inactivated by pertussis toxin. In a control experiment, pertussis toxin treatment (1 microgram/5 microliter, i.c.v.; 48 hr before the 8-OH-DPAT challenge) did not inhibit the ACTH response, potentiated the oxytocin response, and eliminated the prolactin response to 8-OH-DPAT. Thus, pertussis toxin-sensitive G(i)/G(o)-proteins do not mediate the 5-HT(1A) receptor-mediated increase in ACTH and oxytocin secretion. Combined, these studies provide the first in vivo evidence for a key role of G(z)-proteins in coupling hypothalamic 5-HT(1A) receptors to effector mechanisms.

Characterization of the functional heterologous desensitization of hypothalamic 5-HT(1A) receptors after 5-HT(2A) receptor activation.

Desensitization of 5-HT(1A) receptors could be involved in the long-term therapeutic effect of anxiolytic and antidepressant drugs. Pretreatment of rats with the 5-HT(2A/2C) agonist DOI induces an attenuation of hypothalamic 5-HT(1A) receptor-G(z)-protein signaling, measured as the ACTH and oxytocin responses to an injection of the 5-HT(1A) agonist 8-OH-DPAT. We characterized this functional heterologous desensitization of 5-HT(1A) receptors in rats and examined some of the mechanisms that are involved. A time course experiment revealed that DOI produces a delayed and reversible reduction of the ACTH and oxytocin responses to an 8-OH-DPAT challenge. The maximal desensitization occurred at 2 hr, and it disappeared 24 hr after DOI injection. The desensitization was dose-dependent, and it shifted the oxytocin and ACTH dose-response curves of 8-OH-DPAT to the right (increased ED(50)) with no change in their maximal responses (E(max)). The 5-HT(2A) receptor antagonist MDL 100,907 prevented the DOI-induced desensitization, indicating that 5-HT(2A) receptors mediate the effect of DOI. Analysis of the components of the 5-HT(1A) receptor-G(z)-protein signaling system showed that DOI did not alter the level of membrane-associated G(z)-proteins in the hypothalamus. Additionally, DOI did not alter the binding of [(3)H]8-OH-DPAT or the inhibition by GTPgammaS of [(3)H]8-OH-DPAT binding in the hypothalamus. In conclusion, the activation of 5-HT(2A) receptors induces a transient functional desensitization of 5-HT(1A) receptor signaling in the hypothalamus, which may occur distal to the 5-HT(1A) receptor-G(z)-protein interface.

Transglutaminase-induced cross-linking of tau proteins in progressive supranuclear palsy.

The mechanisms leading to the abnormal self-polymerization of tau into straight and paired helical filaments (PHFs) and neurofibrillary tangles (NFT) in Alzheimer disease (AD) and progressive supranuclear palsy (PSP) are not known. However, transglutaminase-induced cross-linking of PHF-tau was observed in AD and thus may also contribute to the formation of NFT in other neurodegenerative disorders including PSP. Tissue homogenates from PSP and normal age-matched controls were used to immunoaffinity-purify proteins containing transglutaminase-induced epsilon-(gamma-glutamyl) lysine cross-links. The immunoaffinity-purified proteins were then examined on immunoblots with a PHF-tau antibody, PHF-1. There were significantly higher levels of epsilon-(gamma-glutamyl) lysine cross-linking of PHF-tau in globus pallidus and pons regions of PSP cases compared to barely detectable cross-links in controls. The occipital cortex, an area spared from neurofibrillary pathology in PSP, showed no detectable cross-linking of PHF-tau protein in either PSP cases or control cases. Double-label immunofluorescence demonstrated the colocalization of the cross-link and PHF-tau in NFT in pons of PSP Previous studies and present data are consistent with the hypothesis that transglutaminase-induced cross-linking may be a factor contributing to the abnormal polymerization and stabilization of tau in straight and PHFs leading to neurofibrillary tangle formation in neurodegenerative diseases, including PSP and AD.

Treatment of cycling female rats with fluoxetine induces desensitization of hypothalamic 5-HT(1A) receptors with no change in 5-HT(2A) receptors.

Although women constitute the majority of patients who receive treatment with selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, most animal studies of SSRIs are conducted on males. The present study investigated whether long-term treatment of cycling female rats with fluoxetine alters their estrous cycle and the sensitivity of hypothalamic serotonin (5-HT) 5-HT(1A) and 5-HT(2A) receptor systems. Adult female rats received daily injections of fluoxetine (10 mg/kg, i.p.) for three consecutive estrous cycles (15.2+/-0.2 days) with the first injection beginning on metestrus (when circulating estrogen levels are low and stable). Fluoxetine did not alter basal plasma estradiol levels at metestrus, nor did it alter the pattern of estrous cyclicity. Rats treated with fluoxetine showed a loss in body weight. On the morning of metestrus of the fourth cycle (18 h after the last fluoxetine injection), the rats were injected with a sub-maximal dose of the 5-HT(1A) agonist (+/-)-8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT, 50 MICRO/kg, s.c.) or a maximal dose of the 5-HT(2A) agonist [(+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] (DOI). Plasma levels of oxytocin, ACTH and corticosterone were measured as peripheral indicators of hypothalamic 5-HT(1A) and 5-HT(2A) receptor sensitivity. Injecting 8-OH-DPAT to saline pretreated rats produced a significant increase in plasma oxytocin (299%), ACTH (1456%) and corticosterone (170%) levels but not in plasma prolactin or renin concentrations. Greater increases in plasma levels of these hormones were observed after injecting DOI. Fluoxetine treatment completely blocked the oxytocin, ACTH and corticosterone responses to 8-OH-DPAT, but did not inhibit the effect of DOI on any hormone, thus confirming that fluoxetine treatment did not produce a deficit in the functioning of corticotropin releasing hormone or oxytocin containing neurons. These results indicate that in cycling female rats, fluoxetine treatment desensitizes hypothalamic post-synaptic 5-HT(1A) receptor signaling. Understanding the pharmacological effects of fluoxetine in females may lead to more effective treatment of women with mood disorders.

Estrogen desensitizes 5-HT(1A) receptors and reduces levels of G(z), G(i1) and G(i3) proteins in the hypothalamus.

The present study investigated whether estrogen would desensitize hypothalamic serotonin(1A) (5-HT(1A)) receptors by examining the neuroendocrine response to 8-OH-DPAT, a 5-HT(1A) agonist. Rats were ovariectomized, allowed to recover for 5 days, then given 2 daily injections of estradiol benzoate or vehicle (10 microg/day, s.c.). Twenty-four hours after the second injection, rats were challenged with a sub-maximal dose of 8-OH-DPAT (50 microg/kg, sc) or saline 15 min prior to sacrifice. 8-OH-DPAT produced a significant increase in plasma oxytocin, ACTH and corticosterone levels in ovariectomized rats. While estrogen treatment for 2 days did not alter basal hormone levels, it did significantly reduce the magnitude of oxytocin, ACTH and corticosterone responses to 8-OH-DPAT. The reduction in hormone responses was accompanied by a significant reduction in hypothalamic levels of G(z), G(i1) and G(i3) proteins (by 50%, 30% and 50%, respectively). These findings suggest that a reduction in these G proteins may contribute to the mechanisms underlying estrogen-induced desensitization of 5-HT(1A) receptors. The desensitization of 5-HT(1A) receptors has been suggested to underlie the therapeutic effects of antidepressant 5-HT uptake inhibitors (SSRIs). Thus, the present results suggest that estrogen or estrogen-like substances in combination with SSRIs may prove effective in developing novel therapeutic strategies for neuropsychiatric disorders in women.

Estrogen treatment increases the levels of regulator of G protein signaling-Z1 in the hypothalamic paraventricular nucleus: possible role in desensitization of 5-hydroxytryptamine1A receptors.

Desensitization of post-synaptic serotonin1A (5-HT1A) receptors may underlie the clinical improvement of neuropsychiatric disorders. In the hypothalamic paraventricular nucleus, Galphaz proteins mediate the 5-HT1A receptor-stimulated increases in hormone release. Regulator of G protein signaling-Z1 (RGSZ1) is a GTPase-activating protein selective for Galphaz proteins. RGSZ1 regulates the duration of interaction between Galphaz proteins and effector systems. The present investigation determined the levels of RGSZ1 in the hypothalamic paraventricular nucleus of rats subjected to four different treatment protocols that produce desensitization of 5-HT1A receptors. These protocols include: daily administration of beta estradiol 3-benzoate (estradiol) for 2 days; daily administration of fluoxetine for 3 and 14 days; daily administration of cocaine for 7 or 14 days; and acute administration of (+/-)-1-(2,5 dimethoxy-4-iodophenyl)-2-amino-propane HCl (DOI; a 5-HT2A/2C receptor agonist). Estradiol treatment was the only protocol that increased the levels of RGSZ1 protein in the hypothalamic paraventricular nucleus in a dose-dependent manner (46%-132% over control). Interestingly, previous experiments indicate that only estradiol produces a decreased Emax of 5-HT1A receptor-stimulation of hormone release, whereas fluoxetine, cocaine and DOI produce a shift to the right (increased ED50). Thus, the desensitization of 5-HT1A receptors by estradiol might be attributable to increased levels of RGSZ1 protein. These findings may provide insight into the adaptation of 5-HT1A receptor signaling during pharmacotherapies of mood disorders in women and the well-established gender differences in the vulnerability to depression.

Tau mRNA isoforms following sciatic nerve axotomy with and without regeneration.

The microtubule-associated protein tau promotes the polymerization and stabilization of microtubules in normal neurons and is the main component of paired helical filaments, one of the pathological structures characteristic of Alzheimer's disease (AD). In adult neurons alternative splicing generates tau isoforms with 4 microtubule binding domains (4R tau) while tau in developing neurons contains only 3 such domains (3R tau). The extra microtubule binding domain confers adult tau with an increased ability to interact with and stabilize microtubules. We hypothesized that tau gene expression would revert to the developmental pattern following nerve injury. The sciatic nerve of adult rats was unilaterally crushed or transected and tau mRNA isoform expression in the spinal cord was examined by reverse transcriptase-polymerase chain reaction. At 2 and 3 days post-crush, both the 3R and 4R tau mRNA isoform levels on the injured side had decreased compared to the contralateral side. However, the ratio of 4R to 3R tau mRNA was not significantly different between the two sides at any post-crush time point examined. Following nerve transection, a significant increase in the 3R tau mRNA isoform on the transected compared to the contralateral side occurred at 14 days; the ratio of 4R to 3R tau mRNA was significantly decreased on the transected compared to the contralateral side at 7, 14 and 42 days. These results suggest that a recapitulation of the developmental pattern of 3R tau gene expression occurs following nerve transection but not nerve crush. Our results combined with the recent findings that the 3R tau protein isoform preferentially forms paired helical filament-like structures in vitro suggests that an increased expression of the 3R tau mRNA isoform may also occur in AD.

Neuronal responses to injury and aging: lessons from animal models.

Alzheimer's disease (AD), the most common type of adult-onset dementia, is characterized by a variety of brain abnormalities, including degeneration of certain populations of nerve cells, alterations in the neuronal cytoskeleton, and the abnormal deposition of amyloid within brain parenchyma. Pathogenetic processes that lead to these brain abnormalities are difficult to study in humans. Recently, investigators have begun to utilize animal models to examine some of the mechanisms that cause cellular/molecular alterations in transmitter systems, cytoskeletal elements, and APP. These investigations have helped to clarify issues related to the lesions that occur in aged humans and individuals with AD.

A sensitive and specific radioenzymatic assay for the simultaneous determination of choline and phosphatidylcholine.

A radioenzymatic procedure for the simultaneous measurement of picomol quantities of phosphatidylcholine and choline is described. Phospholipase-D from Streptomyces chromofuscus hydrolyzes phosphatidylcholine to choline and phosphatidic acid. Choline kinase in the presence of [32P]ATP phosphorylates the choline to form [32P]phosphorylcholine. The phosphorylcholine, isolated by ion exchange chromatography, is measured by scintillation spectroscopy. Using a chloroform: methanol separation, phosphatidylcholine and choline can be measured from the same sample. Different sources of phospholipase-D were compared for their ability to hydrolyze phosphatidylcholine to choline and phosphatidic acid. Phospholipase-D from Streptomyces chromofuscus was found to result in almost complete hydrolysis. As a measure of specificity, lysophosphatidylcholine and sphingomyelin were assayed using this method and were found to result in, at most, only 2% of the amount of phosphorylcholine produced compared to phosphatidylcholine. This procedure allows for the simultaneous measurement of choline and phosphatidylcholine in brain and serum samples with a very high degree of sensitivity and specificity.

Corticosteroids alter G protein inwardly rectifying potassium channels protein levels in hippocampal subfields.

Corticosterone or cortisol, stress hormones in rat and human, respectively, alter neurotransmitter receptor-mediated responses in the brain. Corticosterone could alter these responses by modifying any component of the receptor-effector pathway. Many of these receptors are linked to guanine nucleotide regulatory proteins (G proteins) which, in turn, can activate second messenger systems and/or ion channels, such as G protein inwardly rectifying potassium channels (GIRK). The aim of these experiments was to determine whether corticosterone treatment altered the levels of GIRK proteins in rat hippocampus. Corticosterone treatment selectively altered the levels of GIRK1 and GIRK2 (measured on immunoblots) depending on the subfield of the hippocampus examined. These data lend credence to the hypothesis that corticosterone differentially alters neurotransmitter receptor-mediated responses dependent on the brain area.

Corticosterone alters G protein alpha-subunit levels in the rat hippocampus.

The hypothalamic-pituitary-adrenal axis regulates the synthesis and secretion of corticosteroid hormones. The hippocampus, a component of the limbic system, contains the highest concentration of corticosteroid receptors in the brain and may play an important role in regulating hypothalamic-pituitary-adrenal axis activity and mediating physiological responses to stress. The corticosteroid hormone corticosterone alters the response elicited by activation of several different G protein-linked neurotransmitter receptors in the hippocampus. In the present study we used Western blot and immunohistochemical techniques to determine the effects of chronic adrenalectomy (ADX), low basal (CT) and high (HCT) corticosterone treatments on Gs, Gi1 and 2 and Go alpha-subunit levels and intracellular location in the rat hippocampus. CT treatment increased Gs alpha-subunit levels and HCT treatment increased the levels of Gs, Gi1 and 2 and Go alpha-subunits when compared to sham as detected on Western blots. No change in the intracellular location of the G protein alpha-subunits was detected using immunohistochemistry. Based on our results, we conclude that corticosterone alters G protein alpha-subunit levels in the rat hippocampus without altering their intracellular location. These results provide an important piece of information towards understanding how corticosteroids alter G protein-linked neurotransmitter receptor-mediated responses.

Fluoxetine gradually increases [125I]DOI-labelled 5-HT2A/2C receptors in the hypothalamus without changing the levels of Gq- and G11-proteins.

The time course of fluoxetine-induced supersensitivity of hypothalamic 5-HT2A/2C receptors was examined. Daily injections of fluoxetine (7 or 14 days) significantly increased agonist ([125I]DOI)-labeled high-affinity-state 5-HT2A/2C receptors in the hypothalamus, but not frontal cortex. No change was observed in the density of [3H]ketanscrin-labeled 5-HT2A receptors in either brain region. The levels of Gq- and G11- proteins in the hypothalamus and cortex were not altered by fluoxetine. These results suggest that fluoxetine gradually increases the G-protein coupling of 5-HT2A/2C receptors without altering the levels of Gq- or G11-proteins.

Elevated transglutaminase-induced bonds in PHF tau in Alzheimer's disease.

Transglutaminase-induced epsilon-(gamma-glutamyl)lysine bonds covalently cross-link and polymerize peptides into insoluble high molecular weight protein aggregates resistant to degradation and proteolytic digestion. We investigated the hypothesis that excessive deposition of epsilon-(gamma-glutamyl)lysine bonds is a neuropathological mechanism which induces the polymerization of tau protein into stable aggregates leading to the formation of paired helical filaments (PHFs) which deposit into neurofibrillary tangles in Alzheimer's disease (AD) brain. We demonstrate a significant (45%) elevation in epsilon-(gamma-glutamyl)lysine cross-links in AD cortex as compared to control cortex. In vivo, PHF tau, and high and medium molecular weight neurofilament proteins have significantly greater cross-linking by epsilon-(gamma-glutamyl)lysine bonds in AD brains as compared to controls. The cross-linking of PHF tau occurs both intra-molecularly and inter-molecularly. The inter-molecular cross-linking of tau could account for the formation of high molecular weight tau polymers. These results suggest that transglutaminase-induced cross-linking of tau protein could play a role in the formation and stabilization of neurofibrillary tangles. Inhibition of transglutaminase-induced cross-linking may therefore, provide a novel strategy for the treatment of AD.

Regulation of peripherin and neurofilament expression in regenerating rat motor neurons.

Northern blotting, in situ hybridization and immunocytochemistry were used to study the changes in levels of mRNA coding for peripherin and in immunoreactivity of peripherin, a type III neuronal intermediate filament, in rat spinal motor neurons following axotomy of the sciatic nerve. For comparison, parallel studies examined the biology of neurofilament (NF) proteins in this model. The sciatic nerve was crushed at the junction of the L4-L5 spinal nerves. Levels of messenger RNA (mRNA) coding for peripherin in the motor neurons doubled by 4 days postaxotomy and remained elevated for a period of 6 weeks. Within 4-7 days of injury peripherin immunoreactivity increased significantly in cell bodies of motor neurons and remained elevated through 6 weeks. In contrast, no changes were detected in NF-M immunoreactivity over the same time period. By 8 weeks postaxotomy, levels of peripherin mRNA and protein returned to control values. The increases in the expression of peripherin parallel those of beta-tubulin and actin, and these changes are quite different from the alterations in neurofilament mRNA that decrease after axotomy. The contrasting responses of peripherin and NF to nerve injury indicates that each of these intermediate filaments may play distinct roles in nerve growth and regeneration.

Aluminum neurotoxicity: altered expression of cytoskeletal genes.

To better understand perturbations of the neuronal cytoskeleton that occur in several mammalian disorders, we have focused on an animal model in which neurofibrillary pathology follows the administration of aluminum salts. In susceptible species, the injection of aluminum produces accumulations of neurofilaments (NFs) in cell bodies and proximal axons of certain populations of neurons. Mechanisms involved in the production of these abnormalities are unclear; in particular, the role of gene expression in the genesis of this type of neurofibrillary pathology has not been examined. In this study of aluminum-intoxicated rabbits, the expression of genes coding for several cytoskeletal proteins was studied in the spinal cord and dorsal root ganglia (DRG)--tissues with and without neurofibrillary pathology, respectively. In aluminum-treated rabbits, in situ hybridization using a cDNA probe demonstrated the presence of mRNA coding for the 68-kDa NF (NF-L) protein in spinal cord motor neurons with NF accumulations as well in unaffected neurons. On Northern blots, the expression of genes coding for the NF-L protein and tubulin was reduced by approximately 3.5-fold and 3-fold, respectively, in spinal cords of aluminum-intoxicated rabbits as compared to controls. On blots, levels of actin mRNA were not significantly different in spinal cords of aluminum-treated rabbits as compared to controls, but there was a trend for a slight reduction. In DRG of intoxicated animals, the expression of genes coding for these cytoskeletal proteins was not altered.

Neuronal gene expression in aluminum-induced neurofibrillary pathology: an in situ hybridization study.

Alterations in cytoskeletal proteins such as the perikaryal accumulation of neurofilaments (NFs) occur in a number of human neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis and may contribute to their debilitating effects. The administration of aluminum salts to rabbits induces the aberrant accumulation of NFs within the proximal axons and perikarya of vulnerable neurons and is one animal model which has been extensively studied in an attempt to gain insight into the mechanism(s) of NF perturbations in human disease. Previous studies using Northern blotting techniques to examine mRNA levels in the aluminum-induced neuropathy model have led to seemingly contradictory results. We have used in situ hybridization which provides the cellular resolution needed to: 1) determine whether there are generalized decreases in the levels of mRNA expression or decreases in mRNA encoding specific proteins; 2) determine whether alterations in mRNA levels occur specifically in neurons with NF accumulations; and 3) begin to resolve some of the apparent contradictions in the literature. A moderate dose of aluminum lactate administered on two consecutive days produced neurofibrillary tangles in spinal cord neurons seven days after the first dose. Polyadenylated mRNA levels were not altered in spinal cord neurons in aluminum-treated compared to saline-treated control animals or in tangle-bearing compared to non tangle-bearing neurons in aluminum-treated animals. Middle and high NF subunit (NFH) mRNA levels were not significantly different from polyadenylated mRNA levels in spinal cord neurons in aluminum-treated/control animals. NFH mRNA levels were decreased in neurons containing aluminum-induced NF accumulations. These results suggest that NFH gene expression may be down regulated by an inhibitory feedback mechanism induced by perikaryal accumulations of NFs. This inhibitory feedback regulation for NFH may have implications for neurodegenerative diseases in which NFs accumulate in neuronal perikarya.