Role of microglial amylin receptors in mediating beta amyloid (Aß)-induced inflammation.

BACKGROUND: Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer's disease (AD) pathology. Amyloid beta (Aß) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether Aß activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors. METHODS: The presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca2+]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric Aß1-42 prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain. RESULTS: Acute applications of human amylin or Aß1-42 resulted in an increase in [Ca2+]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNFα and IL-1ß, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNFα, and IL-1ß. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls. CONCLUSION: Microglial amylin receptors mediate Aß-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.

Beta amyloid-induced depression of hippocampal long-term potentiation is mediated through the amylin receptor.

Alzheimer's disease (AD) is characterized by accumulation of amyloid-ß peptide (Aß) in the brain regions that subserve memory and cognition. The amylin receptor is a potential target receptor for expression of the deleterious actions of soluble oligomeric Aß species. We investigated whether the amylin receptor antagonist, AC253, neutralizes the depressant effects of Aß(1-42) and human amylin on hippocampal long-term potentiation (LTP). Furthermore, we examined whether depressed levels of LTP observed in transgenic mice, which overexpress amyloid precursor protein (TgCRND8), could be restored with AC253. In mouse hippocampal brain slices, field EPSPs were recorded from the stratum radiatum layer of the CA1 area (cornu ammonis 1 region of the hippocampus) in response to electrical stimulation of Schaeffer collateral afferents. LTP was induced by 3-theta burst stimulation protocols. Aß(1-42) (50 nM) and human amylin (50 nM), but not Aß(42-1) (50 nM), depressed LTP evoked using both stimulation protocols. Preapplication of AC253 (250 nM) blocked Aß- and human amylin-induced reduction of LTP without affecting baseline transmission or LTP on its own. In contrast to wild-type controls, where robust LTP is observed, 6- to 12-month-old TgCRND8 mice show blunted LTP that is significantly enhanced by application of AC253. Our data demonstrate that the effects of Aß(1-42) and human amylin on LTP are expressed via the amylin receptor, and moreover, blockade of this receptor increases LTP in transgenic mice that show increased brain amyloid burden. Amylin receptor antagonists could serve as potentially useful therapeutic agents in AD.

Amyloid ß (Aß) peptide directly activates amylin-3 receptor subtype by triggering multiple intracellular signaling pathways.

The two age-prevalent diseases Alzheimer disease and type 2 diabetes mellitus share many common features including the deposition of amyloidogenic proteins, amyloid ß protein (Aß) and amylin (islet amyloid polypeptide), respectively. Recent evidence suggests that both Aß and amylin may express their effects through the amylin receptor, although the precise mechanisms for this interaction at a cellular level are unknown. Here, we studied this by generating HEK293 cells with stable expression of an isoform of the amylin receptor family, amylin receptor-3 (AMY3). Aß1-42 and human amylin (hAmylin) increase cytosolic cAMP and Ca(2+), trigger multiple pathways involving the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Aß1-42 and hAmylin also induce cell death during exposure for 24-48 h at low micromolar concentrations. In the presence of hAmylin, Aß1-42 effects on HEK293-AMY3-expressing cells are occluded, suggesting a shared mechanism of action between the two peptides. Amylin receptor antagonist AC253 blocks increases in intracellular Ca(2+), activation of protein kinase A, MAPK, Akt, cFos, and cell death, which occur upon AMY3 activation with hAmylin, Aß1-42, or their co-application. Our data suggest that AMY3 plays an important role by serving as a receptor target for actions Aß and thus may represent a novel therapeutic target for development of compounds to treat neurodegenerative conditions such as Alzheimer disease.

ß-Amyloid protein (Aß) and human amylin regulation of apoptotic genes occurs through the amylin receptor.

Deposition of amyloid-beta (Aß) protein, a 39-43 amino acid peptide, in the brain is a major pathological feature of Alzheimer's disease (AD). We have previously provided evidence that in primary cultures of rat basal forebrain and human fetal neurons (HFNs), neurotoxic effects of oligomeric Aß are expressed through the amylin receptor. In this study, we utilized RT-PCR arrays to compare RNA expression levels of 84 markers for pro and anti- apoptotic signalling pathways following exposure of HFNs to either Aß(1-42) (20 µM) or human amylin (2 µM). Oligomeric Aß(1-42) or human amylin was applied to HFNs alone or after pre-treatment of cultures with the amylin receptor antagonist, AC253. Changes in RNA levels were then quantified and compared to each other in order to identify increases or decreases in gene expression of apoptotic markers. Applications of Aß(1-42) or human amylin, but not the inactive inverse sequence Aß(42-1) or rat amylin, resulted in a time-dependent marked increase in mediators of apoptosis including a 10- to 30-fold elevations in caspases 3, 6, 9, BID and XIAP levels. Amylin receptor antagonists, AC253 (10 µM) or AC187 (10 µM), significantly attenuated the induction of several pro-apoptotic mediators up-regulated following exposure to Aß(1-42) or human amylin and increased the expression of several anti-apoptotic markers. These data allow us to identify key elements in the Aß-induced apoptosis that are blocked by antagonism of the amylin receptor and further support the potential for amylin receptor blockade as a potential therapeutic avenue in AD.

Actions of ß-amyloid protein on human neurons are expressed through the amylin receptor.

Disruption of neurotoxic effects of amyloid ß protein (Aß) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aß in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aß. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aß-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, amylin receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aß actions through the amylin receptor in human neurons and temporospatial interrelationship of Aß and the amylin receptor in an in vivo model of AD together provide a persuasive rationale for this receptor as a novel therapeutic target in the treatment of AD.

Genetic Depletion of Amylin/Calcitonin Receptors Improves Memory and Learning in Transgenic Alzheimer's Disease Mouse Models.

Based upon its interactions with amyloid ß peptide (Aß), the amylin receptor, a class B G protein-coupled receptor (GPCR), is a potential modulator of Alzheimer's disease (AD) pathogenesis. However, past pharmacological approaches have failed to resolve whether activation or blockade of this receptor would have greater therapeutic benefit. To address this issue, we generated compound mice expressing a human amyloid precursor protein gene with familial AD mutations in combination with deficiency of amylin receptors produced by hemizygosity for the critical calcitonin receptor subunit of this heterodimeric GPCR. These compound transgenic AD mice demonstrated attenuated responses to human amylin- and Aß-induced depression of hippocampal long-term potentiation (LTP) in keeping with the genetic depletion of amylin receptors. Both the LTP responses and spatial memory (as measured with Morris water maze) in these mice were improved compared to AD mouse controls and, importantly, a reduction in both the amyloid plaque burden and markers of neuroinflammation was observed. Our data support the notion of further development of antagonists of the amylin receptor as AD-modifying therapies.

Ionic mechanisms of action of prion protein fragment PrP(106-126) in rat basal forebrain neurons.

Prion diseases are neurodegenerative disorders that are characterized by the presence of the misfolded prion protein (PrP). Neurotoxicity in these diseases may result from prion-induced modulation of ion channel function, changes in neuronal excitability, and consequent disruption of cellular homeostasis. We therefore examined PrP effects on a suite of potassium (K(+)) conductances that govern excitability of basal forebrain neurons. Our study examined the effects of a PrP fragment [PrP(106-126), 50 nM] on rat neurons using the patch clamp technique. In this paradigm, PrP(106-126) peptide, but not the "scrambled" sequence of PrP(106-126), evoked a reduction of whole-cell outward currents in a voltage range between -30 and +30 mV. Reduction of whole-cell outward currents was significantly attenuated in Ca(2+)-free external media and also in the presence of iberiotoxin, a blocker of calcium-activated potassium conductance. PrP(106-126) application also evoked a depression of the delayed rectifier (I(K)) and transient outward (I(A)) potassium currents. By using single cell RT-PCR, we identified the presence of two neuronal chemical phenotypes, GABAergic and cholinergic, in cells from which we recorded. Furthermore, cholinergic and GABAergic neurons were shown to express K(v)4.2 channels. Our data establish that the central region of PrP, defined by the PrP(106-126) peptide used at nanomolar concentrations, induces a reduction of specific K(+) channel conductances in basal forebrain neurons. These findings suggest novel links between PrP signalling partners inferred from genetic experiments, K(+) channels, and PrP-mediated neurotoxicity.

Prolactin-releasing peptide effects in the rat brain are mediated through the Neuropeptide FF receptor.

Prolactin-releasing peptide (PrRP), an RF amide peptide present in the brain, generates a wide variety of centrally generated autonomic responses, including increases in arterial blood pressure and heart rate. The identity of the receptor mediating the effects of PrRP is unknown. In addition to GPR10, which is its putative endogenous receptor, PrRP demonstrates a high binding affinity for Neuropeptide FF (NPFF) receptors, specifically the NPFF2 receptor. In the present study, we examined whether the central cardiovascular effects of PrRP in the intact animal and its cellular effects on parvocellular paraventricular nucleus (PVN) neurons are mediated via NPFF receptors. In conscious rats, intracerebroventricular (i.c.v.) PrRP caused an increase in arterial blood pressure and heart rate, which was blocked with RF9, a specific NPFF receptor antagonist. These PrRP-evoked cardiovascular effects were preserved in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat strain, in which the GRP10 receptor gene was mutated. In rat brain slices, whole-cell patch clamp recordings of parvocellular paraventricular nucleus neurons show PrRP caused a decrease in evoked and miniature GABAergic inhibitory postsynaptic currents (IPSCs), effects that were antagonized by RF9, but not neuropeptide Y, a putative GPR10 receptor antagonist. The effects of PrRP on IPSCs in OLETF rats were similar to those in wild-type rats. Both in vivo and in vitro data strongly suggest that certain PrRP effects in the brain are expressed via NPFF receptors, probably NPFF2, rather than the GPR10 receptor. These observations may assume clinical relevance as RF amide peptides such NPFF and PrRP become therapeutic targets for a variety of autonomically related disorders.

Amyloid beta protein modulates glutamate-mediated neurotransmission in the rat basal forebrain: involvement of presynaptic neuronal nicotinic acetylcholine and metabotropic glutamate receptors.

Amyloid beta (Abeta) protein, a 39-43 amino acid peptide deposited in brains of individuals with Alzheimer's disease (AD), has been shown to interact directly with a number of receptor targets including neuronal nicotinic acetylcholine receptors (nAChRs) and glutamate receptors. In this study, we investigated the synaptic effects of Abeta(1-42) on glutamate-mediated neurotransmission in the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. Glutamatergic miniature EPSCs (mEPSCs) were recorded using whole-cell patch-clamp recordings from identified cholinergic DBB neurons in rat forebrain slices. In 54% of DBB neurons, bath application of Abeta(1-42) (100 nM), but not Abeta(42-1) (inverse fragment), significantly increased the frequency of mEPSCs without affecting amplitude or kinetic parameters (rise or decay time). In 32% of DBB neurons, bath application of Abeta(1-42) significantly decreased only the frequency but not amplitude of mEPSCs. Application of dihydro-beta-erythroidine (DHbetaE) (an antagonist for the alpha4beta2 subtype of nAChRs) but not alpha-bungarotoxin (an antagonist for the alpha7 subtype of nAChRs) blocked Abeta(1-42)-mediated increases in mEPSC frequency. The Abeta(1-42)-mediated increase in glutamatergic transmission is thus presynaptic and mediated via non-alpha7 AChRs. In contrast, Abeta(1-42)-mediated decreases in mEPSC frequency could not be antagonized by either DHbetaE or alpha-bungarotoxin. However, the Abeta(1-42)-evoked depression in mEPSC frequency was antagonized by (RS)-alpha-methyl-4-carboxyphenyglycine, a nonselective group I/II metabotropic glutamate receptor antagonist. These observations provide further insight into the mechanisms whereby Abeta affects synaptic function in the brain and may be relevant in the context of synaptic failure observed in AD.

Cyclic AC253, a novel amylin receptor antagonist, improves cognitive deficits in a mouse model of Alzheimer's disease.

INTRODUCTION: Amylin receptor serves as a portal for the expression of deleterious effects of amyloid ß-protein (Aß), a key pathologic hallmark of Alzheimer's disease. Previously, we showed that AC253, an amylin receptor antagonist, is neuroprotective against Aß toxicity in vitro and abrogates Aß-induced impairment of hippocampal long-term potentiation. METHODS: Amyloid precursor protein-overexpressing TgCRND8 mice received intracerebroventricularly AC253 for 5 months. New cyclized peptide cAC253 was synthesized and administered intraperitoneally three times a week for 10 weeks in the same mouse model. Cognitive functions were monitored, and pathologic changes were quantified biochemically and immunohistochemically. RESULTS: AC253, when administered intracerebroventricularly, improves spatial memory and learning, increases synaptic integrity, reduces microglial activation without discernible adverse effects in TgCRND8 mice. cAC253 demonstrates superior brain permeability, better proteolytic stability, and enhanced binding affinity to brain amylin receptors after a single intraperitoneal injection. Furthermore, cAC253 administered intraperitoneally also demonstrates improvement in spatial memory in TgCRND8 mice. DISCUSSION: Amylin receptor is a therapeutic target for Alzheimer's disease and represents a disease-modifying therapy for this condition.

Neuropeptide FF distribution in the human and rat forebrain: a comparative immunohistochemical study.

Neuropeptide FF (NPFF) is an octapeptide implicated in a variety of physiological functions, including nociception, cardiovascular responses, and neuroendocrine regulation. The NPFF gene and its mRNA are highly conserved across species. A comparative study of NPFF distribution in the human and rat forebrain was carried out by using single NPFF and double NPFF + vasopressin (VP) immunohistochemistry. NPFF is extensively localized within neurochemical circuits of human and rat forebrain. Semiquantitative analysis revealed that the densities of NPFF cells and fibers in many forebrain nuclei in the human correlate well with those observed for the same structures in the rat. High numbers of NPFF positive neurons in the dorsomedial hypothalamic nucleus and a dense plexus of NPFF fibers surrounding the fornix within the bed nucleus of the stria terminalis were identified in the human and rat forebrain. Within the hypothalamus of both species, dense NPFF innervation was observed in the perinuclear zone of the supraoptic nucleus (SO) just dorsolateral to the VP-positive neurons. Extensive NPFF innervation of ventricular ependyma and brain microvasculature were common for both species. At the same time, obvious differences in NPFF localization between the two species were also apparent. For example, in contrast to the rat SO, no NPFF- or NPFF- + VP-immunostained cells were observed in the human SO. Knowledge of NPFF neuroanatomical localization in the human brain and the relationship of these observations to those in the rat brain may provide insight into the role of this peptide in central cardiovascular and neuroendocrine regulation.

Neuropeptide FF (NPFF) control of magnocellular neurosecretory cells of the rat hypothalamic paraventricular nucleus (PVN).

Neuropeptide FF (NPFF) is an octapeptide belonging to an extended family of RF amide peptides that have been implicated in a wide variety of physiological functions in the brain. NPFF and its receptors are abundantly expressed in the rat brain and spinal cord including the hypothalamic paraventricular nucleus (PVN), an autonomic nucleus critical for the secretion of neurohormones and the regulation of sympathetic outflow. In this study, we sought to examine the effects of NPFF on GABAergic inhibitory synaptic input to magnocellular neurosecretory cells (MNCs) of the PVN, which secrete the neurohormones, vasopressin and oxytocin from their terminals in the neurohypophysis. Whole cell patch clamp recordings under voltage clamp conditions were performed from PVN MNCs in the brain slice. Bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) were isolated in the presence of glutamate receptor blockers. In tetrodotoxin, NPFF (5 microM) caused an increase in frequency, but not amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in MNCs indicating a presynaptic locus of action for this peptide. Intracerebroventricular application of NPFF resulted in an activation of GABAergic neurons located adjacent to the PVN as revealed by immunohistochemistry for Fos protein and in situ hybridization for glutamic acid decarboxylase (GAD67) mRNA. Based on these observations we conclude that NPFF facilitates inhibitory input to MNCs of the PVN via GABAergic interneurons located in immediate vicinity of the nucleus. These findings provide a cellular and anatomic basis for the NPFF-induced inhibition of vasopressin release has been reported consequent to hypovolemia and hyperosmolar stimulation.

Histamine induces the production of matrix metalloproteinase-9 in human astrocytic cultures via H1-receptor subtype.

Accumulation of ß-amyloid (Aß) protein within the brain is a neuropathological hallmark of Alzheimer's disease (AD). One strategy to facilitate Aß clearance from the brain is to promote Aß catabolism. Matrix metalloproteinase-9 (MMP-9), a member of the family of Zn(+2)-containing endoproteases, known to be expressed and secreted by astrocytes, is capable of degrading Aß. Histamine, a major aminergic brain neurotransmitter, stimulates the production of MMP-9 in keratinocytes through the histamine H1 receptor (H1R). In the present study, we show that histamine evokes a concentration- and calcium-dependent release of MMP-9 from human astrocytic U373 cells and primary cultures of human and rat astrocytes through the H1R subtype. Activation of H1R on astrocytes elevated intracellular levels of Ca(2+) that was accompanied by time-dependent increases in MAP kinase p44/p42 and PKC. In-cell western blots revealed dose-dependent increases in both enzymes, confirming involvement of these signal transduction pathways. We next investigated the extent of recombinant human MMP-9 (rhMMP-9) proteolytic activity on soluble oligomeric Aß (soAß). Mass spectrometry demonstrated time-dependent cleavage of soAß (20 µM), but not another amyloidogenic protein amylin, upon incubation with rhMMP-9 (100 nM) at 1, 4 and 17 h. Furthermore, Western blots showed a shift in soAß equilibrium toward lower order, less toxic monomeric species. In conclusion, both MAPK p44/p42 and PKC pathways appear to be involved in histamine-upregulated MMP-9 release via H1Rs in astrocytes. Furthermore, MMP-9 appears to cleave soAß into less toxic monomeric species. Given the key role of histamine in MMP-9 release, this neurotransmitter may serve as a potential therapeutic target for AD.

Antagonist of the amylin receptor blocks beta-amyloid toxicity in rat cholinergic basal forebrain neurons.

Salvage of cholinergic neurons in the brain through a blockade of the neurotoxic effects of amyloidbeta protein (Abeta) is one of the major, but still elusive, therapeutic goals of current research in Alzheimer's disease (AD). To date, no receptor has been unequivocally identified for Abeta. Human amylin, which acts via a receptor composed of the calcitonin receptor-like receptor and a receptor-associated membrane protein, possesses amyloidogenic properties and has a profile of neurotoxicity that is strikingly similar to Abeta. In this study, using primary cultures of rat cholinergic basal forebrain neurons, we show that acetyl-[Asn30, Tyr32] sCT(8-37) (AC187), an amylin receptor antagonist, blocks Abeta-induced neurotoxicity. Treatment of cultures with AC187 before exposure to Abeta results in significantly improved neuronal survival as judged by MTT and live-dead cell assays. Quantitative measures of Abeta-evoked apoptotic cell death, using Hoechst and phosphotidylserine staining, confirm neuroprotective effects of AC187. We also demonstrate that AC187 attenuates the activation of initiator and effector caspases that mediate Abeta-induced apoptotic cell death. These data are the first to show that expression of Abeta toxicity may occur through the amylin receptor and suggest a novel therapeutic target for the treatment of AD.

Central administration of neuropeptide FF (NPFF) causes increased neuronal activation and up-regulation of NPFF gene expression in the rat brainstem.

Neuropeptide FF (NPFF) is a morphine modulatory peptide that plays an important role in a wide variety of physiological functions, including those related to nociception and central autonomic regulation. NPFF fibers and cells have been shown to be discretely localized in key autonomic centers within the brain, including the brainstem nucleus of the solitary tract (NTS). Central applications of NPFF evoke a number of important biological effects through activation of central neuronal circuits whose identities remain unknown at present. NPFF administered in this manner may also be capable of up- or down-regulating its own gene expression. In this study, we investigated the effects of intracerebroventricular (i.c.v.) administration of NPFF on the activation and the gene expression of NPFF in NTS neurons. Conscious rats received saline or NPFF (8 or 10 microg i.c.v.), with concomitant monitoring of arterial blood pressure. Brains were prepared for Fos immunohistochemistry to identify neuronal activation and NPFF in situ hybridization to determine cells expressing NPFF mRNA in the NTS. At a dose of 8 microg, i.c.v., NPFF did not evoke alterations in blood pressure, but, at 10 microg, there was an increase in arterial blood pressure of 30-40 mmHg. Image analysis showed a dose-dependent increase in number of NPFF neurons that were activated in rats receiving i.c.v. NPFF compared with saline controls. NPFF gene expression in the NTS showed a similar dose-dependent increase following i.c.v. administration of either 8 or 10 microg of NPFF. Significantly greater numbers of activated neurons expressing the NPFF gene (double labeled) were observed in the NTS at the level of the area postrema in animals receiving i.c.v. NPFF compared with saline controls. These data indicate that centrally administered NPFF is capable of up-regulating its own gene expression in the NTS and that this effect appears in part to be independent of elevations in arterial blood pressure that this peptide can evoke when administered i.c.v. at the higher dose. The up-regulation of NPFF may play a homeostatic role in response to specific cardiovascular challenges, such as hypotension.

Distribution of the neuropeptide FF1 receptor (hFF1) in the human hypothalamus and surrounding basal forebrain structures: immunohistochemical study.

Neuropeptides with C-terminal RFamide and their receptors NPFF1 (FF1) and NPFF2 (FF2) have been implicated in a wide variety of functions, including nociception and autonomic and neuroendocrine regulation. Recent studies indicate that the FF1, but not FF2, mRNA is highly expressed in the human hypothalamus. In the present study, localization of FF1 in the human hypothalamus and surrounding regions was studied immunohistochemically by using an antibody against human FF1 (hFF1). Brain sections from healthy 30-50-year-old individuals were used for hFF1 immunohistochemistry. The highest density of hFF1-stained cells was found in the posterior division of the bed nucleus of the stria terminalis and in the zona incerta. A moderate density of cells was observed in the perifornical nucleus, infundibular nucleus, tuberomammillary nucleus, and lateral tuberal nucleus. A lesser density was revealed in the dorsomedial hypothalamic nucleus, basal nucleus of Meynert, and anterior amygdaloid area. Only scattered hFF1 cells were found in the suprachiasmatic nucleus and hypothalamic paraventricular nucleus. hFF1 cells and fibers were absent in the supraoptic and mammillary nuclei. Single and double strands of hFF1-immunopositive punctate varicosities marked cellular processes of different caliber. The density of hFF1-immunostained fiber networks did not always coincide with that of hFF1-immunostained cells. hFF1 immunoreactivity was also found in the wall of blood vessels within most brain areas studied. Localization of hFF1 in discrete regions of the hypothalamus and extended amygdala may provide important insights into the role of amidated neuropeptides in central autonomic and neuroendocrine control in the human brain.

Galanin attenuates beta-amyloid (Abeta) toxicity in rat cholinergic basal forebrain neurons.

In brains of Alzheimer's disease (AD) patients, expression of the neuropeptide galanin is significantly upregulated and galanin-immunoreactive fibers hypertrophy and hyperinnervate cholinergic neurons of the basal forebrain. However, the role of galanin in AD, whether it is detrimental or neuroprotective, remains controversial. In this study, using primary cultured neurons from the rat basal forebrain, we show that pretreatment with galanin protects cholinergic neurons against beta-amyloid-induced apoptotic cell death as judged by visual observation, MTT assay, Live/dead cell assay, TUNEL and cleaved caspase-3 staining. These effects are mimicked by the galanin receptor 2 (GALR2) agonist, AR-M1896. Western blot analysis revealed Abeta-induced decrease in phospho-PKC and phospho-Akt levels was reversed by galanin. Galanin also attenuated cleavage of caspases-3 and -9 following exposure to Abeta. These findings support a neuroprotective role for galanin and may have implications for development of compounds based on this peptide to treat AD.

RF9, a potent and selective neuropeptide FF receptor antagonist, prevents opioid-induced tolerance associated with hyperalgesia.

Neuropeptide FF (NPFF) has been proposed to play a role in pain modulation, opioid tolerance, and several other physiological processes. However, pharmacological agents that would help define physiological roles for this peptide are still missing. Here we report the discovery of a potent and selective NPFF receptor antagonist, RF9, that can be administered systemically. This compound does not show any effects by itself but can block efficiently the increase in blood pressure and heart rate evoked by NPFF. When chronically coinjected with heroin, RF9 completely blocks the delayed and long-lasting paradoxical opioid-induced hyperalgesia and prevents the development of associated tolerance. Our data indicate that NPFF receptors are part of a bona fide antiopioid system and that selective antagonists of these receptors could represent useful therapeutic agents for improving the efficacy of opioids in chronic pain treatment.

Fucoidan inhibits cellular and neurotoxic effects of beta-amyloid (A beta) in rat cholinergic basal forebrain neurons.

The deposition of beta-amyloid protein (A beta), a 39-43 amino acid peptide, in the brain and a loss of cholinergic neurons in the basal forebrain are pathological hallmarks of Alzheimer's disease (AD). Seaweeds consumed in Asia contain Fucoidan, a sulfated polysaccharide. Fucoidan has been known to exhibit various biological actions, such as an anti-inflammatory and antioxidant action. In this study, using whole-cell patch clamp recordings we examined the effects of Fucoidan on A beta-induced whole-cell currents in acutely dissociated rat basal forebrain neurons. We further investigated whether Fucoidan is capable of blocking A beta neurotoxicity in primary neuronal cultures. In dissociated cells, bath application of A beta(25-35) (1 microM) caused a reduction of the whole-cell currents by 16%. Fucoidan, in a dose-dependent manner, blocks the A beta(25-35) reduction of whole-cell currents. Exposure of A beta(25-35) (20 microM) or A beta(1-42) (20 microM) to rat cholinergic basal forebrain cultures for 48 h resulted in 40-60% neuronal death, which was significantly decreased by pretreatment of cultures with Fucoidan (0.1-1.0 microM). Fucoidan also attenuated A beta-induced down-regulation of phosphorylated protein kinase C. A beta(1-42)-induced generation of reactive oxygen species was blocked by prior exposure of cultures to Fucoidan. Furthermore, A beta activation of caspases 9 and 3, which are signaling pathways implicated in apoptotic cell death, is blocked by pretreatment of cultures with Fucoidan. These results show that Fucoidan is able to block A beta-induced reduction in whole-cell currents in basal forebrain neurons and has neuroprotective effects against A beta-induced neurotoxicity in basal forebrain neuronal cultures.

Human amylin actions on rat cholinergic basal forebrain neurons: antagonism of beta-amyloid effects.

Human amylin (hAmylin), a 37-amino acid pancreatic peptide, and amyloid beta protein (A beta), a 39-43 amino acid peptide, abundantly deposited in the brains of Alzheimer's patients, induce neurotoxicity in hippocampal and cortical cultures. Although the mechanism of this neurotoxicity is unknown, both peptides are capable of modulating ion channel function that may result in a disruption of cellular homeostasis. In this study, we examined the effects of hAmylin on whole cell currents in chemically identified neurons from the rat basal forebrain and the interactions of hAmylin-induced responses with those of A beta. Whole cell patch-clamp recordings were performed on enzymatically dissociated neurons of the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. Bath application of hAmylin (1 nM to 5 microM) resulted in a dose-dependent reduction in whole cell currents in a voltage range between -30 and +30 mV. Single-cell RT-PCR analysis reveal that all DBB neurons responding to hAmylin or A beta were cholinergic. Using specific ion channel blockers, we identified hAmylin and A beta effects on whole cell currents to be mediated, in part, by calcium-dependent conductances. Human amylin also depressed the transient outward (IA) and the delayed rectifier (IK) potassium currents. The hAmylin effects on whole cell currents could be occluded by A beta and vice versa. Human amylin and A beta responses could be blocked with AC187 (50 nM to 1 microM), a specific antagonist for the amylin receptor. The present study indicates that hAmylin, like A beta, is capable of modulating ion channel function in cholinergic basal forebrain neurons. Furthermore, the two peptides may share a common mechanism of action. The ability of an amylin antagonist to block the responses evoked by hAmylin and A beta may provide a novel therapeutic approach for Alzheimer's disease.