David MacTavish

Amyloid β Protein Modulates Glutamate-Mediated Neurotransmission in the Rat Basal Forebrain: Involvement of Presynaptic Neuronal Nicotinic Acetylcholine and Metabotropic Glutamate Receptors

Amyloid β (Aβ) protein, a 39–43 amino acid peptide deposited in brains of individuals with Alzheimers 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 Aβ1–42 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 Aβ1–42 (100 nm), but not Aβ42–1 (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 Aβ1–42 significantly decreased only the frequency but not amplitude of mEPSCs. Application of dihydro-β-erythroidine (DHβE) (an antagonist for the α4β2 subtype of nAChRs) but not α-bungarotoxin (an antagonist for the α7 subtype of nAChRs) blocked Aβ1–42-mediated increases in mEPSC frequency. The Aβ1–42-mediated increase in glutamatergic transmission is thus presynaptic and mediated via non-α7 AChRs. In contrast, Aβ1–42-mediated decreases in mEPSC frequency could not be antagonized by either DHβE or α-bungarotoxin. However, the Aβ1–42 -evoked depression in mEPSC frequency was antagonized by (RS)-α-methyl-4-carboxyphenyglycine, a nonselective group I/II metabotropic glutamate receptor antagonist. These observations provide further insight into the mechanisms whereby Aβ affects synaptic function in the brain and may be relevant in the context of synaptic failure observed in AD.

Activation by hypotension of neurons in the hypothalamic paraventricular nucleus that project to the brainstem

To investigate the involvement of neuronal nitric oxide (NO) in the response of the brain to changes in blood pressure, we studied the activation of putative NO‐producing neurons in the paraventricular nucleus of the hypothalamus (PVN) in rats whose mean arterial pressures (MAPs) were decreased by 40–50% with hemorrhage (HEM) or infusion of sodium nitroprusside (NP). Activation was assessed on the basis of expression of the immediate early gene, c‐fos; putative NO‐producing neurons were identified with the histochemical stain for nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d); and the proportions of neurons projecting to the nucleus of the tractus solitarius (NTS) and/or caudal ventrolateral medulla (CVLM) were determined with retrograde tracing techniques.

Fucoidan inhibits cellular and neurotoxic effects of β-amyloid (Aβ) in rat cholinergic basal forebrain neurons

The deposition of β‐amyloid protein (Aβ), a 39–43 amino acid peptide, in the brain and a loss of cholinergic neurons in the basal forebrain are pathological hallmarks of Alzheimers 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 Aβ‐induced whole‐cell currents in acutely dissociated rat basal forebrain neurons. We further investigated whether Fucoidan is capable of blocking Aβ neurotoxicity in primary neuronal cultures. In dissociated cells, bath application of Aβ25−35 (1 µm) caused a reduction of the whole‐cell currents by 16%. Fucoidan, in a dose‐dependent manner, blocks the Aβ25−35 reduction of whole‐cell currents. Exposure of Aβ25−35 (20 µm) or Aβ1−42 (20 µm) 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 µm). Fucoidan also attenuated Aβ‐induced down‐regulation of phosphorylated protein kinase C. Aβ1−42‐induced generation of reactive oxygen species was blocked by prior exposure of cultures to Fucoidan. Furthermore, Aβ 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 Aβ‐induced reduction in whole‐cell currents in basal forebrain neurons and has neuroprotective effects against Aβ‐induced neurotoxicity in basal forebrain neuronal cultures.

Changes in blood volume and pressure induce c-fos expression in brainstem neurons that project to the paraventricular nucleus of the hypothalamus

Immunohistochemistry for c-fos was combined with retrograde tracing techniques to study the effects of acute reductions in arterial blood pressure due to hemorrhage (HEM) in conscious rats on activated neurons in the brainstem nucleus of the tractus solitarius (NTS) or ventrolateral medulla (VLM) which project to the paraventricular nucleus (PVN) of the hypothalamus. In an attempt to separate blood pressure effects from those associated with changes in blood volume, a similar approach was used to study the effects of drug-evoked hypotension using peripheral infusions of sodium nitroprusside (NP). Few differences were found in patterns or numbers of activated neurons (Fos-immunoreactive) in the NTS or VLM after HEM or NP treatment; only in the NTS at the level of the area postrema were significantly higher numbers of neurons that expressed Fos found in NP rats. In addition, a large proportion of PVN-projecting neurons in the NTS and VLM was activated whereas many activated neurons in the NTS and VLM did not project to the PVN. These results show that a decrease in blood pressure leads to the activation of NTS and VLM neurons but that a change in blood volume does not activate significantly greater numbers of neurons in these areas that project to the PVN or to other targets. Whereas substantial numbers of neurons in the NTS and VLM appear to transmit cardiovascular information to the PVN, many others likely transmit this information to other central targets.

Galanin attenuates β-amyloid (Aβ) toxicity in rat cholinergic basal forebrain neurons

Abstract In brains of Alzheimers 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 β-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 Aβ-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 Aβ. These findings support a neuroprotective role for galanin and may have implications for development of compounds based on this peptide to treat AD.

Antagonist of the Amylin Receptor Blocks β-Amyloid Toxicity in Rat Cholinergic Basal Forebrain Neurons

Salvage of cholinergic neurons in the brain through a blockade of the neurotoxic effects of amyloidβ protein (Aβ) is one of the major, but still elusive, therapeutic goals of current research in Alzheimers disease (AD). To date, no receptor has been unequivocally identified for Aβ. 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 Aβ. 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 Aβ-induced neurotoxicity. Treatment of cultures with AC187 before exposure to Aβ results in significantly improved neuronal survival as judged by MTT and live-dead cell assays. Quantitative measures of Aβ-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 Aβ-induced apoptotic cell death. These data are the first to show that expression of Aβ toxicity may occur through the amylin receptor and suggest a novel therapeutic target for the treatment of AD.

Amyloid β (Aβ) Peptide Directly Activates Amylin-3 Receptor Subtype by Triggering Multiple Intracellular Signaling Pathways

Background: Aβ and human amylin peptides share similar biophysical and neurotoxic properties. Results: Aβ directly activates amylin-3 receptor (AMY3) isoform and triggers multiple signaling pathways. Conclusion: Aβ actions are expressed via AMY3 receptors. Significance: AMY3 could serve as a therapeutic target for attenuating Aβ toxicity. The two age-prevalent diseases Alzheimer disease and type 2 diabetes mellitus share many common features including the deposition of amyloidogenic proteins, amyloid β protein (Aβ) and amylin (islet amyloid polypeptide), respectively. Recent evidence suggests that both Aβ 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). Aβ1–42 and human amylin (hAmylin) increase cytosolic cAMP and Ca2+, trigger multiple pathways involving the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Aβ1–42 and hAmylin also induce cell death during exposure for 24–48 h at low micromolar concentrations. In the presence of hAmylin, Aβ1–42 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 Ca2+, activation of protein kinase A, MAPK, Akt, cFos, and cell death, which occur upon AMY3 activation with hAmylin, Aβ1–42, or their co-application. Our data suggest that AMY3 plays an important role by serving as a receptor target for actions Aβ and thus may represent a novel therapeutic target for development of compounds to treat neurodegenerative conditions such as Alzheimer disease.

Beta Amyloid-Induced Depression of Hippocampal Long-Term Potentiation Is Mediated through the Amylin Receptor

Alzheimers disease (AD) is characterized by accumulation of amyloid-β peptide (Aβ) 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 Aβ species. We investigated whether the amylin receptor antagonist, AC253, neutralizes the depressant effects of Aβ1–42 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. Aβ1–42 (50 nm) and human amylin (50 nm), but not Aβ42–1 (50 nm), depressed LTP evoked using both stimulation protocols. Preapplication of AC253 (250 nm) blocked Aβ- 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 Aβ1–42 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.

Hypotension induces fos immunoreactivity in NADPH-diaphorase positive neurons in the paraventricular and supraoptic hypothalamic nuclei of the rat

Double staining for Fos and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D) was used to study the distribution of activated neurons that synthesize nitric oxide in the paraventricular (PVN) and supraoptic nuclei (SON) following hypotensive stimulation in conscious rats. Fos was detected in many magno- and parvocellular NADPH-D positive neurons in response to haemorrhage or drug-evoked hypotension using i.v. infusions of sodium nitroprusside. However, quantitative analysis did not reveal any differences in the number of Fos positive PVN neurons following either mode of stimulation. These results suggest that a subpopulation of hypothalamic NADPH-D positive neurons is activated following hypotensive challenge. This activation of NADPH-D neurons may occur indirectly through other CNS structures that influence the excitability of hypothalamic SON and PVN. Furthermore, the lack of a difference in activated neurons within the PVN following either haemorrhage or nitroprusside infusion suggests that while a drop in blood pressure causes activation of neurons that produce nitric oxide, a decrease in blood volume, which accompanies haemorrhage, does not.

Actions of β-Amyloid Protein on Human Neurons Are Expressed through the Amylin Receptor

Disruption of neurotoxic effects of amyloid β protein (Aβ) is one of the major, but as yet elusive, goals in the treatment of Alzheimers disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aβ in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aβ. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aβ-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 Aβ actions through the amylin receptor in human neurons and temporospatial interrelationship of Aβ 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.