Cannabidiol (CBD), one of the most studied phytocannabinoids, is non-psychotropic and can induce protective effects on the central nervous system against acute and chronic brain injury. Interestingly, CBD inhibits processes relating to amyloid beta (Aβ)-induced neurotoxicity in mouse models of Alzheimer’s disease, though the detailed molecular mechanism underlying the CBD neurotoxicity modulation is not fully understood. In this study, using atomic force microscopy, we find that CBD promotes the aggregation of Aβ peptides, enhancing the formation of Aβ oligomers, also known as Aβ-derived diffusible ligands (ADDLs). The CBD-mediated sequestration of Aβ monomers in soluble ADDLs could reduce neurotoxicity. This study highlights a possible role of CBD in modulating the formation of ADDL aggregates and provides insight into potentially neuroprotective properties of CBD in Alzheimer’s disease.

In Alzheimer’s disease (AD), Amyloid-beta (Aβ) oligomers are considered an appealing therapeutic- and diagnostic target. However, to date, the molecular mechanisms associated with the pathological accumulation or structure of Aβ oligomers remains an enigma to the scientific community. Here we demonstrate the strong seeding properties of unique Aβ fragment signatures and show that the truncated Aβ peptides of residues Aβ1-23, Aβ1-24 and Aβ1-25, rapidly seed to form small, SDS-PAGE stable assemblies of ∼5kDa to ∼14kDa molecular mass range. Mass spectrometry analysis of SDS-PAGE fractionated and gel extracted oligomers revealed that the truncated Aβ isoforms of residues 1-23 to 1-25 form stable entities with low molecular weight (LMW) oligomers, which strongly resemble the regularly reported Aβ entities of putative dimeric or trimeric assemblies found in human post-mortem AD and Tg mouse brain extracts. Furthermore, electrophysiological recordings in the mouse hippocampus indicate that LMW Aβ assemblies formed by fragments Aβ1-23 to Aβ1-25 significantly impair long-term-potentiation (LTP) in the absence of full-length Aβ1-42. Extensive antibody screening highlights the important observation, that the LMW Aβ assemblies formed by these truncated Aβ peptides escape immuno-detection using conventional, conformation specific antibodies but, more importantly, the clinical antibody aducanumab. Our novel findings suggest that there are new Aβ target “loopholes” which can be exploited for the development of therapeutic antibodies with binding properties against stable target hotspots present in Aβ oligomers. We provide here a first example of a new class of monoclonal antibody with unique binding properties against LMW Aβ oligomers, in the absence of binding to large fibrillar Aβ assemblies, or dense amyloid plaques. Our research supports a novel, unparalleled approach for targeting early, pathological Aβ species during the insidious phase of AD and prior to the appearance of large oligomeric or protofibrilar assemblies.

Here we demonstrate for the first time that cannabidiol (CBD) acts to protect synaptic plasticity in an in vitro model of Alzheimer’s disease (AD). The non-psycho active component of Cannabis sativa , CBD has previously been shown to protect against the neurotoxic effects of beta amyloid peptide (Aβ) in cell culture and cognitive behavioural models of neurodegeneration. Hippocampal long-term potentiation (LTP) is an activity dependent increase in synaptic efficacy often used to study cellular mechanisms related to memory. Here we show that acute application of soluble oligomeric beta amyloid peptide (Aβ 1–42 ) associated with AD, attenuates LTP in the CA 1 region of hippocampal slices from C57Bl/6 mice. Application of CBD alone did not alter LTP, however pre-treatment of slices with CBD rescued the Aβ 1–42 mediated deficit in LTP. We found that the neuroprotective effects of CBD were not reversed by WAY100635, ZM241385 or AM251, demonstrating a lack of involvement of 5HT 1A , adenosine (A 2A ) or Cannabinoid type 1 (CB 1 ) receptors respectively. However in the presence of the PPARγ antagonist GW9662 the neuroprotective effect of CBD was prevented. Our data suggests that this major component of Cannabis sativa , which lacks psychoactivity may have therapeutic potential for the treatment of AD.

Epidemiological evidence suggests that chronic treatment with simvastatin may protect against the development of Alzheimer's disease (AD), but as yet it is unclear how this effect is mediated. Extensive data also indicates that the amyloid β-protein (Aβ) plays a central role in the disease process, and it has been suggested that the protective effects of simvastatin may be mediated by reducing Aβ production or by counteracting the toxic effects of Aβ. Accordingly, using the AβPPswe/PS1dE9 mouse model of AD, we investigated the effects of simvastatin on long-term potentiation (LTP), amyloid biology, and two key kinases involved in Aβ-mediated toxicity. Since burgeoning data indicate that both fibrillar and non-fibrillar forms of Aβ play a prominent role in AD pathogenesis, we were careful to investigate the effects of simvastatin on three biochemically distinct pools of Aβ. In untreated AβPPswe/PS1dE9 mice, there was a dramatic and significant increase in the levels of water-soluble Aβ between 6 and 8 months, but this remained constant between 8 and 18 months. In contrast, the concentrations of detergent-soluble and formic acid (FA)-soluble Aβ species increased across all ages examined, thus demonstrating that while amyloid deposition continued, the levels of water-soluble Aβ remained relatively constant. LTP was normal at 6 months, but was significantly impaired at 8 and 18 months. Importantly, a diet supplemented with 0.04% simvastatin for one month (at 7 months) positively affected synaptic plasticity in AβPPswe/PS1dE9 mice and did not significantly alter levels of water-soluble, detergent-soluble, or FA-soluble Aβ, but did increase phosphorylation of both Akt and GSK-3, while tau and tau phosphorylation were unaltered. These results indicate that the protective effects of simvastatin may be mediated by maintaining signaling pathways that help to protect and rescue LTP.

Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-β protein (Aβ) have important roles in Alzheimer's disease with toxicities mimicked by synthetic Aβ(1-42). However, no defined toxic structures acting via specific receptors have been identified and roles of proposed receptors, such as prion protein (PrP), remain controversial. Here we quantify binding to PrP of Aβ(1-42) after different durations of aggregation. We show PrP-binding and PrP-dependent inhibition of long-term potentiation (LTP) correlate with the presence of protofibrils. Globular oligomers bind less avidly to PrP and do not inhibit LTP, whereas fibrils inhibit LTP in a PrP-independent manner. That only certain transient Aβ assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in Aβ-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of Aβ nanotubes or their interaction with PrP might have a role in treatment of Alzheimer's disease.

Recent studies have investigated the potential therapeutic properties of statins; agents commonly used to treat hypercholesterolemia. Behavioural studies in Tg2576 mice, demonstrated that chronic treatment with simvastatin improved learning and memory (Li et al., 2006; Ann Neurol 60: 729-739). In cultured neurones, chronic mevastatin treatment however depressed NMDA receptor-mediated current yet enhanced voltage-gated sodium channel current (Kannan et al., Neurobiology of Aging 2008). Here we have investigated the effects of acute and chronic simvastatin treatment on synaptic transmission, action potential regulation and synaptic plasticity in the APPswe/PS1dE9 mouse model. Mice were treated chronically by including simvastatin in their diet (0.04%) for periods of 3 to 6 months. Alternatively simvastatin was applied via the perfusion solution. Electrophysiological recordings were performed in transverse hippocampal slices from APPswe/PS1dE9 mice and age matched controls. Antidromic compound action potentials (cAPs) were recorded in the CA1 cell body region by stimulating the alveus (in DNQX). Excitatory post synaptic potentials (EPSPs) were evoked in the CA1 region every 30s and paired pulse facilitation (PPF) examined to assess neurotransmitter release. Stable baseline responses were recorded prior to application of simvastatin or LTP induction. LTP was induced using two trains of stimuli at 100Hz for one second applied 30s apart. In control mice (18month), acute application of simvastatin (35μM) caused a significant increase in the amplitude of the cAP, a shift in the input/put put curve and a decrease in PPF while in age matched APPswe/PS1dE9 mice acute simvastatin had no affect. A lower concentration (10μM) also caused an increased in cAP amplitude which was absent in slices from APPswe/PS1dE9 mice.This increased excitability is blocked by antagonists of PI3 kinase. Acute simvastatin application also had no effect in slices from animals previously treated chronically with simvastatin in their diet. In addition, chronic simvastatin treatment rescued LTP in APPSwe/PS1dE9 mice (18 and 8 months old) however it failed to rescue STP. Chronic simvastatin treatment can therefore rescue LTP in hippocampal CA1 in APPswe/PS1dE9 mice. In addition, acute simvastatin enhances neuronal excitability via a PI3 kinase dependent mechanism which is absent in slices from APPswe/PS1dE9 mice.

Nonfibrillar, water-soluble low-molecular weight assemblies of the amyloid β-protein (Aβ) are believed to play an important role in Alzheimer's disease (AD). Aqueous extracts of human brain contain Aβ assemblies that migrate on SDS-polyacrylamide gels and elute from size exclusion as dimers (∼8 kDa) and can block long-term potentiation and impair memory consolidation in the rat. Such species are detected specifically and sensitively in extracts of Alzheimer brain suggesting that SDS-stable dimers may be the basic building blocks of AD-associated synaptotoxic assemblies. Consequently, understanding the structure and properties of Aβ dimers is of great interest. In the absence of sufficient brain-derived dimer to facilitate biophysical analysis, we generated synthetic dimers designed to mimic the natural species. For this, Aβ(1-40) containing cysteine in place of serine 26 was used to produce disulphide cross-linked dimer, (AβS26C)2. Such dimers had no detectable secondary structure, produced an analytical ultracentrifugation profile consistent for an ∼8.6 kDa protein, and had no effect on hippocampal long-term potentiation (LTP). However, (AβS26C)2 aggregated more rapidly than either AβS26C or wild-type monomers and formed parastable β-sheet rich, thioflavin T-positive, protofibril-like assemblies. Whereas wild-type Aβ aggregated to form typical amyloid fibrils, the protofibril-like structures formed by (AβS26C)2 persisted for prolonged periods and potently inhibited LTP in mouse hippocampus. These data support the idea that Aβ dimers may stabilize the formation of fibril intermediates by a process distinct from that available to Aβ monomer and that higher molecular weight prefibrillar assemblies are the proximate mediators of Aβ toxicity.

Beta‐amyloid (Aβ) is a neuro‐peptide implicated in the pathogenesis of Alzheimer's disease (AD). Aβ‐peptide is known to disrupt cellular processes, including synaptic plasticity. To date, the precise mechanisms leading to the Aβ‐mediated impairment of normal neurophysiological function still remains elusive. A rise in the pro‐inflammatory cytokine interleukin‐1‐β (IL‐1β) has been previously reported, following Aβ peptide insult. IL‐1β in turn, activates a cascade of pro‐apoptotic markers, gradually leading to cell death. In this work, we have investigated the possible protective effects of interleukin‐1 receptor antagonist (IL‐1ra) on the effects of Aβ‐peptide on long‐term potentiation (LTP) in the CA1 region of the rat hippocampus in vivo. We observed a significant depression of LTP in the group of animals that received intracerebroventricular (icv) injection of Aβ‐peptide (1–40) compared with control animals injected with vehicle. Administration of IL‐1ra alone (icv) also resulted in a depression of LTP; however, there was no change in the baseline synaptic response. Combined injection of Aβ(1–40) + IL‐1ra caused an attenuation of the effects observed with Aβ(1–40) alone for a period of up to 15 min following LTP induction; rescuing post‐tetanicpotentiation (PTP). Gradually however, EPSP‐values declined to produce a level of LTP similar to that observed following treatment with Aβ(1–40) alone. These results suggest that the acute Aβ‐mediated impairment of PTP and LTP may be partial as a result of activation of an inflammatory response and the release of IL‐1β. The attenuation of plasticity by IL‐1ra alone supports the theory that low levels of IL‐1β are required for normal synaptic plasticity. The limited rescue of the Aβ‐mediated effects on LTP, in the presence of IL‐1ra, may represent the short half life found with this receptor antagonist in vivo. © 2008 Wiley‐Liss, Inc.

PURPOSE:Metabotropic receptor agonists that signal through G(q)-coupled pathways increase Ca(2+) in mammalian Müller cells by release from intracellular stores and Ca(2+) influx pathways that have not been well described. The authors examined the involvement of voltage-dependent and non-voltage-dependent Ca(2+) channels in metabotropic muscarinic receptor-activated Ca(2+) increases and store-operated Ca(2+) influx in cultured mouse Müller cells.METHODS:Intracellular Ca(2+) was measured using fluorescence imaging with the ratiometric dye fura-2. Currents were recorded using the whole-cell patch-clamp recording method: mRNA and protein were identified using reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical approaches.RESULTS:The muscarinic receptor agonist carbachol (3-20 microM) produced increases in Ca(2+) that were blocked by the muscarinic receptor antagonists atropine and pirenzepine. RT-PCR confirmed mRNA for metabotropic M1 muscarinic receptors. Depletion of Ca(2+) stores by the sarcoplasmic/endoplasmic Ca(2+) ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid or the inhibition of phospholipase C occluded the carbachol-activated increase in Ca(2+). Carbachol-activated Ca(2+) increases in Müller cells were enhanced by the diacylglycerol derivative 1-oleyl-2-acetyl-sn-glycerol and were blocked by transient receptor potential (TRP) channel blockers Gd(3+), La(3+), 2-APB, and flufenamic acid. Both muscarinic receptor activation and thapsigargin treatment depleted Ca(2+) stores and produced Ca(2+) entry that was attenuated by La(3+), 2-APB, Gd(3+), and flufenamic acid. mRNA and protein for TRPC1 and TRPC6 were present in mouse Müller cells, and carbachol activated a Gd(3+)-sensitive, TRP-like cation channel.CONCLUSIONS:Metabotropic muscarinic receptor-activated Ca(2+) increases in mouse Müller cells require the release of Ca(2+) from intracellular stores and the activation of Ca(2+) entry that involves TRP-like cation channels but is independent of voltage-dependent Ca(2+) channels.

We have investigated changes in the morphological structure of A beta 1-40 during different incubation time periods at 37 degrees C ranging from 1 h to 7 days using Thioflavin T, Congo red binding and electron microscopy. We found distinctive changes in A assembly demonstrating the formation of beta pleated sheets following 7-day incubation. Here we demonstrate that samples of the same A beta 1-40 peptide that are morphologically distinct can both attenuate hippocampal long-term potentiation (LTP) in the CA1 in vivo. The peptides were applied via intracerebroventricular injection and the effects on synaptic transmission, post-tetanic potentiation (PTP) and LTP were observed. The effects of A beta 1-40 that had either been freshly solubilized (FS-A beta) or incubated at 37 degrees C for 7 days (7D-A beta) were examined. FS-A beta and 7D-A beta peptide were both found to significantly attenuate LTP, although the assembly states of these peptides appeared to be completely different. Paired pulse facilitation (PPF) with an inter-stimulus interval of So ins was also monitored prior to, following peptide injection and 60 min following LTP induction. 7D-A beta caused an increase in PPF prior to LTP induction and also depressed PTP. Our data demonstrate that, while both forms of the peptide can attenuate LTP, the fibrillar form of the peptide may also influence transmitter release. (C) 2007 Elsevier B.V. All rights reserved.

The age-related deficit in long-term potentiation (LTP) in the dentate gyrus is positively correlated with hippocampal concentration of the pro-inflammatory cytokine, interleukin-1 beta (IL-1 beta). Previous evidence also indicates that the inhibition of LTP induced by intracerebroventricular injection of amyloid-beta(1-40) (A beta) is accompanied by increased hippocampal IL-1 beta concentration and IL-1 beta-stimulated signalling, specifically activation of the stress-activated protein kinase, c-jun N-terminal kinase (JNK). We considered that the underlying age-related neuroinflammation may render older rats more susceptible to A beta administration and, to investigate this, young, middle-aged and aged rats were injected intracerebroventricularly with A beta or vehicle. Hippocampal IL-1 beta concentration, JNK phosphorylation, expression of the putative A beta receptor, Receptor for advanced glycation end products (RAGE) and the microglial cell surface marker, CD40 were assessed. We report that A beta inhibited LTP in a concentration-dependent manner in young rats and that this was accompanied by concentration-dependent increases in hippocampal IL-1 beta and expression of phosphorylated JNK, RAGE and CD40. While 20 mu mol/L A beta exerted no significant effect on LTP in young rats, it inhibited LTP in middle-aged and aged rats and the increased vulnerability of aged rats was associated with increased IL-1 beta concentration. Treatment of rats with eicosapentaenoic acid attenuated the inhibitory effect of 60 mu mol/L A beta on LTP in young rats and the effect of 20 mu mol/L A beta in middle-aged and aged rats. We present evidence which indicates that the effect of eicosapentaenoic acid may be linked with its ability to stimulate activation of peroxisome proliferator-activated receptor gamma.

The data we present here suggest that agonists of peroxisome proliferator-activated receptor-γ (PPARγ) can attenuate the effects of beta-amyloid peptide (Aβ). Alzheimer's disease is associated with elevated levels of Aβ, and enhanced expression of PPARγ. In this study, we determined that application of Aβ[1–40] could impair hippocampal post-tetanic potentiation (PTP) and long-term potentiation (LTP) in vitro. We investigated the effects of PPARγ agonists; troglitazone, ciglitazone and 15-deoxy-Δ12,14 prostaglandin J2 (PGJ2) on synaptic transmission and plasticity in area CA1. Both ciglitazone and PGJ2 increased baseline synaptic transmission significantly, without altering paired-pulse facilitation. PGJ2 produced a significant reduction in LTP, whereas ciglitazone and troglitazone had no significant effect. In addition, prior application of each ligand attenuated the previously observed Aβ[1–40]-mediated impairment of LTP. The effect of troglitazone on the Aβ[1–40]-mediated impairment of LTP was not reversed by the PPARγ antagonist, GW-9662. These findings demonstrate that PPARγ agonists attenuate the effects of Aβ on LTP, and support the potential use of these agents to alleviate the symptoms of Alzheimer's disease. We also suggest that PPARγ agonists may regulate expression of hippocampal LTP in vitro.

The effects of the beta-amyloid peptide (Aβ) fragment 25–35 were investigated on hippocampal synaptic transmission and long-term potentiation (LTP) in vitro. Aβ[25–35] was found to impair both post-tetanic potentiation (PTP) and LTP in the hippocampal CA1. The anthra[1,9-cd]pyrazol-6(2H)-one, SP600125, was used to inhibit c-Jun N-terminal kinase (JNK) activity, which is believed to mediate cell death. Prior application of SP600125 attenuated the Aβ[25–35]-mediated impairment of PTP and LTP, when measured from the pre-drug baseline. In the presence of SP600125 alone, we observed an increase in baseline synaptic transmission and reduction in paired-pulse facilitation, consistent with an increase in synaptic transmission. There was no alteration in the level of PTP and LTP obtained, when measured from the pre-drug baseline. In the presence of both SP600125 and Aβ, however, PTP was greatly enhanced compared with controls. We therefore suggest that the activation of the JNK signalling pathway mediates the effects of Aβ on synaptic plasticity. Our data also indicate that endogenous JNK activity may regulate neurotransmitter release in the hippocampal CA1 in vitro.

Hippocampal long-term potentiation (LTP) is a form of synaptic plasticity used as a cellular model of memory. Beta amyloid (A beta) is involved in Alzheimer's disease (AD), a neurode-generative disorder leading to cognitive deficits. Nicotine is also claimed to act as a cognitive enhancer. A beta is known to bind with high affinity to the alpha 7-nicotinic acetylcholine receptor (nAChR). Here we have investigated the effect of intracerebroventricular (i.c.v.) injection of the endogenous peptide A beta 1-40 on LTP in area CA1 of urethananesthetized rats. We also examined the effect of A beta 12-28 (i.c.v.), which binds with high affinity to the alpha 7-nAChR and the specific alpha 7-nAChR antagonist methyllycaconitine (MLA) on LTP. We found that A beta 12-28 had no effect on LTP, whereas MLA depressed significantly LTP, suggesting that activation of the alpha 7-nAChR is a requirement for LTP. Within the in vivo environment, where other factors may compete with A beta 12-28 for binding to alpha 7-nAChR, it does not appear to modulate LTP. To determine if the depressive action of A beta 1-40 on LTP could be modulated by nicotine, these agents were also co-applied. Injection of 1 or 10 nmol A beta 1-40 caused a significant depression of LTP, whereas nicotine alone (3 mg/kg) had no effect on LTP. Co-injection of nicotine with A beta 1-40 1 h prior to LTP induction caused a further significant depression of LTP compared with A beta 1-40 alone. These results demonstrate that nicotine enhances the deficit in LTP produced by A beta 1-40. This then suggests that nicotine may exacerbate the depressive actions of A beta on synaptic plasticity in AD.

Long-term potentiation (LTP), in the hippocampal CA1 region is dependent on postsynaptic calcium influx. It is generally accepted that calcium influx occurs via activation of the NMDA receptor channel complex. However, studies in vitro using a high-frequency stimulus protocol (≥200 Hz) demonstrated previously an NMDA receptor-independent form of LTP that is dependent upon activation of L-type voltage-dependent calcium channels (VDCCs). Here we have investigated a role for L-type VDCCs in LTP in vivo. Two structurally different, L-type VDCC blockers, verapamil (1, 3 and 10 mg/kg) and diltiazem (1, 10 and 20 mg/kg), depressed the induction of LTP in a dose-dependent manner. Increased activation of L-type VDCCs by Bay K 8644, an L-type agonist, however, did not enhance LTP. The NMDA receptor antagonist d-AP5 (5 and 20 mM injected i.c.v) impaired, but failed to block fully LTP in vivo. A reduced level of LTP could still be recorded following co-administration of verapamil and d-AP5. The level of LTP recorded was similar to that observed in the presence of either verapamil (10 mg/kg) or d-AP5 alone. These results suggest that activation of the NMDA receptor/channel and L-type VDCCs are involved in the induction of LTP in area CA1 in vivo. However, it appears that activation of other receptor/channels may also play a role in this form of LTP.

Amyloid-beta (Abeta) is a major constituent of the neuritic plaque found in the brain of Alzheimer's disease patients, and a great deal of evidence suggests that the neuronal loss that is associated with the disease is a consequence of the actions of Abeta. In the past few years, it has become apparent that activation of c-Jun N-terminal kinase (JNK) mediates some of the effects of Abeta on cultured cells; in particular, the evidence suggests that Abeta-triggered JNK activation leads to cell death. In this study, we investigated the effect of intracerebroventricular injection of Abeta(1-40) on signaling events in the hippocampus and on long term potentiation in Schaffer collateral CA1 pyramidal cell synapses in vivo. We report that Abeta(1-40) induced activation of JNK in CA1 and that this was coupled with expression of the proapoptotic protein, Bax, cytosolic cytochrome c, poly-(ADP-ribose) polymerase cleavage, and Fas ligand expression in the hippocampus. These data indicate that Abeta(1-40) inhibited expression of long term potentiation, and this effect was abrogated by administration of the JNK inhibitor peptide, D-JNKI1. In parallel with these findings, we observed that Abeta-induced changes in caspase-3 activation and TdT-mediated dUTP nick-end labeling staining in neuronal cultured cells were inhibited by D-JNKI1. We present evidence suggesting that interleukin (IL)-1beta plays a significant role in mediating the effects of Abeta(1-40) because Abeta(1-40) increased hippocampal IL-1beta and because several effects of Abeta(1-40) were inhibited by the caspase-1 inhibitor Ac-YVAD-CMK. On the basis of our findings, we propose that Abeta-induced changes in hippocampal plasticity are likely to be dependent upon IL-1beta-triggered activation of JNK.