Cannabidiol Reverses Deficits in Hippocampal LTP in a Model of Alzheimer's Disease.

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 (Aß) 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 (Aß1-42) associated with AD, attenuates LTP in the CA1 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 Aß1-42 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 5HT1A, adenosine (A2A) or Cannabinoid type 1 (CB1) 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.

Simvastatin treatment preserves synaptic plasticity in AßPPswe/PS1dE9 mice.

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 ß-protein (Aß) plays a central role in the disease process, and it has been suggested that the protective effects of simvastatin may be mediated by reducing Aß production or by counteracting the toxic effects of Aß. Accordingly, using the AßPPswe/PS1dE9 mouse model of AD, we investigated the effects of simvastatin on long-term potentiation (LTP), amyloid biology, and two key kinases involved in Aß-mediated toxicity. Since burgeoning data indicate that both fibrillar and non-fibrillar forms of Aß play a prominent role in AD pathogenesis, we were careful to investigate the effects of simvastatin on three biochemically distinct pools of Aß. In untreated AßPPswe/PS1dE9 mice, there was a dramatic and significant increase in the levels of water-soluble Aß 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 Aß species increased across all ages examined, thus demonstrating that while amyloid deposition continued, the levels of water-soluble Aß 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 AßPPswe/PS1dE9 mice and did not significantly alter levels of water-soluble, detergent-soluble, or FA-soluble Aß, 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.

Amyloid-ß nanotubes are associated with prion protein-dependent synaptotoxicity.

Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-ß protein (Aß) have important roles in Alzheimer's disease with toxicities mimicked by synthetic Aß(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 Aß(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 Aß assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in Aß-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of Aß nanotubes or their interaction with PrP might have a role in treatment of Alzheimer's disease.

Amyloid beta-protein dimers rapidly form stable synaptotoxic protofibrils.

Nonfibrillar, water-soluble low-molecular weight assemblies of the amyloid ß-protein (Aß) are believed to play an important role in Alzheimer's disease (AD). Aqueous extracts of human brain contain Aß 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 Aß 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, Aß(1-40) containing cysteine in place of serine 26 was used to produce disulphide cross-linked dimer, (AßS26C)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, (AßS26C)2 aggregated more rapidly than either AßS26C or wild-type monomers and formed parastable ß-sheet rich, thioflavin T-positive, protofibril-like assemblies. Whereas wild-type Aß aggregated to form typical amyloid fibrils, the protofibril-like structures formed by (AßS26C)2 persisted for prolonged periods and potently inhibited LTP in mouse hippocampus. These data support the idea that Aß dimers may stabilize the formation of fibril intermediates by a process distinct from that available to Aß monomer and that higher molecular weight prefibrillar assemblies are the proximate mediators of Aß toxicity.

The effects of IL-1 receptor antagonist on beta amyloid mediated depression of LTP in the rat CA1 in vivo.

Beta-amyloid (Abeta) is a neuro-peptide implicated in the pathogenesis of Alzheimer's disease (AD). Abeta-peptide is known to disrupt cellular processes, including synaptic plasticity. To date, the precise mechanisms leading to the Abeta-mediated impairment of normal neurophysiological function still remains elusive. A rise in the pro-inflammatory cytokine interleukin-1-beta (IL-1beta) has been previously reported, following Abeta peptide insult. IL-1beta 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 Abeta-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 Abeta-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 Abeta(1-40) + IL-1ra caused an attenuation of the effects observed with Abeta(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 Abeta(1-40) alone. These results suggest that the acute Abeta-mediated impairment of PTP and LTP may be partial as a result of activation of an inflammatory response and the release of IL-1beta. The attenuation of plasticity by IL-1ra alone supports the theory that low levels of IL-1beta are required for normal synaptic plasticity. The limited rescue of the Abeta-mediated effects on LTP, in the presence of IL-1ra, may represent the short half life found with this receptor antagonist in vivo.

Metabotropic receptor-activated calcium increases and store-operated calcium influx in mouse Müller cells.

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.

Inhibition of LTP in vivo by beta-amyloid peptide in different conformational states.

We have investigated changes in the morphological structure of Abeta1-40 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 Abeta assembly demonstrating the formation of beta pleated sheets following 7-day incubation. Here we demonstrate that samples of the same Abeta1-40 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 Abeta1-40 that had either been freshly solubilized (FS-Abeta) or incubated at 37 degrees C for 7 days (7D-Abeta) were examined. FS-Abeta and 7D-Abeta 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 50 ms was also monitored prior to, following peptide injection and 60 min following LTP induction. 7D-Abeta 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.

Modulation of amyloid-beta-induced and age-associated changes in rat hippocampus by eicosapentaenoic acid.

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-1beta (IL-1beta). Previous evidence also indicates that the inhibition of LTP induced by intracerebroventricular injection of amyloid-beta(1-40) (Abeta) is accompanied by increased hippocampal IL-1beta concentration and IL-1beta-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 Abeta administration and, to investigate this, young, middle-aged and aged rats were injected intracerebroventricularly with Abeta or vehicle. Hippocampal IL-1beta concentration, JNK phosphorylation, expression of the putative Abeta receptor, Receptor for advanced glycation end products (RAGE) and the microglial cell surface marker, CD40 were assessed. We report that Abeta inhibited LTP in a concentration-dependent manner in young rats and that this was accompanied by concentration-dependent increases in hippocampal IL-1beta and expression of phosphorylated JNK, RAGE and CD40. While 20 micromol/L Abeta 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-1beta concentration. Treatment of rats with eicosapentaenoic acid attenuated the inhibitory effect of 60 micromol/L Abeta on LTP in young rats and the effect of 20 micromol/L Abeta 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.

Agonists of peroxisome proliferator-activated receptor-gamma attenuate the Abeta-mediated impairment of LTP in the hippocampus in vitro.

The data we present here suggest that agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma) can attenuate the effects of beta-amyloid peptide (Abeta). Alzheimer's disease is associated with elevated levels of Abeta, and enhanced expression of PPARgamma. In this study, we determined that application of Abeta([1-40]) could impair hippocampal post-tetanic potentiation (PTP) and long-term potentiation (LTP) in vitro. We investigated the effects of PPARgamma agonists; troglitazone, ciglitazone and 15-deoxy-delta(12,14) 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 Abeta([1-40])-mediated impairment of LTP. The effect of troglitazone on the Abeta([1-40])-mediated impairment of LTP was not reversed by the PPARgamma antagonist, GW-9662. These findings demonstrate that PPARgamma agonists attenuate the effects of Abeta on LTP, and support the potential use of these agents to alleviate the symptoms of Alzheimer's disease. We also suggest that PPARgamma agonists may regulate expression of hippocampal LTP in vitro.

The role of c-Jun N-terminal kinase in the A beta-mediated impairment of LTP and regulation of synaptic transmission in the hippocampus.

The effects of the beta-amyloid peptide (Abeta) fragment 25-35 were investigated on hippocampal synaptic transmission and long-term potentiation (LTP) in vitro. Abeta([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 Abeta([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 Abeta, however, PTP was greatly enhanced compared with controls. We therefore suggest that the activation of the JNK signalling pathway mediates the effects of Abeta on synaptic plasticity. Our data also indicate that endogenous JNK activity may regulate neurotransmitter release in the hippocampal CA1 in vitro.

Activation of the c-Jun N-terminal kinase signaling cascade mediates the effect of amyloid-beta on long term potentiation and cell death in hippocampus: a role for interleukin-1beta?

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.

Inhibition of L-type voltage dependent calcium channels causes impairment of long-term potentiation in the hippocampal CA1 region in vivo.

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 (> or =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.