The Familial British Dementia Mutation Promotes Formation of Neurotoxic Cystine Cross-linked Amyloid Bri (ABri) Oligomers.

Familial British dementia (FBD) is an inherited neurodegenerative disease believed to result from a mutation in the BRI2 gene. Post-translational processing of wild type BRI2 and FBD-BRI2 result in the production of a 23-residue long Bri peptide and a 34-amino acid long ABri peptide, respectively, and ABri is found deposited in the brains of individuals with FBD. Similarities in the neuropathology and clinical presentation shared by FBD and Alzheimer disease (AD) have led some to suggest that ABri and the AD-associated amyloid ß-protein (Aß) are molecular equivalents that trigger analogous pathogenic cascades. But the sequences and innate properties of ABri and Aß are quite different, notably ABri contains two cysteine residues that can form disulfide bonds. Thus we sought to determine whether ABri was neurotoxic and if this activity was regulated by oxidation and/or aggregation. Crucially, the type of oxidative cross-linking dramatically influenced both ABri aggregation and toxicity. Cyclization of Bri and ABri resulted in production of biologically inert monomers that showed no propensity to assemble, whereas reduced ABri and reduced Bri aggregated forming thioflavin T-positive amyloid fibrils that lacked significant toxic activity. ABri was more prone to form inter-molecular disulfide bonds than Bri and the formation of covalently stabilized ABri oligomers was associated with toxicity. These results suggest that extension of the C-terminal of Bri causes a shift in the type of disulfide bonds formed and that structures built from covalently cross-linked oligomers can interact with neurons and compromise their function and viability.

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.

Interaction between prion protein and toxic amyloid ß assemblies can be therapeutically targeted at multiple sites.

A role for PrP in the toxic effect of oligomeric forms of Aß, implicated in Alzheimer's disease (AD), has been suggested but remains controversial. Here we show that PrP is required for the plasticity-impairing effects of ex vivo material from human AD brain and that standardized Aß-derived diffusible ligand (ADDL) preparations disrupt hippocampal synaptic plasticity in a PrP-dependent manner. We screened a panel of anti-PrP antibodies for their ability to disrupt the ADDL-PrP interaction. Antibodies directed to the principal PrP/Aß-binding site and to PrP helix-1, were able to block Aß binding to PrP suggesting that the toxic Aß species are of relatively high molecular mass and/or may bind multiple PrP molecules. Two representative and extensively characterized monoclonal antibodies directed to these regions, ICSM-35 and ICSM-18, were shown to block the Aß-mediated disruption of synaptic plasticity validating these antibodies as candidate therapeutics for AD either individually or in combination.

Aß oligomers inhibit synapse remodelling necessary for memory consolidation.

Extensive research has implicated the amyloid-ß protein (Aß) in the aetiology of Alzheimer's disease (AD). This protein has been shown to produce memory deficits when injected into rodent brain and in mouse models of AD Aß production is associated with impaired learning and/or recall. Here we examined the effects of cell-derived SDS-stable 7PA2-derived soluble Aß oligomers on consolidation of avoidance learning. At 0, 3, 6, 9 or 12h after training, animals received an intracerebroventricular injection of Aß-containing or control media and recall was tested at 24 and 48 h. Immediately after 48 h recall animals were transcardially perfused and the brain removed for sectioning and EM analysis. Rats receiving injections of Aß at 6 or 9h post-training showed a significant impairment in memory consolidation at 48 h. Importantly, impaired animals injected at 9h had significantly fewer synapses in the dentate gyrus. These data suggest that Aß low-n oligomers target specific temporal facets of consolidation-associated synaptic remodelling whereby loss of functional synapses results in impaired consolidation.

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.

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.

Amyloid beta peptide as a physiological modulator of neuronal 'A'-type K+ current.

Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K(+) channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid beta protein (Abeta) can increase the 'A'-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Abeta is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones. A-type K(+) currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1-40 and 1-42 forms of Abeta (100nM, 2-24h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in 'A'-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Abeta, rather than protein degredation. Endogenous Abeta production in cerebellar granule neurones was blocked using inhibitors of either gamma- or beta-secretase and resulted in decreased K(+) current. Crucially this could be prevented by co-application of exogenous Abeta (1nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred. These data show that Abeta is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Abeta is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

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.