Bruce L. Kagan

Impairment of hippocampal long-term potentiation by Alzheimer amyloid ?-peptides

Although it is generally believed that amyloid β (Aβ) peptides contribute to the pathogenesis of Alzheimers disease, the precise role of these peptides in the development of memory loss of Alzheimers disease, has not been fully understood. The present study examined the effect of several synthetic Aβ peptides on long‐term potentiation (LTP), a cellular model of learning and memory, in rat hippocampal slices. Brief perfusion of slices with low concentrations (200 nM or 1 μM) of Aβ1–42, Aβ1–40 or their active fragment Aβ25‐–35 significantly inhibited LTP induction without affecting the basal synaptic transmission and posttetanic potentiation in the dentate medial perforant path. A similar effect of Aβ25–35 was also observed in the Schaffer colleteral‐CA1 pathway. When comparing actions of several Aβ variants derived from Aβ25–35, the N‐terminal sequence of Aβ25–35 was found necessary for inhibiting LTP. In addition, Aβ variants lacking neurotoxic action and aggregating property were also able to block LTP, suggesting that this effect was neurotoxicity independent. Our findings demonstrated that subneurotoxic concentrations of Aβ peptides could strongly suppress long‐term synaptic plasticity in the hippocampus. Such an effect might underlie the memory deficits seen in Alzheimers disease before neuronal cell loss. J. Neurosci. Res. 60:65–72, 2000

The channel hypothesis of Alzheimer’s disease: current status

The channel hypothesis of Alzheimers disease (AD) proposes that the beta-amyloid (Abeta) peptides which accumulate in plaques in the brain actually damage and/or kill neurons by forming ion channels. Evidence from a number of laboratories has demonstrated that Abeta peptides can form ion channels in lipid bilayers, liposomes, neurons, oocyctes, and endothelial cells. These channels possess distinct physiologic characteristics that would be consistent with their toxic properties. Abeta channels are heterogeneous in size, selectivity, blockade, and gating. They are generally large, voltage-independent, and relatively poorly selective amongst physiologic ions, admitting calcium ion (Ca(2+)), Na(+), K(+), Cs(+), Li(+), and possibly Cl(-). They are reversibly blocked by zinc ion (Zn(2+)), and tromethamine (tris), and irreversibly by aluminum ion (Al(3+)). Congo red inhibits channel formation, but does not block inserted channels. Although much evidence implicates Abeta peptides in the neurotoxicity of AD, no other toxic mechanism has been demonstrated to be the underlying etiology of AD. Channel formation by several other amyloid peptides lends credence to the notion that this is a critical mechanism of cytotoxicity.

Ion channel activity of the BH3 only Bcl-2 family member, BID.

BID is a member of the BH3-only subgroup of Bcl-2 family proteins that displays pro-apoptotic activity. The NH2-terminal region of BID contains a caspase-8 (Casp-8) cleavage site and the cleaved form of BID translocates to mitochondrial membranes where it is a potent inducer of cytochromec release. Secondary structure and fold predictions suggest that BID has a high degree of α-helical content and structural similarity to Bcl-XL, which itself is highly similar to bacterial pore-forming toxins. Moreover, circular dichroism analysis confirmed a high α-helical content of BID. Amino-terminal truncated BIDΔ1–55, mimicking the Casp-8-cleaved molecule, formed channels in planar bilayers at neutral pH and in liposomes at acidic pH. In contrast, full-length BID displayed channel activity only at nonphysiological pH 4.0 (but not at neutral pH) in planar bilayers and failed to form channels in liposomes even under acidic conditions. On a single channel level, BIDΔ1–55 channels were voltage-gated and exhibited multiconductance behavior at neutral pH. When full-length BID was cleaved by Casp-8, it too demonstrated channel activity similar to that seen with BIDΔ1–55. Thus, BID appears to share structural and functional similarity with other Bcl-2 family proteins known to have channel-forming activity, but its activity exhibits a novel form of activation: proteolytic cleavage.

Defensins : a family of antimicrobial and cytotoxic peptides

Defensins are antimicrobial and cytotoxic peptides that contain 29-35 amino acid residues, including 6 invariant cysteines that form 3 intramolecular disulfide bonds. They constitute more than 5% of the total cellular protein of human and rabbit neutrophils (PMN), and are also produced by rabbit lung macrophages and by murine and human small intestinal Paneth cells. Defensins exerted antimicrobial effects in vitro against many Gram-positive and Gram-negative bacteria, fungi, mycobacteria and some enveloped viruses, and were cytotoxic to a wide range of normal and malignant targets, including cells resistant to TNF-alpha and NK-cytolytic factor. Human and rabbit defensins formed voltage-sensitive channels in a variety of planar lipid bilayers when a negative voltage of approximately 70-90 mV was applied to the contralateral side. These channels showed modest anion selectivity and their formation was strongly influenced by defensin concentration. Although most other channel-forming peptides have prominent alpha-helical domains, the structure of defensin molecules is primarily composed of antiparallel beta-sheets. Studies with various prokaryotic and eukaryotic cells provided convincing evidence that defensins killed these targets by forming voltage-regulated channels in the susceptible cells membrane. The broad spectrum of defensin-susceptible targets and the abundance of defensins in specialized host defense cells of the blood, lungs and intestinal tract suggest that defensins could play a significant role in innate immunity to infection and neoplasia.

Amyloid peptide channels.

At least 16 distinct clinical syndromes including Alzheimer’s disease (AD), Parkinson’s disease (PD), rheumatoid arthritis, type II diabetes mellitus (DM), and spongiform encephelopathies (prion diseases), are characterized by the deposition of amorphous, Congo red-staining deposits known as amyloid. These “misfolded” proteins adopt β-sheet structures and aggregate spontaneously into similar extended fibrils despite their widely divergent primary sequences. Many, if not all, of these peptides are capable of forming ion-permeable channels in vitro and possibly in vivo. Common channel properties include irreversible, spontaneous insertion into membranes, relatively large, heterogeneous single-channel conductances, inhibition of channel formation by Congo red, and blockade of inserted channels by Zn2+. Physiologic effects of amyloid, including Ca2+ dysregulation, membrane depolarization, mitochondrial dysfunction, inhibition of long-term potentiation (LTP), and cytotoxicity, suggest that channel formation in plasma and intracellular membranes may play a key role in the pathophysiology of the amyloidoses.

Truncated β-amyloid peptide channels provide an alternative mechanism for Alzheimer’s Disease and Down syndrome

Full-length amyloid beta peptides (Aβ1–40/42) form neuritic amyloid plaques in Alzheimer’s disease (AD) patients and are implicated in AD pathology. However, recent transgenic animal models cast doubt on their direct role in AD pathology. Nonamyloidogenic truncated amyloid-beta fragments (Aβ11–42 and Aβ17–42) are also found in amyloid plaques of AD and in the preamyloid lesions of Down syndrome, a model system for early-onset AD study. Very little is known about the structure and activity of these smaller peptides, although they could be the primary AD and Down syndrome pathological agents. Using complementary techniques of molecular dynamics simulations, atomic force microscopy, channel conductance measurements, calcium imaging, neuritic degeneration, and cell death assays, we show that nonamyloidogenic Aβ9–42 and Aβ17–42 peptides form ion channels with loosely attached subunits and elicit single-channel conductances. The subunits appear mobile, suggesting insertion of small oligomers, followed by dynamic channel assembly and dissociation. These channels allow calcium uptake in amyloid precursor protein-deficient cells. The channel mediated calcium uptake induces neurite degeneration in human cortical neurons. Channel conductance, calcium uptake, and neurite degeneration are selectively inhibited by zinc, a blocker of amyloid ion channel activity. Thus, truncated Aβ fragments could account for undefined roles played by full length Aβs and provide a unique mechanism of AD and Down syndrome pathologies. The toxicity of nonamyloidogenic peptides via an ion channel mechanism necessitates a reevaluation of the current therapeutic approaches targeting the nonamyloidogenic pathway as avenue for AD treatment.

Alzheimer amyloid aβ1–42 channels: Effects of solvent, pH, and congo red

Substantial genetic and biochemical evidence implicates amyloid peptides (A|fg) in the etiology of Alzheimers Disease (AD). Recent evidence indicates that A|fg1–42 is the predominant species in the hallmark senile amyloid plaque of AD. Furthermore, A|fg1–42 forms aggregates inside lysosomes of cultured neurons leading to lysosomal disruption and cell death. We report here that A|fg1–42 forms slightly cation selective, voltage‐independent ion channels with multiple conductance levels at neurotoxic concentrations in planar bilayer membranes. The channels show substantial irregularity of activity, and the size of conductances and the length of open lifetimes depend on solvent history. Formation of channels requires anionic lipids, is enhanced in acidic solutions, and is inhibited by Congo Red. These properties suggest that the channels are formed by aggregates of A|fg1–42. In addition, the channels are reversibly blocked by zinc in a voltage‐independent manner. The properties of these channels would likely render them neurotoxic to relevant neurons in vivo. These results are consistent with the channel hypothesis of A|fg neurotoxicity. J. Neurosci. Res. 57:467–478, 1999.

Membrane channel formation by antimicrobial protegrins.

Protegrins are small, arginine- and cysteine-rich, beta-sheet peptides with potent activity against bacteria, fungi, and certain enveloped viruses. We report that protegrins form weakly anion-selective channels in planar phospholipid bilayers, induce potassium leakage from liposomes and form moderately cation-selective channels in planar lipid membranes that contain bacterial lipopolysaccharide. The disruption of microbial membranes may be a central attribute related to the host defense properties of protegrins.

Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.

Antimicrobial Properties of Amyloid Peptides

More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous Congo red staining deposits known as amyloids exhibit a characteristic green birefringence and cross-β structure. Substantial evidence implicates oligomeric intermediates of amyloids as toxic species in the pathogenesis of these chronic disease states. A growing body of data has suggested that these toxic species form ion channels in cellular membranes causing disruption of calcium homeostasis, membrane depolarization, energy drainage, and in some cases apoptosis. Amyloid peptide channels exhibit a number of common biological properties including the universal U-shape β-strand-turn-β-strand structure, irreversible and spontaneous insertion into membranes, production of large heterogeneous single-channel conductances, relatively poor ion selectivity, inhibition by Congo red, and channel blockade by zinc. Recent evidence has suggested that increased amounts of amyloids not only are toxic to its host target cells but also possess antimicrobial activity. Furthermore, at least one human antimicrobial peptide, protegrin-1, which kills microbes by a channel-forming mechanism, has been shown to possess the ability to form extended amyloid fibrils very similar to those of classic disease-forming amyloids. In this paper, we will review the reported antimicrobial properties of amyloids and the implications of these discoveries for our understanding of amyloid structure and function.