Raik Rönicke

Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-β

Extracellular plaques of amyloid-β and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer’s disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer’s disease. Despite the importance of plaques to Alzheimer’s disease, oligomers are considered to be the principal toxic forms of amyloid-β. Interestingly, many adverse responses to amyloid-β, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-β are strongly associated with Alzheimer’s disease, are more toxic than amyloid-β, residues 1–42 (Aβ1–42) and Aβ1–40, and have been proposed as initiators of Alzheimer’s disease pathogenesis. Here we report a mechanism by which pE-Aβ may trigger Alzheimer’s disease. Aβ3(pE)–42 co-oligomerizes with excess Aβ1–42 to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aβ1–42 alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aβ3(pE)–42 plus 95% Aβ1–42 (5% pE-Aβ) seed new cytotoxic LNOs through multiple serial dilutions into Aβ1–42 monomers in the absence of additional Aβ3(pE)–42. LNOs isolated from human Alzheimer’s disease brain contained Aβ3(pE)–42, and enhanced Aβ3(pE)–42 formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aβ3(pE)–42 confers tau-dependent neuronal death and causes template-induced misfolding of Aβ1–42 into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aβ3(pE)–42 acts similarly at a primary step in Alzheimer’s disease pathogenesis.

Mechanism of amyloid plaque formation suggests an intracellular basis of Aβ pathogenicity

The formation of extracellular amyloid plaques is a common patho-biochemical event underlying several debilitating human conditions, including Alzheimer’s disease (AD). Considerable evidence implies that AD damage arises primarily from small oligomeric amyloid forms of Aβ peptide, but the precise mechanism of pathogenicity remains to be established. Using a cell culture system that reproducibly leads to the formation of Alzheimer’s Aβ amyloid plaques, we show here that the formation of a single amyloid plaque represents a template-dependent process that critically involves the presence of endocytosis- or phagocytosis-competent cells. Internalized Aβ peptide becomes sorted to multivesicular bodies where fibrils grow out, thus penetrating the vesicular membrane. Upon plaque formation, cells undergo cell death and intracellular amyloid structures become released into the extracellular space. These data imply a mechanism where the pathogenic activity of Aβ is attributed, at least in part, to intracellular aggregates.

Early neuronal dysfunction by amyloid β oligomers depends on activation of NR2B-containing NMDA receptors.

Several studies indicate that NMDA receptor signaling is involved in Aβ oligomer-mediated impairment of neuronal function and morphology. Utilizing primary neuronal cell culture and hippocampal slices from rat and mouse, we found that Aβ oligomer administration readily impairs long-term potentiation, reduces baseline synaptic transmission, decreases neuronal spontaneous network activity and induces retraction of synaptic contacts long before major cytotoxic effects are visible. Interestingly, all these effects can be blocked with the NR2B-containing NMDA-receptor antagonist ifenprodil or Ro 25-6981 suggesting that activation of downstream effectors of these receptors is involved in early detrimental actions of Aβ oligomers. In line we found that Jacob, a messenger that can couple extrasynaptic NMDA-receptor activity to CREB dephosphorylation, accumulates in the nucleus after Aβ oligomer administration and that the nuclear accumulation of Jacob can be blocked by a simultaneous application of ifenprodil. We conclude that Aβ oligomers induce early neuronal dysfunction mainly by activation of NR2B-containing NMDA-receptors.

Microglia Cells Protect Neurons by Direct Engulfment of Invading Neutrophil Granulocytes: A New Mechanism of CNS Immune Privilege

Microglial cells maintain the immunological integrity of the healthy brain and can exert protection from traumatic injury. During ischemic tissue damage such as stroke, peripheral immune cells acutely infiltrate the brain and may exacerbate neurodegeneration. Whether and how microglia can protect from this insult is unknown. Polymorphonuclear neutrophils (PMNs) are a prominent immunologic infiltrate of ischemic lesions in vivo. Here, we show in organotypic brain slices that externally applied invading PMNs massively enhance ischemic neurotoxicity. This, however, is counteracted by additional application of microglia. Time-lapse imaging shows that microglia exert protection by rapid engulfment of apoptotic, but, strikingly, also viable, motile PMNs in cell culture and within brain slices. PMN engulfment is mediated by integrin- and lectin-based recognition. Interference with this process using RGDS peptides and N-acteyl-glucosamine blocks engulfment of PMNs and completely abrogates the neuroprotective function of microglia. Thus, engulfment of invading PMNs by microglia may represent an entirely new mechanism of CNS immune privilege.

N-Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long-term potentiation

J. Neurochem. (2012) 121, 774–784.

Molecular basis of β-amyloid oligomer recognition with a conformational antibody fragment

Oligomers are intermediates of the β-amyloid (Aβ) peptide fibrillogenic pathway and are putative pathogenic culprits in Alzheimer’s disease (AD). Here we report the biotechnological generation and biochemical characterization of an oligomer-specific antibody fragment, KW1. KW1 not only discriminates between oligomers and other Aβ conformations, such as fibrils or disaggregated peptide; it also differentiates between different types of Aβ oligomers, such as those formed by Aβ (1–40) and Aβ (1–42) peptide. This high selectivity of binding contrasts sharply with many other conformational antibodies that interact with a large number of structurally analogous but sequentially different antigens. X-ray crystallography, NMR spectroscopy, and peptide array measurements imply that KW1 recognizes oligomers through a hydrophobic and significantly aromatic surface motif that includes Aβ residues 18–20. KW1-positive oligomers occur in human AD brain samples and induce synaptic dysfunctions in living brain tissues. Bivalent KW1 potently neutralizes this effect and interferes with Aβ assembly. By altering a specific step of the fibrillogenic cascade, it prevents the formation of mature Aβ fibrils and induces the accumulation of nonfibrillar aggregates. Our data illuminate significant mechanistic differences in oligomeric and fibril recognition and suggest the considerable potential of KW1 in future studies to detect or inhibit specific types of Aβ conformers.

Combining Independent Drug Classes into Superior, Synergistically Acting Hybrid Molecules

Increasing the potency of synthesized drugs has been a stepwise process accomplished by progressively modifying the chemical scaffold of a single parent lead compound. To date, there has been no basis for thinking that the combination of pharmacological effects of independently acting drugs could be achieved beyond mere simultaneous administration. We reasoned that if the target molecule of two independent classes of drugs was the same, chemical synthesis of a hybrid compound where these drugs presented moieties within one molecule might yield synergistic effects; that is, a new quality might emerge that would be more than the sum of the singlemoiety compounds. Such multifunctional hybrid compounds that assign different functions to its different moieties to achieve a synergistic pharmacodynamic effect have successful predecessors in nature: for example, bleomycin is a natural compound with three different moieties acting in concert to cleave DNA.

K-Lysine acetyltransferase 2a regulates a hippocampal gene expression network linked to memory formation

Neuronal histone acetylation has been linked to memory consolidation, and targeting histone acetylation has emerged as a promising therapeutic strategy for neuropsychiatric diseases. However, the role of histone‐modifying enzymes in the adult brain is still far from being understood. Here we use RNA sequencing to screen the levels of all known histone acetyltransferases (HATs) in the hippocampal CA1 region and find that K‐acetyltransferase 2a (Kat2a)—a HAT that has not been studied for its role in memory function so far—shows highest expression. Mice that lack Kat2a show impaired hippocampal synaptic plasticity and long‐term memory consolidation. We furthermore show that Kat2a regulates a highly interconnected hippocampal gene expression network linked to neuroactive receptor signaling via a mechanism that involves nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB). In conclusion, our data establish Kat2a as a novel and essential regulator of hippocampal memory consolidation.

Amyloid-Beta Induced Changes in Vesicular Transport of BDNF in Hippocampal Neurons

The neurotrophin brain derived neurotrophic factor (BDNF) is an important growth factor in the CNS. Deficits in transport of this secretory protein could underlie neurodegenerative diseases. Investigation of disease-related changes in BDNF transport might provide insights into the cellular mechanism underlying, for example, Alzheimers disease (AD). To analyze the role of BDNF transport in AD, live cell imaging of fluorescently labeled BDNF was performed in hippocampal neurons of different AD model systems. BDNF and APP colocalized with low incidence in vesicular structures. Anterograde as well as retrograde transport of BDNF vesicles was reduced and these effects were mediated by factors released from hippocampal neurons into the extracellular medium. Transport of BDNF was altered at a very early time point after onset of human APP expression or after acute amyloid-beta(1-42) treatment, while the activity-dependent release of BDNF remained unaffected. Taken together, extracellular cleavage products of APP induced rapid changes in anterograde and retrograde transport of BDNF-containing vesicles while release of BDNF was unaffected by transgenic expression of mutated APP. These early transport deficits might lead to permanently impaired brain functions in the adult brain.

Oligomer-targeting with a conformational antibody fragment promotes toxicity in Aβ-expressing flies

IntroductionThe self-assembly of Aβ peptides into a range of conformationally heterogeneous amyloid states represents a fundamental event in Alzheimer’s disease. Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized in vivo by state-specific antibody fragments.ResultsWe show that oligomer targeting with the KW1 antibody fragment, but not fibril targeting with the B10 antibody fragment, affects toxicity in Aβ-expressing Drosophila melanogaster. The effect of KW1 is observed to occur selectively with flies expressing Aβ(1–40) and not with those expressing Aβ(1–42) or the arctic variant of Aβ(1–42) This finding is consistent with the binding preference of KW1 for Aβ(1–40) oligomers that has been established in vitro. Strikingly, and in contrast to the previously demonstrated in vitro ability of this antibody fragment to block oligomeric toxicity in long-term potentiation measurements, KW1 promotes toxicity in the flies rather than preventing it. This result shows the crucial importance of the environment in determining the influence of antibody binding on the nature and consequences of the protein misfolding and aggregation.ConclusionsWhile our data support to the pathological relevance of oligomers, they highlight the issues to be addressed when developing inhibitory strategies that aim to neutralize these states by means of antagonistic binding agents.