Although soluble oligomeric and protofibrillar assemblies of Aβ-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Aβ-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds ‘forward' in a near-irreversible manner from the monomeric Aβ peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Aβ amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Aβ but rather toward soluble amyloid protofibrils. We characterized these ‘backward' Aβ protofibrils generated from mature Aβ fibers and compared them with previously identified ‘forward' Aβ protofibrils obtained from the aggregation of fresh Aβ monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Aβ-aggregates that could readily be activated by exposure to biological lipids.
ADAM10 is involved in the proteolytic processing and shedding of proteins such as the amyloid precursor protein (APP), cadherins, and the Notch receptors, thereby initiating the regulated intramembrane proteolysis (RIP) of these proteins. Here, we demonstrate that the sheddase ADAM10 is also subject to RIP. We identify ADAM9 and -15 as the proteases responsible for releasing the ADAM10 ectodomain, and Presenilin/␥-Secretase as the protease responsible for the release of the ADAM10 intracellular domain (ICD). This domain then translocates to the nucleus and localizes to nuclear speckles, thought to be involved in gene regulation. Thus, ADAM10 performs a dual role in cells, as a metalloprotease when it is membrane-bound, and as a potential signaling protein once cleaved by ADAM9/15 and the ␥-Secretase. ADAMs8 (A disintegrin and metalloprotease) are type 1 transmembrane proteins related to snake venom integrin ligands and metalloproteases. All 38 different family members feature a common modular ectodomain structure (1-4) (Fig. 1A). In addition to the membrane-bound, full-length prototype, soluble ADAM variants have also been identified, consisting of only the ectodomain or fragments thereof that are released into the intercellular space. Such variants are generated by partial gene duplication (ADAM9) (5), alternative splicing (ADAM12) (6, 7), or proteolysis (ADAMs 8, 13, and 19) (8 -10). ADAMs can be grouped either by their tissue distribution and/or functional properties. One major group (ADAMs 2, 3, 5, 6, 16, 18, 20, 21, 24, 25, 26, 29, and 30) is expressed exclusively in the male gonad, with an emerging role in sperm maturation. A second group (ADAMs 2,7,11,18,22,23,and 29) is characterized by an inactive protease domain, and they seem to be mainly important for cell adhesion and fusion. A large third group of ADAMs displays a broad expression profile and has demonstrated (ADAMs 8,9,10,12,17,19, and 28) or predicted (ADAMs 15,20,21,30, and 33) proteolytic activity. These proteases play a major role in the ectodomain shedding of proteins involved in paracrine signaling, cell adhesion, and intracellular signaling (reviewed in Refs. 11 and 12). The site specificity of the cleavage of these substrates is rather relaxed, and apparently different family members can mutually compensate for each other. This has been illustrated particularly well for the amyloid precursor protein (APP) (13-17).ADAM10 is one of the proteolytically active ADAM members (15, 18 -21). The list of ADAM10 substrates is still growing, confirming the central role of ADAM10 in many important biological processes, such as cell migration and axonal navigation (robo receptors and ephrins (22, 23), cell adhesion (cadherins (19, 21), CD44 and L1 (24)), and regulation of immune reactions, and control of apoptosis (FasL) (25). Importantly, genetic ablation of ADAM10 in vertebrates (15) and invertebrates (26 -29) mainly results in loss of Notch phenotypes, indicating the crucial role for this protease in the Notch signaling pathway (30,31). Finally, AD...
With the discovery of the blocking effect, learning theory took a huge leap forward, because blocking provided a crucial clue that surprise is what drives learning. This in turn stimulated the development of novel association-formation theories of learning. Eventually, the ability to explain blocking became nothing short of a touchstone for the validity of any theory of learning, including propositional and other nonassociative theories. The abundance of publications reporting a blocking effect and the importance attributed to it suggest that it is a robust phenomenon. Yet, in the current article we report 15 failures to observe a blocking effect despite the use of procedures that are highly similar or identical to those used in published studies. Those failures raise doubts regarding the canonical nature of the blocking effect and call for a reevaluation of the central status of blocking in theories of learning. They may also illustrate how publication bias influences our perspective toward the robustness and reliability of seemingly established effects in the psychological literature.
Therapeutic hypothermia (HT) is standard care for moderate and severe neonatal hypoxic-ischaemic encephalopathy (HIE), the leading cause of permanent brain injury in term newborns. However, the optimal temperature for HT is still unknown, and few preclinical studies have compared multiple HT treatment temperatures. Additionally, HT may not benefit infants with severe encephalopathy. In a neonatal rat model of unilateral hypoxia-ischaemia (HI), the effect of five different HT temperatures was investigated after either moderate or severe injury. At postnatal-day seven, rat pups underwent moderate or severe HI followed by 5 h at normothermia (37 °C), or one of five HT temperatures: 33.5 °C, 32 °C, 30 °C, 26 °C, and 18 °C. One week after treatment, neuropathological analysis of hemispheric and hippocampal area loss, and CA1 hippocampal pyramidal neuron count, was performed. After moderate injury, a significant reduction in hemispheric and hippocampal loss on the injured side, and preservation of CA1 pyramidal neurons, was seen in the 33.5 °C, 32 °C, and 30 °C groups. Cooling below 33.5 °C did not provide additional neuroprotection. Regardless of treatment temperature, HT was not neuroprotective in the severe HI model. Based on these findings, and previous experience translating preclinical studies into clinical application, we propose that milder cooling should be considered for future clinical trials.
The ␥-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu 333 in human) serving as the anionic binding site for the ␣-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu 332 is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu 332 could bind the ␣-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic ␣-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased ␥-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu 332 crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally ␥-secretase activity. Consequently, these studies indicate that the main role of the Glu 332 is in the maturation and assembly of ␥-secretase rather than in the recognition of the substrates.
Introduction: Bacterial lipopolysaccharide (LPS) injection prior to hypoxia-ischaemia significantly increases hypoxia-ischaemic brain injury in 7-day-old (P7) rats. In addition, therapeutic hypothermia (HT) is not neuroprotective in this setting. However, the mechanistic aspects of this therapeutic failure have yet to be elucidated. This study was designed to investigate the underlying cellular mechanisms in this double-hit model of infection-sensitised hypoxia-ischaemic brain injury. Material and Methods: P7 rat pups were injected with either vehicle or LPS, and after a 4-hour delay were exposed to left carotid ligation followed by global hypoxia inducing a unilateral stroke-like hypoxia-ischaemic injury. Pups were randomised to the following treatments: (1) vehicle-treated pups receiving normothermia treatment (NT) (Veh-NT; n = 40), (2) LPS-treated pups receiving NT treatment (LPS-NT; n = 40), (3) vehicle-treated pups receiving HT treatment (Veh-HT; n = 38) and (4) LPS-treated pups receiving HT treatment (LPS-HT; n = 35). On postnatal day 8 or 14, Western blot analysis or immunohistochemistry was performed to examine neuronal death, apoptosis, astrogliosis and microglial activation. Results: LPS sensitisation prior to hypoxia-ischaemia significantly exacerbated apoptotic neuronal loss. NeuN, a neuronal biomarker, was significantly reduced in the LPS-NT and LPS-HT groups (p = 0.008). Caspase-3 activation was significantly increased in the LPS-sensitised groups (p < 0.001). Additionally, a significant increase in astrogliosis (glial fibrillary acidic expression, p < 0.001) was seen, as well as a trend towards increased microglial activation (Iba 1 expression, p = 0.051) in LPS-sensitised animals. Treatment with HT did not counteract these changes. Conclusion: LPS-sensitised hypoxia-ischaemic brain injury in newborn rats is mediated through neuronal death, apoptosis, astrogliosis and microglial activation. In this double-hit model, treatment with HT does not ameliorate these changes.
Humans can spontaneously create rules that allow them to efficiently generalize what they have learned to novel situations. An enduring question is whether rule-based generalization is uniquely human or whether other animals can also abstract rules and apply them to novel situations. In recent years, there have been a number of high-profile claims that animals such as rats can learn rules. Most of those claims are quite weak because it is possible to demonstrate that simple associative systems (which do not learn rules) can account for the behavior in those tasks. Using a procedure that allows us to clearly distinguish feature-based from rule-based generalization (the Shanks–Darby procedure), we demonstrate that adult humans show rule-based generalization in this task, while generalization in rats and pigeons was based on featural overlap between stimuli. In brief, when learning that a stimulus made of two components (“AB”) predicts a different outcome than its elements (“A” and “B”), people spontaneously abstract an opposites rule and apply it to new stimuli (e.g., knowing that “C” and “D” predict one outcome, they will predict that “CD” predicts the opposite outcome). Rats and pigeons show the reverse behavior—they generalize what they have learned, but on the basis of similarity (e.g., “CD” is similar to “C” and “D”, so the same outcome is predicted for the compound stimulus as for the components). Genuinely rule-based behavior is observed in humans, but not in rats and pigeons, in the current procedure. Electronic supplementary materialThe online version of this article (doi:10.1007/s10071-015-0895-8) contains supplementary material, which is available to authorized users.
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