Mutations in nicastrin protein differentially affect amyloid beta-peptide production and Notch protein processing.

The γ-secretase complex is responsible for intramembrane processing of over 60 substrates and is involved in Notch signaling as well as in the generation of the amyloid ß-peptide (Aß). Aggregated forms of Aß have a pathogenic role in Alzheimer disease and, thus, reducing the Aß levels by inhibiting γ-secretase is a possible treatment strategy for Alzheimer disease. Regrettably, clinical trials have shown that inhibition of γ-secretase results in Notch-related side effects. Therefore, it is of great importance to find ways to inhibit amyloid precursor protein (APP) processing without disturbing vital signaling pathways such as Notch. Nicastrin (Nct) is part of the γ-secretase complex and has been proposed to be involved in substrate recognition and selection. We have investigated how the four evenly spaced and conserved cysteine residues in the Nct ectodomain affect APP and Notch processing. We mutated these cysteines to serines and analyzed them in cells lacking endogenous Nct. We found that two mutants, C213S (C2) and C230S (C3), differentially affected APP and Notch processing. Both the formation of Aß and the intracellular domain of amyloid precursor protein (AICD) were reduced, whereas the production of Notch intracellular domain (NICD) was maintained on a high level, although C230S (C3) showed impaired complex assembly. Our data demonstrate that single residues in a γ-secretase component besides presenilin are able to differentially affect APP and Notch processing.

The large hydrophilic loop of presenilin 1 is important for regulating gamma-secretase complex assembly and dictating the amyloid beta peptide (Abeta) Profile without affecting Notch processing.

Gamma-secretase is an enzyme complex that mediates both Notch signaling and beta-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid beta peptide (Abeta), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of gamma-secretase gives rise to Abeta peptides of different lengths, where Abeta42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320-374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of gamma-secretase, for gamma-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, gamma-secretase complex formation, and had a differential effect on Abeta-peptide production. Although the production of Abeta38, Abeta39, and Abeta40 was severely impaired, the effect on Abeta42 was affected to a lesser extent, implying that the production of the AD-related Abeta42 peptide is separate from the production of the Abeta38, Abeta39, and Abeta40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the epsilon/S3 and gamma sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Abeta peptides without affecting Notch processing, two parameters of significance when considering gamma-secretase as a target for pharmaceutical intervention in AD.

Is placebo acupuncture what it is intended to be?

Randomized, placebo-controlled clinical trials are recommended for evaluation of a treatment's efficacy with the goal of separating the specific effects (verum) from the non-specific ones (placebo). In order to be able to carry out placebo-controlled acupuncture trials, minimal/sham acupuncture procedures and a sham acupuncture needle has been used with the intention of being inert. However, clinical and experimental results suggest that sham/minimal acupuncture is not inert since it is reported that both verum acupuncture and sham/minimal acupuncture induce a significant alleviation of pain. This alleviation is as pronounced as the alleviation obtained with standard treatment and more obvious than the one obtained with placebo medication or by the use of waiting list controls. These results also suggest that sham acupuncture needles evoke a physiological response. In healthy individuals sham acupuncture results in activation of limbic structures, whereas a deactivation is seen in patients with pain, i.e. results from healthy individuals do not reflect what is seen in clinical conditions. Also, depending on the etiology of pain (or any under clinical condition under investigation), the response to sham acupuncture is varying. The acupuncture ritual may also be seen as an emotional focused therapy allowing for psychological re-orientation. Sham needling in such context may be as powerful as verum acupuncture. We recommend that the evaluated effects of acupuncture could be compared with those of standard treatment, also taking the individual response into consideration, before its use or non-use is established.

Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease.

Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

Gamma-secretase: a complex target for Alzheimer's disease.

Data accumulated during the past two decades place amyloid beta-peptide (Abeta) at center stage as the main perpetrator in initiating the pathological cascade that eventually leads to Alzheimer's disease. Consequently, significant resources have been allocated to identify and develop treatment strategies that alter the metabolism of Abeta. The gamma-secretase protease has deservedly received attention as an attractive drug target, as it is directly involved in Abeta biogenesis and determines the pathogenic potential of Abeta by its heterogeneous catalytic action, generating peptides of various lengths. Despite the complexity of the multi-subunit gamma-secretase and the lack of structural information, drug discovery research has identified small-molecule compounds that inhibit or modulate activity of this enzyme and some of these have already entered clinical trials.

The Emperors sham - wrong assumption that sham needling is sham.

During the last five years a large number of randomised controlled clinical trials (RCTs) have been published on the efficacy of acupuncture in different conditions. In most of these studies verum is compared with sham acupuncture. In general both verum and sham have been found to be effective, and often with little reported difference in outcome. This has repeatedly led to the conclusion that acupuncture is no more effective than placebo treatment. However, this conclusion is based on the assumption that sham acupuncture is inert. Since sham acupuncture evidently is merely another form of acupuncture from the physiological perspective, the assumption that sham is sham is incorrect and conclusions based on this assumption are therefore invalid. Clinical guidelines based on such conclusions may therefore exclude suffering patients from valuable treatments.

Stable insertion of Alzheimer Abeta peptide into the ER membrane strongly correlates with its length.

Alzheimer's disease is characterized by the deposition of amyloid beta-peptide (Abeta) plaques in the brain. Full-length amyloid-beta precursor protein (APP) is processed by alpha- and beta-secretases to yield soluble APP derivatives and membrane-bound C-terminal fragments, which are further processed by gamma-secretase to a non-amyloidogenic 3 kDa product or to Abeta fragments. As different Abeta fragments contain different parts of the APP transmembrane helix, one may speculate that they are retained more or less efficiently in the membrane. Here, we use the translocon-mediated insertion of different APP-derived polypeptide segments into the endoplasmic reticulum membrane to assess the propensities for membrane retention of Abeta fragments. Our results show a strong correlation between the length of an Abeta-derived segment and its ability to integrate into the microsomal membrane.

The Alzheimer's disease-associated gamma-secretase complex: functional domains in the presenilin 1 protein.

Alzheimer's disease is neuropathologically characterized by the presence of neurofibrillary tangles and amyloid plaques in the brain. Amyloid plaques are extracellular deposits primarily composed of the amyloid beta-peptide, which is derived from the amyloid beta-precursor protein (APP) by sequential cleavages at the beta-secretase and gamma-secretase sites. gamma-Secretase cleavage is performed by a high molecular weight protein complex containing presenilin (PS), nicastrin, Aph-1 and Pen-2. The gamma-secretase complex is an unusual transmembrane aspartyl protease that cleaves APP within the transmembrane domain. In addition to APP, a large number of other single membrane-spanning proteins have been shown to be cleaved within their transmembrane domains by the gamma-secretase complex in a process referred to as regulated intramembrane proteolysis. Here we review recent research leading to the identification and understanding of the gamma-secretase complex components with emphasis on PS, which harbors the catalytic site. In addition, we summarize our own work focused on identifying and studying domains in PS1 that are critical for mediating gamma-secretase activity. Biochemical understanding of the gamma-secretase complex is important from a basic biological and physiological point of view, and could help in the development of small molecules that modulate gamma-secretase processing in an APP-specific manner.

Acupuncture--self-appraisal and the reward system.

Acupuncture is an ancient therapy with a variety of different explanatory models. A cascade of physiological effects has been reported, both in the peripheral and the central nervous system, following the insertion of a needle or light tapping of the skin. Clinical trials testing the specific claims of acupuncture have generally tried to focus on testing the efficacy of applying specific techniques and/or specified points. However, different conditions may respond differently to different modes of stimulation. Recently, it was demonstrated that both superficial and deep needling (with de qi/Hibiki) resulted in amelioration of patellofemoral pain and unpleasantness. The pleasurable aspect of the acupuncture experience has largely been ignored as it has been considered secondary to its pain alleviating effects. This aspect of acupuncture treatment is likely to be related to activation of self-appraisal and the reward system. When a patient seeks a therapist there are expectations of a specific effect. These expectations are partly based on self-relevant phenomena and self-referentia introspection and constitute the preference. Also, when asked about the effect of the treatment, processes that orientate pre-attentive anticipatory or mnemonic information and processes that mediate self-reflection and recollection are integrated together with sensory detection to enable a decision about the patient's perception of the effect of acupuncture treatment. These 'self-appraisal' processes are dependent on two integrated networks: a ventral medial prefrontal cortex-paralimbic-limbic 'affective' pathway and a dorsal medial prefrontal cortex-cortical-hippocampal 'cognitive' pathway. The limbic structures are implicated in the reward system and play a key role in most diseases and illness responses including chronic pain and depression, regulating mood and neuromodulatory responses (eg sensory, autonomic, and endocrine). The pleasurable and neuromodulatory aspects of acupuncture as well as 'placebo needling' may partly be explained by the activation or deactivation of limbic structures including the hippocampus, amygdala, and their connections with the hypothalamus. In patients with patellofemoral pain, the effects of superficial and deep needling remained for six months. These long term pain-alleviating effects have been attributed to activation of pain inhibiting systems in cortical and subcortical pathways. When considering long term effects the cortical-cerebellar system needs to be taken into account. The cortical-cerebellar system is probably central to the development of neural models that learn and eventually stimulate routinely executed (eg motor skills) and long term (eg pain alleviation) cognitive processes. These higher order cognitive processes are initially mediated in prefrontal cortical loci but later shift control iteratively to internal cerebellar representations of these processes. Possibly part of the long term healing effects of acupuncture may be attributed to changes in the cerebellar system thereby sparing processing load in cortical and subcortical areas. As cortical and subcortical structures are activated and/or de-activated following stimulation of receptors in the skin, disregarding site, 'placebo or sham needling' does not exist and conclusions drawn on the basis that it is an inert control are invalid. 'Self' may be seen as a shifting illusion, ceaselessly constructed and deconstructed, and the effect of acupuncture may reflect its status (as well as that of the therapist).

Non-invasive continuous estimation of blood flow changes in human patellar bone.

A photoplethysmographic (PPG) technique to assess blood flow in bone tissue has been developed and tested. The signal detected by the PPG consists of a constant-level (DC) component-which is related to the relative vascularization of the tissue-and a pulsatile (AC) component-which is synchronous with the pumping action of the heart. The PPG probe was applied on the skin over the patella. The probe uses near-infrared (804 nm) and green (560 nm) light sources and the AC component of the PPG signals of the two wavelengths was used to monitor pulsatile blood flow in the patellar bone and the overlying skin, respectively. Twenty healthy subjects were studied and arterial occlusion resulted in elimination of PPG signals at both wavelengths, whereas occlusion of skin blood flow by local surface pressure eliminated only the PPG signal at 560 nm. In a parallel study on a physical model with a rigid tube we showed that the AC component of the PPG signal originates from pulsations of blood flow in a rigid structure and not necessarily from volume pulsations. We conclude that pulsatile blood flow in the patellar bone can be assessed with the present PPG technique.

Comparison of symptoms and clinical findings in subgroups of individuals with patellofemoral pain.

Patellofemoral pain syndrome (PFPS) is one of the most common musculoskeletal disorders. However, no consensus on the definition, classification, assessment, diagnosis, or management has been reached. We evaluated symptoms and clinical findings in subgroups of individuals with PFPS, classified on the basis of the findings in radiological examinations and compared the findings with knee-healthy subjects. An orthopedic surgeon and a physical therapist consecutively examined 80 patients clinically diagnosed as having PFPS and referred for physical therapy. The examination consisted of taking a case history and clinical tests. Radiography revealed pathology in 15 patients, and scintigraphic examination revealed focal uptake in 2 patients indicating pathology (group C). Diffusely increased uptake was present in 29 patients (group B). In the remaining 29 patients radiographic and scintigraphic examinations were normal (group A). Knee-healthy controls (group D) reported no clinical symptoms. No symptom could be statistically demonstrated to differ between the three patient groups. Knee-healthy subjects differed significantly from the three patient groups in all clinical tests measuring pain in response to the provocations; compression test, medial and lateral tenderness, passive gliding of the patella, but they also differed in Q angle. Differences in clinical tests between the patient groups were nonsignificant. The main finding in our study on patients clinically diagnosed with PFPS is that possible pathologies cannot be detected from the patient's history or from commonly used clinical tests.

Collagenous Alzheimer amyloid plaque component assembles amyloid fibrils into protease resistant aggregates.

Recently, a novel plaque-associated protein, collagenous Alzheimer amyloid plaque component (CLAC), was identified in brains from patients with Alzheimer's disease. CLAC is derived from a type II transmembrane collagen precursor protein, termed CLAC-P (collagen XXV). The biological function and the contribution of CLAC to the pathogenesis of Alzheimer's disease and plaque formation are unknown. In vitro studies indicate that CLAC binds to fibrillar, but not to monomeric, amyloid beta-peptide (Abeta). Here, we examined the effects of CLAC on Abeta fibrils using assays based on turbidity, thioflavin T binding, sedimentation analysis, and electron microscopy. The incubation of CLAC with preformed Abeta fibrils led to increased turbidity, indicating that larger aggregates were formed. In support of this contention, more Abeta was sedimented in the presence of CLAC, as determined by gel electrophoresis. Moreover, electron microscopy revealed an increased amount of Abeta fibril bundles in samples incubated with CLAC. Importantly, the frequently used thioflavin T-binding assay failed to reveal these effects of CLAC. Digestion with proteinase K or trypsin showed that Abeta fibrils, incubated together with CLAC, were more resistant to proteolytic degradation. Therefore, CLAC assembles Abeta fibrils into fibril bundles that have an increased resistance to proteases. We suggest that CLAC may act in a similar way in vivo.

Molecular identification of AMY, an Alzheimer disease amyloid-associated protein.

One of the neuropathological lesions characteristic of Alzheimer disease (AD) is the cerebral accumulation of the amyloid beta-peptide (A beta). Although numerous studies have demonstrated that A beta spontaneously forms amyloid in vitro, the molecular events underlying A beta amyloid formation in vivo are less well understood. Immunohistochemical studies have shown that other proteins colocalize with A beta in amyloid deposits in brain. The identity of one of these proteins, AMY, has so far remained elusive; therefore we attempted to purify AMY. The AMY protein was found to co-purify with A beta in insoluble fractions from human AD brain, and was absent in brains from control subjects. AMY immunoreactivity was primarily restricted to a 50-kDa and 100-kDa protein species. Interestingly, the chromatographic and immunological profile of AMY resembled the recently identified amyloid-associated protein CLAC, derived from a transmembrane collagen-like precursor, CLAC-P. Antibodies against AMY recognized CLAC-P expressed in mammalian cells. In addition, side-by-side comparisons of AD brain sections and extracts, using antibodies against both AMY and CLAC, respectively, resulted in almost identical staining patterns. Therefore, we conclude that the AMY immunoreactivity seen in association with amyloid in AD brain is due to the presence of the CLAC protein.

Amyloid beta pathology in Alzheimer's disease and schizophrenia.

OBJECTIVE: Severe cognitive impairment is common in elderly patients with schizophrenia. Alzheimer's disease is the main cause of dementia among the elderly. Biochemical and genetic studies suggest that amyloid beta-peptide is central in Alzheimer's disease. The authors examined the possible involvement of amyloid beta-peptide in cognitive impairment in schizophrenia. METHOD: Specific antibodies against two major forms of amyloid beta-peptide, Abetax-40 and Abetax-42, were used in sandwich enzyme-linked immunosorbent assays to determine the levels of amyloid beta-peptide in postmortem brain samples from Alzheimer's disease patients (N=10), normal elderly comparison subjects (N=11), and schizophrenia patients with (N=7) or without (N=26) Alzheimer's disease. RESULTS: The levels of amyloid beta-peptide were highest in the Alzheimer's disease patients, followed by the patients with schizophrenia and comparison subjects. The mean Abetax-42 level in the schizophrenia patients without Alzheimer's disease was similar to that in the comparison subjects, but the level in the schizophrenia patients with Alzheimer's disease was significantly higher than in those without Alzheimer's disease or the comparison subjects. The Abetax-42 level in the schizophrenia patients with Alzheimer's disease was significantly lower than the level in the Alzheimer's disease cohort. CONCLUSIONS: In contrast to elderly schizophrenia patients with Alzheimer's disease pathology, those without Alzheimer's disease had amyloid beta-peptide levels that were not significantly different from those of normal subjects; hence amyloid beta-peptide does not account for the cognitive deficits in this group. These results suggest that the causes of cognitive impairment in "pure" schizophrenia are different from those in Alzheimer's disease.

Characterization of long-term mouse brain aggregating cultures: evidence for maintenance of neural precursor cells.

An extensive characterization of fetal mouse brain cell aggregates has been performed using immunohistochemical and stereological methods. Single cell suspensions from mechanically dissociated cortex and hippocampus were cultured in serum-free, B27-supplemented medium under constant gyratory agitation for up to 56 days. Three-dimensional aggregates started to form immediately after seeding and reached a final average size of 500 microm in diameter. Among the cell types identified, neurons were the most abundant cells in the aggregates, followed by astrocytes, microglia, and oligodendrocytes. Western blotting for synaptophysin and immunostaining for neurotransmitter-related molecules indicated the presence of well-defined phenotypic characteristics of the neurons in this culture system, suggesting functionality. Proliferating cells, many with neural precursor cell properties, were seen throughout the culture period and could be isolated from the aggregates even after 2 months in culture. Neural precursor cells were isolated from the aggregates after more than 1 month in culture; these cells were successfully differentiated into neurons, astrocytes, and oligodendrocytes. The aggregate culture system may provide a versatile tool for molecular dissection of processes identified in mouse models, including transgenic animals and manipulation of neural precursor cells.

The Arctic mutation interferes with processing of the amyloid precursor protein.

The Arctic amyloid precursor protein (APP) Alzheimer mutation, is located inside the beta-amyloid (Abeta) domain. Here, hybrid APP mutants containing both the Swedish and the Arctic APP mutations were investigated. ELISA measurements of cell media showed decreased levels of both Abeta40 and Abeta42. Similar results were obtained for the Dutch and Italian mutations, whereas the Flemish mutation displayed increased amounts of Abeta40 and Abeta42. Immunoprecipitation studies revealed increased Abeta40/p3 and Abeta42/p3 ratios for the Arctic mutation. These results were further verified by quantification revealing decreased levels of alphaAPPs accompanied by increased betaAPPs levels in the media. Thus, the pathogenic effects of the Arctic mutation may not only be due to the changed properties of the peptide but also altered processing of Arctic APP.

Sensory stimulation (acupuncture) for the treatment of idiopathic anterior knee pain.

A randomized controlled study was conducted to evaluate the effect of acupuncture treatment in idiopathic anterior knee pain, a pain syndrome without known aetiology. Fifty-eight patients, clinically and radiologically examined, were randomly assigned to either deep or minimal superficial acupuncture treatment. The patients were treated twice weekly for a total of 15 treatments. The main outcome measurements were one leg vertical jump, functional score, daily VAS recording and skin temperature. Fifty-seven patients completed the study. Pain measurements on VAS decreased significantly within both groups; in the deep acupuncture group from 25 before treatments to 10 afterwards, and in the superficial (placebo) acupuncture group from 30 to 10. There was no significant difference between the groups. The improvement on the VAS recordings remained significant even after 3 and 6 months. Even though the pain decreased after sensory stimulation, neither the ability to jump on one leg, the functional score nor the skin temperature changed. This study shows that patients with idiopathic anterior knee pain benefit from both electroacupuncture treatment and subcutaneous needling. The pain-relieving effect remains for at least 6 months. Central pain inhibition, caused by either afferent stimulation or by non-specific therapeutic (placebo) effects, is a plausible explanation behind the treatment effects.

Amyloid neuropathology in the single Arctic APP transgenic model affects interconnected brain regions.

The Arctic APP mutation (E693G) within the amyloid ß (Aß) domain of amyloid precursor protein (APP) leads to dementia with clinical features similar to Alzheimer's disease (AD), which is believed to be mediated via increased formation of protofibrils. We have generated a transgenic mouse model, TgAPParc, with neuron-specific expression of human amyloid precursor protein with the Arctic mutation (hAPParc), showing mild amyloid pathology with a relatively late onset. Here we performed a detailed analysis of the spatiotemporal progression of neuropathology in homozygous TgAPParc, focusing on intracellular Aß and diffuse Aß aggregates rather than amyloid plaques. We show that the neuropathology in homozygous TgAPParc mice starts with intracellular Aß aggregates, which is followed by diffuse extracellular Aß deposits in subiculum that later expands to brain regions receiving neuronal projections from regions already affected. Together this suggests that the pathology in TgAPParc mice affects interconnected brain regions and may represent a valuable tool to study the spread and progression of neuropathology in Alzheimer's disease.

Progressive neuropathology and cognitive decline in a single Arctic APP transgenic mouse model.

The Arctic APP mutation (E693G) leads to dementia with clinical features similar to Alzheimer disease (AD), but little is known about the pathogenic mechanism of this mutation. To address this question, we have generated a transgenic mouse model, TgAPParc, with neuron-specific expression of human APP with the Arctic mutation (hAPParc). Heterozygous mice from two separate founder lines with different levels of expression of hAPParc were analyzed with respect to brain morphology and behavior every 3 months until the age of 18 months. Standard histological stainings and immunohistochemistry using a panel of Aß antibodies showed an age- and dose-dependant progression of amyloid deposition in the brain, starting in the subiculum and spreading to the thalamus. Cognitive behavioral testing revealed deficits in hippocampus-dependent spatial learning and memory in the Barnes maze test. This study demonstrates that the Arctic APP mutation is sufficient to cause amyloid deposition and cognitive dysfunction, and thus the TgAPParc mouse model provides a valuable tool to study the effect of the Arctic mutation in vivo without possible confounding effect of other APP mutations.

Minimal acupuncture is not a valid placebo control in randomised controlled trials of acupuncture: a physiologist's perspective.

Placebo-control of acupuncture is used to evaluate and distinguish between the specific effects and the non-specific ones. During 'true' acupuncture treatment in general, the needles are inserted into acupoints and stimulated until deqi is evoked. In contrast, during placebo acupuncture, the needles are inserted into non-acupoints and/or superficially (so-called minimal acupuncture). A sham acupuncture needle with a blunt tip may be used in placebo acupuncture. Both minimal acupuncture and the placebo acupuncture with the sham acupuncture needle touching the skin would evoke activity in cutaneous afferent nerves. This afferent nerve activity has pronounced effects on the functional connectivity in the brain resulting in a 'limbic touch response'. Clinical studies showed that both acupuncture and minimal acupuncture procedures induced significant alleviation of migraine and that both procedures were equally effective. In other conditions such as low back pain and knee osteoarthritis, acupuncture was found to be more potent than minimal acupuncture and conventional non-acupuncture treatment. It is probable that the responses to 'true' acupuncture and minimal acupuncture are dependent on the aetiology of the pain. Furthermore, patients and healthy individuals may have different responses. In this paper, we argue that minimal acupuncture is not valid as an inert placebo-control despite its conceptual brilliance.