Compounding artefacts with uncertainty, and an amyloid cascade hypothesis that is 'too big to fail'

With each failure of anti-amyloid-β therapy in clinical trials, new trials are initiated with no hint of slowing down. This may be due, in part, to the fact that the amyloid cascade hypothesis has been so modified over time that it is now impossible to confirm or deny. The hypothesis now states, in effect, that invisible molecules target invisible structures. Still relevant, however, are multiple factors that surely cast some doubt but have either been rationalized or overlooked. Among these are the poor correlation between amyloid-β deposits and disease, the substantial differences between familial and sporadic disease, pathological assessment that indicates the secondary nature of lesions/proteins/cascades, the fact that soluble species are poorly reproducible laboratory phenomena, and the irrelevance of synaptic assessment to pathological interpretation. Although not yet dogma, the premature addition of mild cognitive impairment as the implied in vivo homologue to the soluble toxin-synapse interaction is also problematic. In either case, the amyloid cascade hypothesis continues to dominate the Alzheimer's disease literature and grant applications. The more the neuroscience community perseverates along these lines in the face of accumulating outcome data to the contrary, the more one is left to wonder whether the hypothesis is too big to fail.     

Mechanisms of AD neurodegeneration may be independent of Aβ and its derivatives

Alzheimer's disease (AD) is the most common cause of dementia in the aged population. Most cases are sporadic although a small percent are familial (FAD) linked to genetic mutations. AD is caused by severe neurodegeneration in the hippocampus and neocortical regions of the brain but the cause of this neuronal loss is unclear. A widely discussed theory posits that amyloid depositions of Aβ peptides or their soluble forms are the causative agents of AD. Extensive research in the last 20 years however, failed to produce convincing evidence that brain amyloid is the main cause of AD neurodegeneration. Moreover, a number of observations, including absence of correlations between amyloid deposits and cognition, detection in normal individuals of amyloid loads similar to AD, and animal models with behavioral abnormalities independent of amyloid, are inconsistent with this theory. Other theories propose soluble Aβ peptides or their oligomers as agents that promote AD. These peptides, however, are normal components of human CSF and serum and there is little evidence of disease-associated increases in soluble Aβ and oligomers. That mutants of amyloid precursor protein (APP) and presenilin (PS) promote FAD suggests these proteins play crucial roles in neuronal function and survival. Accordingly, PS regulates production of signaling peptides and cell survival pathways while APP functions in cell death and may promote endosomal abnormalities. Evidence that FAD mutations inhibit the biological functions of PS combined with absence of haploinsufficiency mutants, support a model of allelic interference where inactive FAD mutant alleles promote autosomal dominant neurodegeneration by also inhibiting the functions of wild type alleles.     

Resting microglia react to Aβ42 fibrils but do not detect oligomers or oligomer-induced neuronal damage

In Alzheimer's disease (AD), amyloid-β (Aβ) deposits accumulate in the brain parenchyma and contain fibrils of aggregated heterogeneous Aβ peptides. In addition to fibrils, Aβ aggregates into stable soluble species (termed Aβ oligomers), which are increasingly viewed as the key drivers of early neurodegenerative events in AD. Aβ aggregates stimulate microglia recruitment and activation. In the AD brain, microglia surround Aβ deposits, activate, and abnormally produce inflammatory mediators, contributing to AD pathogenesis. However, it remains unclear to which of the conformationally diverse Aβ species microglia specifically react. Here, we explore the “sensor” capability of murine microglia. We examine whether they can detect and discriminate the toxic Aβ oligomers, Aβ fibrils, and Aβ-induced neuronal damage and investigate whether they are activated by diverse human Aβ species cell autonomously or through neuron-derived factors. We find that, on aggregation in vitro, Aβ42 peptides form stable oligomers and fibrils, which are neurotoxic and trigger dendritic spine loss in mature primary mouse hippocampal neurons. Further, in resting primary murine microglia, Aβ42 fibrils induce a pattern of expression of inflammatory genes typical of the classical inflammatory response induced by infectious agents (e.g., the bacterial toxin lipopolysaccharide). Conversely, Aβ42 oligomers never elicit a microglia inflammatory response, whether applied alone, in combination with neuron-derived secreted factors, or in contact with neurons. Thus, microglia strongly react to Aβ42 fibrils, but do not sense Aβ oligomers or oligomer-induced neuronal damage. This suggests that early neurotoxic species can escape detection by microglia, leading to the chronic unfolding of amyloid pathology in AD.     

Failure of Perivascular Drainage of β‐amyloid in Cerebral Amyloid Angiopathy

In Alzheimer's disease, amyloid‐β (Aβ) accumulates as insoluble plaques in the brain and deposits in blood vessel walls as cerebral amyloid angiopathy (CAA). The severity of CAA correlates with the degree of cognitive decline in dementia. The distribution of Aβ in the walls of capillaries and arteries in CAA suggests that Aβ is deposited in the perivascular pathways by which interstitial fluid drains from the brain. Soluble Aβ from the extracellular spaces of gray matter enters the basement membranes of capillaries and drains along the arterial basement membranes that surround smooth muscle cells toward the leptomeningeal arteries. The motive force for perivascular drainage is derived from arterial pulsations combined with the valve effect of proteins present in the arterial basement membranes. Physical and biochemical changes associated with arteriosclerosis, aging and possession of apolipoprotein E4 genotype lead to a failure of perivascular drainage of soluble proteins, including Aβ. Perivascular cells associated with arteries and the lymphocytes recruited in the perivenous spaces contribute to the clearance of Aβ. The failure of perivascular clearance of Aβ may be a major factor in the accumulation of Aβ in CAA and may have significant implications for the design of therapeutics for the treatment of Alzheimer's disease.     

AD synapses contain abundant Aβ monomer and multiple soluble oligomers, including a 56-kDa assembly

Much evidence indicates that soluble amyloid beta (Aβ) oligomers are key mediators of early cognitive loss, but the localization and key peptide species remain unclear. We have used flow cytometry analysis to demonstrate that surviving Alzheimer's disease (AD) synapses accumulate both Aβ and phosphorylated tau (p-tau). The present experiments use peptide-specific X-map assays and Western blot analyses to identify the Aβ peptide species in synaptosome-enriched samples from normal human subjects, neurologic controls, and AD cases. Aβ40 peptide levels did not vary, but both Aβ42 and Aβ oligomers were increased in soluble AD extracts, with oligomer levels 20-fold higher in aqueous compared with detergent extracts. In Western blot analysis, a ladder of sodium dodecyl sulfate (SDS)-stable oligomers was observed in AD cases, varying in size from monomer, the major peptide observed, to larger assemblies up to about 200 kDa and larger. Multiple oligomers, including monomer, small oligomers, a 56-kDa assembly, and amyloid precursor protein (APP) were correlated with the Aβ level measured in flow cytometry-purified synaptosomes. These results suggest that multiple amyloid precursor protein processing pathways are active in AD synapses and multiple soluble oligomeric assemblies may contribute to synaptic dysfunction.     

Rapid amyloid‐β oligomer and protofibril accumulation in traumatic brain injury

Deposition of amyloid‐β (Aβ) is central to Alzheimer's disease (AD) pathogenesis and associated with progressive neurodegeneration in traumatic brain injury (TBI). We analyzed predisposing factors for Aβ deposition including monomeric Aβ40, Aβ42 and Aβ oligomers/protofibrils, Aβ species with pronounced neurotoxic properties, following human TBI. Highly selective ELISAs were used to analyze N‐terminally intact and truncated Aβ40 and Aβ42, as well as Aβ oligomers/protofibrils, in human brain tissue, surgically resected from severe TBI patients (n = 12; mean age 49.5 ± 19 years) due to life‐threatening brain swelling/hemorrhage within one week post‐injury. The TBI tissues were compared to post‐mortem AD brains (n = 5), to post‐mortem tissue of neurologically intact (NI) subjects (n = 4) and to cortical biopsies obtained at surgery for idiopathic normal pressure hydrocephalus patients (iNPH; n = 4). The levels of Aβ40 and Aβ42 were not elevated by TBI. The levels of Aβ oligomers/protofibrils in TBI were similar to those in the significantly older AD patients and increased compared to NI and iNPH controls (P < 0.05). Moreover, TBI patients carrying the AD risk genotype Apolipoprotein E epsilon3/4 (APOE ε3/4; n = 4) had increased levels of Aβ oligomers/protofibrils (P < 0.05) and of both N‐terminally intact and truncated Aβ42 (P < 0.05) compared to APOE ε3/4‐negative TBI patients (n = 8). Neuropathological analysis showed insoluble Aβ aggregates (commonly referred to as Aβ plaques) in three TBI patients, all of whom were APOE ε3/4 carriers. We conclude that soluble intermediary Aβ aggregates form rapidly after TBI, especially among APOE ε3/4 carriers. Further research is needed to determine whether these aggregates aggravate the clinical short‐ and long‐term outcome in TBI.     

Synaptic transmission is impaired prior to plaque formation in amyloid precursor protein–overexpressing mice without altering behaviorally-correlated sharp wave–ripple complexes

One of the hallmarks of Alzheimer's disease is the accumulation of amyloid plaques in brains of affected patients. Several recent studies provided evidence that soluble oligomer forms of amyloid-β (Aβ) rather than plaques determine cognitive decline. In vitro studies using artificial Aβ oligomer preparations suggest that such pathophysiology is caused by a specific impairment of synaptic function. We examined whether synaptic deficits occur before deposition of insoluble fibrillar Aβ by analyzing brain slices taken from young Tg2576 mice overexpressing mutant amyloid precursor protein. Excitatory synaptic transmission in the hippocampal CA1 region was strongly impaired before plaque development, suggesting a dissociation of an early synaptic impairment, probably caused by soluble oligomeric amyloid-β, from subsequent plaque formation. At higher age neurotransmission was also decreased in wild type mice, paralleling a cognitive decline of normal aged animals. Memory formation in rats is accompanied by distinct hippocampal network oscillations. It has recently been shown that hippocampal gamma oscillations, a network correlate of exploratory behavior, are impaired in amyloid precursor protein (APP)–overexpressing mice. We determined whether sharp wave–ripple complexes, which contribute to memory consolidation during slow wave-sleep, are modified in Tg2576 mice. Interestingly, neither sharp waves nor superimposed ripples were changed at pre-plaque or plaque stages. During aging, however, there was a strong reduction of sharp wave frequency and ripple energy in wild type and APP-overexpressing animals. This indicates that the reported changes in network oscillations following APP-overexpression are specific for gamma oscillations, whereas aging has a more general effect on network properties. Taken together our data suggest that non-fibrillar forms of Aβ—possibly Aβ oligomers—specifically interfere with synaptic function in Tg2576, but do not globally alter memory-related network properties. We propose that mechanisms leading to Aβ-related cognitive decline are different from those related to aging.     

Reducing iron in the brain: a novel pharmacologic mechanism of huperzine A in the treatment of Alzheimer's disease

Huperzine A (HupA), a natural inhibitor of acetylcholinesterase derived from a plant, is a licensed anti-Alzheimer's disease (AD) drug in China and a nutraceutical in the United States. In addition to acting as an acetylcholinesterase inhibitor, HupA possesses neuroprotective properties. However, the relevant mechanism is unknown. Here, we showed that the neuroprotective effect of HupA was derived from a novel action on brain iron regulation. HupA treatment reduced insoluble and soluble beta amyloid levels, ameliorated amyloid plaques formation, and hyperphosphorylated tau in the cortex and hippocampus of APPswe/PS1dE9 transgenic AD mice. Also, HupA decreased beta amyloid oligomers and amyloid precursor protein levels, and increased A Disintegrin And Metalloprotease Domain 10 (ADAM10) expression in these treated AD mice. However, these beneficial effects of HupA were largely abolished by feeding the animals with a high iron diet. In parallel, we found that HupA decreased iron content in the brain and demonstrated that HupA also has a role to reduce the expression of transferrin-receptor 1 as well as the transferrin-bound iron uptake in cultured neurons. The findings implied that reducing iron in the brain is a novel mechanism of HupA in the treatment of Alzheimer's disease.     

Aβ oligomers trigger and accelerate Aβ seeding

Aggregation of amyloid‐β (Aβ) that leads to the formation of plaques in Alzheimer's disease (AD) occurs through the stepwise formation of oligomers and fibrils. An earlier onset of aggregation is obtained upon intracerebral injection of Aβ‐containing brain homogenate into human APP transgenic mice that follows a prion‐like seeding mechanism. Immunoprecipitation of these brain extracts with anti‐Aβ oligomer antibodies or passive immunization of the recipient animals abrogated the observed seeding activity, although induced Aβ deposition was still evident. Here, we establish that, together with Aβ monomers, Aβ oligomers trigger the initial phase of Aβ seeding and that the depletion of oligomeric Aβ delays the aggregation process, leading to a transient reduction of seed‐induced Aβ deposits. This work extends the current knowledge about the role of Aβ oligomers beyond its cytotoxic nature by pointing to a role in the initiation of Aβ aggregation in vivo. We conclude that Aβ oligomers are important for the early initiation phase of the seeding process.     

以β淀粉样蛋白作为阿尔茨海默症治疗靶点的研究进展

β淀粉样前体蛋白(APP)是一种单次跨膜天冬氨酸蛋白质,其被β和γ分泌酶相继水解后会产生具有毒性作用的β淀粉样蛋白(Aβ).阿尔茨海默症(AD)最主要的病理特征是Aβ在神经元胞外大量聚集形成淀粉样斑及tau蛋白在胞内过度磷酸化形成纤维化缠结.长期以来,AD的发病被认为与Aβ聚集具有较大的相关性,以Aβ为靶点的抗体性治疗...     

Cathepsin protease activity modulates amyloid load in extracerebral amyloidosis

In cerebral amyloidoses, such as Alzheimer's disease, proteolytic processing of the precursor protein is a fundamental mechanism of the disease, since it generates the amyloid protein. However, the putative significance of proteases in extracerebral amyloidoses is less well defined. In this study, we investigated the biological significance of cathepsin (Cath) B, CathK, and CathL in the pathology and pathogenesis of extracerebral amyloidoses by using the murine model of reactive or secondary AA amyloidosis with three different cathepsin-deficient mouse strains. Extracerebral AA amyloid was induced by injecting amyloid-enhancing factor and silver nitrate into CathB(-/-), CathK(-/-), and CathL(-/-) mice. Wild-type mice served as a control. CathK(-/-) mice deposited over 90% more amyloid and CathL(-/-) mice 60% less amyloid than the control (p < 0.0001). The amyloid load in CathB(-/-) mice did not differ from that in wild-type mice. In vitro degradation experiments with recombinant human and murine serum amyloid A (SAA) 1.1 and CathK and CathL showed that CathL generates a large number of differently sized SAA cleavage products. One of these fragments spans the heparin/heparan sulphate binding site and the neutral cholesterol ester hydrolase activating region of SAA. CathK showed only endoproteolytic activity and did not generate any AA amyloid-like peptides. This study provides unequivocal evidence that proteases modulate amyloid load in extracerebral amyloidosis. CathL was identified as an amyloid-promoting and CathK as an amyloid-retarding cysteine protease. CathB may only modulate the primary structure of the amyloid peptide without affecting amyloid load.     

A role for astrocyte‐derived amyloid β peptides in the degeneration of neurons in an animal model of temporal lobe epilepsy

Kainic acid, an analogue of the excitatory neurotransmitter glutamate, can trigger seizures and neurotoxicity in the hippocampus and other limbic structures in a manner that mirrors the neuropathology of human temporal lobe epilepsy (TLE). However, the underlying mechanisms associated with the neurotoxicity remain unclear. Since amyloid‐β (Aβ) peptides, which are critical in the development of Alzheimer's disease, can mediate toxicity by activating glutamatergic NMDA receptors, it is likely that the enhanced glutamatergic transmission that renders hippocampal neurons vulnerable to kainic acid treatment may involve Aβ peptides. Thus, we seek to establish what role Aβ plays in kainic acid‐induced toxicity using in vivo and in vitro paradigms. Our results show that systemic injection of kainic acid to adult rats triggers seizures, gliosis and loss of hippocampal neurons, along with increased levels/processing of amyloid precursor protein (APP), resulting in the enhanced production of Aβ‐related peptides. The changes in APP levels/processing were evident primarily in activated astrocytes, implying a role for astrocytic Aβ in kainic acid‐induced toxicity. Accordingly, we showed that treating rat primary cultured astrocytes with kainic acid can lead to increased Aβ production/secretion without any compromise in cell viability. Additionally, we revealed that kainic acid reduces neuronal viability more in neuronal/astrocyte co‐cultures than in pure neuronal culture, and this is attenuated by precluding Aβ production. Collectively, these results indicate that increased production/secretion of Aβ‐related peptides from activated astrocytes can contribute to neurotoxicity in kainic acid‐treated rats. Since kainic acid administration can lead to neuropathological changes resembling TLE, it is likely that APP/Aβ peptides derived from astrocytes may have a role in TLE pathogenesis.     

Selective benefits of simvastatin in bitransgenic APPSwe,Ind/TGF-β1 mice

Cognitive and cerebrovascular deficits are 2 landmarks of Alzheimer's disease (AD) to target for effective therapy. Here, we evaluated the efficacy of simvastatin in bitransgenic A/T mice overexpressing a mutated form of the human amyloid precursor protein (APP_Swe,Ind) and a constitutively active form of transforming growth factor-β1. These mice feature the AD amyloid beta (Aβ) and cerebrovascular pathology. Simvastatin significantly decreased insoluble Aβ peptide levels and Aβ plaque load despite no effect on β-site amyloid precursor protein-cleaving enzyme and Aβ-degrading enzyme neprilysin protein levels. However, simvastatin failed to improve spatial learning and memory deficits and the decreased baseline levels of the memory-related protein early growth response-1 (Egr-1) in the hippocampus CA1 area. The impaired hyperemic response to whisker stimulation in A/T mice was not improved with treatment, but simvastatin fully restored constitutive nitric oxide synthesis in vessel walls and exacerbated agonist-mediated dilatory deficits. These findings point to the efficacy of simvastatin on selective AD features in a complex model of the disease, likely reflecting the challenges faced by recent clinical trials in assessing statin efficacy.     

Plaque-bearing mice with reduced levels of oligomeric amyloid-beta assemblies have intact memory function

The amyloid-beta (Abeta) protein exists in the aging mammalian brain in diverse assembly states, including amyloid plaques and soluble Abeta oligomers. Both forms of Abeta have been shown to impair neuronal function, but their precise roles in Alzheimer's disease (AD) -associated memory loss remain unclear. Both types of Abeta are usually present at the same time in the brain, which has made it difficult to evaluate the effects of plaques and oligomers individually on memory function. Recently, a particular oligomeric Abeta assembly, Abeta 56, was found to impair memory function in the absence of amyloid plaques. Until now it has not been possible to determine the effects of plaques, in the absence of Abeta oligomers, on memory function. We have identified Tg2576 mice with plaques but markedly reduced levels of Abeta oligomers, which enabled us to study the effects of plaques alone on memory function. We found that animals with amyloid plaques have normal memory function throughout an episode of reduced Abeta oligomers, which occurs during a period of accelerated amyloid plaque formation. These observations support the importance of Abeta oligomers in memory loss and indicate that, at least initially, amyloid plaques do not impair memory.     

Aβ induces its own prion protein N-terminal fragment (PrPN1)–mediated neutralization in amorphous aggregates

Plasma membrane cellular prion protein (PrP^C) is a high-affinity receptor for toxic soluble amyloid-β (Aβ) oligomers that mediates synaptic dysfunction. Secreted forms of PrP^C resulting from PrP^C α-cleavage (PrPN1) or shedding (shed PrP^C) display neuroprotective activity in neuronal cultures and in mouse models of Aβ-induced neuronal dysfunction. In vitro, recombinant PrPN1 and PrP inhibit Aβ fibrillization. However, the mechanism by which PrPN1 and shed PrP^C neutralize Aβ oligomers is unclear, and evidence of such neuroprotective activity in Alzheimer's disease (AD) patients is lacking. Here, we show that PrPN1 association with Aβ causes a conformational change resulting in the formation of amorphous and insoluble aggregates that are not compatible with the assembly of Aβs. Using postmortem brain tissues of AD patients, we were able to coimmunoprecipitate Aβ with PrP^C molecules and observed a coaggregation of Aβ and PrPN1 in the guanidine-extractable fraction presumably representing insoluble amyloid plaques. Furthermore, PrP^C α-cleavage is increased in AD brains, and we noticed a significant positive correlation between the levels of α-cleavage and of guanidine-extractable Aβ. These data strongly support the hypothesis that PrP^C α-cleavage is an endogenous neuroprotective mechanism in AD and support the development of PrP^C-derived peptides as therapeutic molecules for AD.     

Reduced cerebrospinal fluid levels of alpha-secretase-cleaved amyloid precursor protein in aged rats: correlation with spatial memory deficits.

The amyloid precursor protein undergoes proteolysis at several sites to yield a number of functionally relevant peptides, including beta-amyloid and the soluble amyloid precursor protein derivatives alpha-soluble amyloid precursor protein and beta-soluble amyloid precursor protein. beta-Amyloid is the primary constituent of senile plaques associated with Alzheimer's disease, while a-soluble amyloid precursor protein promotes synaptogenesis and plays a role in neuroprotective processes. We tested for age-related alterations in these amyloid precursor protein proteolytically derived peptides by measuring the levels of alpha-soluble amyloid precursor protein, total soluble amyloid precursor proteins (alpha- and beta-soluble amyloid precursor protein combined) and beta-amyloid in cerebrospinal fluid from three-, 13- and 23-month-old Fischer-344 rats. Western blot analysis using selective antibodies revealed 50% less total soluble amyloid precursor protein and a-soluble amyloid precursor protein in cisternal cerebrospinal fluid from 23-month-old rats compared with three- and 13-month-old animals. Mass spectrometric analysis indicated, however, that beta-amyloid in cerebrospinal fluid was not different between the three age groups. In a second group of young (five to six months of age) and aged (24-25 months of age) rats, spatial working and reference memory were assessed in a water maze followed by collection of cerebrospinal fluid. As a group, the aged rats consistently performed below the young rats in both working and reference memory tests. The aged rats also had 49% less cerebrospinal fluid alpha-soluble amyloid precursor protein than did their younger counterparts. There was a positive correlation (r= 0.52-0.57, P < 0.001) between performance in spatial memory tasks and cerebrospinal fluid alpha-soluble amyloid precursor protein in these young and aged rats. These results suggest that there is a positive association between cerebrospinal fluid levels of alpha-soluble amyloid precursor protein and cognitive performance in rats, and that alpha-soluble amyloid precursor protein may be involved in the spatial learning and memory changes that accompany ageing.     

Dispersible amyloid β-protein oligomers, protofibrils, and fibrils represent diffusible but not soluble aggregates: their role in neurodegeneration in amyloid precursor protein (APP) transgenic mice

Soluble amyloid β-protein (Aβ) aggregates have been identified in the Alzheimer's disease (AD) brain. Dispersed Aβ aggregates in the brain parenchyma are different from soluble, membrane-associated and plaque-associated solid aggregates. They are in mixture with the extra- or intracellular fluid but can be separated from soluble proteins by ultracentrifugation. To clarify the role of dispersible Aβ aggregates for neurodegeneration we analyzed 2 different amyloid precursor protein (APP)-transgenic mouse models. APP23 mice overexpress human mutant APP with the Swedish mutation. APP51/16 mice express high levels of human wild type APP. Both mice develop Aβ-plaques. Dendritic degeneration, neuron loss, and loss of asymmetric synapses were seen in APP23 but not in APP51/16 mice. The soluble and dispersible fractions not separated from one another were received as supernatant after centrifugation of native forebrain homogenates at 14,000 × g. Subsequent ultracentrifugation separated the soluble, i.e., the supernatant, from the dispersible fraction, i.e., the resuspended pellet. The major biochemical difference between APP23 and APP51/16 mice was that APP23 mice exhibited higher levels of dispersible Aβ oligomers, protofibrils and fibrils precipitated with oligomer (A11) and protofibril/fibril (B10AP) specific antibodies than APP51/16 mice. These differences, rather than soluble Aβ and Aβ plaque pathology were associated with dendritic degeneration, neuron, and synapse loss in APP23 mice in comparison with APP51/16 mice. Immunoprecipitation of dispersible Aβ oligomers, protofibrils, and fibrils revealed that they were associated with APP C-terminal fragments (APP-CTFs). These results indicate that dispersible Aβ oligomers, protofibrils, and fibrils represent an important pool of Aβ aggregates in the brain that critically interact with membrane-associated APP C-terminal fragments. The concentration of dispersible Aβ aggregates, thereby, presumably determines its toxicity.     

Ciliary neurotrophic factor (CNTF) plus soluble CNTF receptor α increases cyclooxygenase-2 expression, PGE2 release and interferon-γ-induced CD40 in murine microglia

Background

Beta amyloid (Aβ) peptides are the major constituents of the senile plaques present in Alzheimer's diseased brain. Pathogenesis has been associated with the aggregated form of the peptide as these fibrils are the conformation readily found in the plaques. However, recent studies have shown that the nonaggregated, soluble assemblies of Aβ have the potential to stimulate neuronal dysfunction and may play a prominent role in the pathogenesis of Alzheimer's disease.

Methods

Soluble, synthetic Aβ1–42 oligomers were prepared producing mainly dimer-trimer conformations as assessed by SDS-PAGE. Similar analysis demonstrated fibril preparations to produce large insoluble aggregates unable to migrate out of the stacking portion of the gels. These peptide preparations were used to stimulate primary murine microglia and cortical neuron cultures. Microglia were analyzed for changes in signaling response and secretory phenotype via Western analysis and ELISA. Viability was examined by quantifying lactate dehydrogenase release from the cultures.

Results

Aβ oligomers and fibrils were used to stimulate microglia for comparison. Both the oligomers and fibrils stimulated proinflammatory activation of primary microglia but the specific conformation of the peptide determined the activation profile. Oligomers stimulated increased levels of active, phosphorylated Lyn and Syk kinase as well as p38 MAP kinase compared to fibrils. Moreover, oligomers stimulated a differential secretory profile for interleukin 6, monocyte chemoattractant protein-1 and keratinocyte chemoattractant when compared to fibrils. Finally, soluble oligomers stimulated death of cultured cortical neurons that was exacerbated by the presence of microglia.

Conclusion

These data suggest that fibrils and oligomers stimulate unique signaling responses in microglia leading to discrete secretory changes and effects on neuron survival. This suggests that inflammation changes during disease may be the consequence of unique peptide-stimulated events and each conformation may represent an individual anti-inflammatory therapeutic target.     

Structural aspects and physiological consequences of APP/APLP trans-dimerization

The amyloid precursor protein (APP) is one of the key proteins in Alzheimer's disease (AD), as it is the precursor of amyloid β (Aβ) peptides accumulating in amyloid plaques. The processing of APP and the pathogenic features of especially Aβ oligomers have been analyzed in detail. Remarkably, there is accumulating evidence from cell biological and structural studies suggesting that APP and its mammalian homologs, the amyloid precursor-like proteins (APLP1 and APLP2), participate under physiological conditions via trans-cellular dimerization in synaptogenesis. This offers the possibility that loss of synapses in AD might be partially explained by dysfunction of APP/APLPs cell adhesion properties. In this review, structural characteristics of APP trans-cellular interaction will be placed critically in context with its putative physiological functions focusing on cell adhesion and synaptogenesis.     

Cerebrovascular miRNAs correlate with the clearance of Aβ through perivascular route in younger 3xTg‐AD mice

The “two‐hit vascular hypothesis for Alzheimer's disease (AD)” and amyloid‐β (Aβ) oligomer hypothesis suggest that impaired soluble Aβ oligomers clearance through the cerebral vasculature may be an initial step of the AD process. Soluble Aβ oligomers are driven into perivascular spaces from the brain parenchyma and toward peripheral blood flow. The underlying vascular‐based mechanism, however, has not been defined. Given that microRNAs (miRNAs), emerging as novel modulators, are involved in numerous physiological and pathological processes, we hypothesized that cerebrovascular miRNAs may regulate the activities of brain blood vessels, which further affects the concentration of Aβ in the AD brain. In this study, perivascular Aβ deposits, higher vascular activation, increased pericyte coverage and up‐regulated capillaries miRNAs at 6 months old (6 mo) were found to correlate with the lower Aβ levels of middle AD stage (9 mo) in 3xTg‐AD (3xTg) mice. It is implicated that at the early stage of AD when intracellular Aβ appeared, higher expression of vessel‐specific miRNAs, elevated pericyte coverage, and activated endothelium facilitate Aβ oligomer clearance through the perivascular route, resulting in a transient reduction of Aβ oligomers at 9 mo. Additionally, ghrelin‐induced upregulation of capillary miRNAs and increased pericyte coverage attenuated Aβ burden at 9 mo, in further support of the relationship between vascular miRNAs and Aβ clearance. This work suggests a cerebral microvessel miRNA may boost endothelial highly activated phenotypes to promote elimination of Aβ oligomers through the perivascular drainage pathway and contribute to AD progression. The targeting of brain vessel‐specific miRNAs may provide a new rationale for the development of innovative therapeutic strategies for AD treatment.