Soluble Abeta oligomers ultrastructurally localize to cell processes and might be related to synaptic dysfunction in Alzheimer's disease brain

Soluble Abeta oligomers have recently been considered to be responsible for cognitive dysfunction prior to senile plaque (SP) formation in Alzheimer's disease (AD) brain. To investigate the ultrastructural localization of soluble Abeta oligomers, we conducted the post-embedding immunoelectron microscopic (IEM) study using an antibody against a molecular mimic of oligomeric Abeta. We examined autopsied brains from AD patients and nondemented subjects. Oligomer-specific immunoreactions detected by IEM tended to be found with higher density (1) in AD than in nondemented brains and (2) at the axon and axon terminal in AD than in nondemented brains. These findings imply that soluble Abeta oligomers might be related to synaptic dysfunction in AD brain.     

Oligomers of beta-amyloid peptide inhibit BDNF-induced arc expression in cultured cortical Neurons

The progressive memory loss observed in Alzheimer's disease (AD) is accompanied by an increase in the levels of amyloid-beta peptide (Abeta) and a block of synaptic plasticity. Both synaptic plasticity and memory require changes in the expression of synaptic proteins such as the activity-regulated cytoskeleton-associated protein, Arc (also termed Arg3.1). Using a model of synaptic plasticity in which BDNF increases Arc expression in cultured cortical neurons, we have found that an oligomeric form of Abeta strongly inhibits the BDNF-induced increase of Arc expression. Given that Abeta oligomers are likely to be involved in the synaptic dysfunction and cognitive impairment observed in amyloid depositing mouse models, we hypothesize that inhibition of Arc induction by BDNF contributes to the synaptic and memory deficits at early stages of AD.     

Fibrillar beta-amyloid impairs the late phase of long term potentiation

Synaptic dysfunction is involved in early stages of Alzheimer's disease (AD). Amyloid-beta peptides (Abeta), a neuropathologic hallmark of the disease, have been shown to alter synaptic function. Given that Abeta is present in different forms including monomeric, oligomeric and fibrillar species, we have investigated whether fibrillar Abeta impairs synaptic function. Here we report that a synthetic fibrillar form of Abeta impairs the late protein-synthesis dependent phase of LTP without affecting the early protein-synthesis independent phase. These findings add to previous reports that Abeta oligomers are highly toxic to cells and might cause synaptic dysfunction, and suggest that a therapeutic intervention in AD should include the use of drugs inhibiting and disassembling fibril formation in addition to drugs inhibiting oligomers formation.     

Soluble oligomeric Abeta disrupts the protein kinase C signaling pathway

Alzheimer's disease (AD) is characterized by selective neurodegeneration of neurons involved in cognitive function. Current hypothesis for AD etiology needs to be reconsidered because fibrillar Abeta cannot explain selective neurodegeneration. Recent evidence suggests oligomeric Abeta may be more relevant to AD etiology. Here we show signaling disruption induced by oligomeric Abeta. Using the MTT assay, NT2 showed greatest susceptibility to soluble oligomeric Abeta. In the kinase assay, treatment with either monomeric Abeta or fibrillar Abeta evoked no response in PKA, PKC and TK. Oligomeric Abeta treatment, however, inactivated membranous PKC but activated cytosolic PKC in NT2 within 24 h. Our data suggest that oligomeric Abeta may cause selective neurodegeneration through a PKC signaling, distinctive from fibrillar Abeta.     

Amyloid beta peptides and glutamatergic synaptic dysregulation

Alzheimer's disease (AD) is a major neurodegenerative disorder in which overproduction and accumulation of amyloid beta (Abeta) peptides result in synaptic dysfunction. Recent reports strongly suggest that in the initial stages of AD glutamate receptors are dysregulated by Abeta accumulation resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits. In the presence of Abeta, 2-amino-3-(3-hydoxy-5-methylisoxazol-4-yl) propionic acid (AMPA) glutamate receptor function is disrupted and the surface expression is reduced. Abeta has also been shown to modulate N-methyl-d-aspartate receptors (NMDARs) and metabotropic glutamate receptors. The Abeta mediated glutamate receptor modifications can lead to synaptic dysfunction resulting in excitotoxic neurodegeneration during the progression of AD. This review discusses the recent findings that glutamatergic signaling could be compromised by Abeta induced modulation of synaptic glutamate receptors in specific brain regions.     

Soluble fibrillar oligomer levels are elevated in Alzheimer's disease brain and correlate with cognitive dysfunction

Recent evidence has suggested a role for soluble oligomeric Aβ species in the pathology of Alzheimer's disease (AD). Fibrillar plaque deposits are present in non-demented individuals and levels of soluble Aβ correlate better with cognitive dysfunction in AD and transgenic mouse models. We have previously reported that there are at least two conformationally distinct types of Aβ oligomers: prefibrillar oligomers that are kinetic intermediates in fibril assembly reactions and are specifically recognized by A11 antibody and fibrillar oligomers that may represent fibril seeds or small pieces of fibrils and are recognized by a fibril specific antibody, OC. We have examined the levels of these two types of oligomers in the PBS soluble fraction of brain tissue from control cases, cases with senile degenerative changes (SDC) and AD patients. We found that the levels of soluble fibrillar oligomers detected by OC antibody are significantly elevated in multiple brain regions of AD patients. The elevated fibrillar oligomer levels were found not to be an artifact of tissue homogenization, nor a result of increased Aβ or APP levels. The concentration of fibrillar oligomers in adjacent brain regions of the same patient can vary widely and were not detected in post-mortem cerebrospinal fluid. In contrast, the level of prefibrillar oligomers are variable in both AD and age matched controls, indicating that they are not correlated with cognitive dysfunction and suggesting that they precede dementia in AD. Significant correlations were found between the levels of fibrillar oligomers and cognitive decline (MMSE scores) as well as the neuropathological hallmarks of AD. These results indicate that fibrillar oligomers may play a key role in the pathology of AD and may be a new target for diagnostic and therapeutic development.     

A focus on the synapse for neuroprotection in Alzheimer disease and other dementias

Synaptic dysfunction and failure are processes that occur early in Alzheimer disease (AD) and are important targets for protective treatments to slow AD progression and preserve cognitive and functional abilities. Synaptic loss is the best current pathologic correlate of cognitive decline, and synaptic dysfunction is evident long before synapses and neurons are lost. Once synaptic function fails, even in the setting of surviving neurons, there may be little chance of effectively interfering with the disease process. This review emphasizes the importance of preserving synaptic structure and function (i.e., "synaptoprotection") in AD. Such "synaptoprotective" therapy will probably need to be administered at a critical early time point, perhaps years before onset of clinical symptoms.     

High mobility group box protein-1 inhibits microglial Abeta clearance and enhances Abeta neurotoxicity

One pathogenic characteristic of Alzheimer's disease (AD) is the formation of extracellular senile plaques with accumulated microglia. According to the amyloid hypothesis, the increase or accumulation of amyloid-beta (Abeta) peptides in the brain parenchyma is the primary event that influences AD pathology. Although the role of microglia in AD pathology has not been clarified, their involvement in Abeta clearance has been noted. High mobility group box protein-1 (HMGB1) is an abundant nonhistone chromosomal protein. We reported recently that HMGB1 was associated with senile plaques and the total protein level significantly increased in AD brain. In this study, diffuse HMGB1 immunoreactivity was observed around dying neurons in the kainic acid- and Abeta1-42 (Abeta42)-injected rat hippocampi. HMGB1 also colocalized with Abeta in the Abeta42-injected rats but not in transgenic mice, which show massive Abeta production without neuronal loss in their brains. Furthermore, coinjection of HMGB1 delayed the clearance of Abeta42 and accelerated neurodegeneration in Abeta42-injected rats. These results suggest that HMGB1 released from dying neurons may inhibit microglial Abeta42 clearance and enhance the neurotoxicity of Abeta42. HMGB1 may thus be another target in the investigation of a therapeutic strategy for AD.     

Intracerebroventricular passive immunization with anti-oligoAbeta antibody in TgCRND8

Based on the central dogma of beta-amyloid (Abeta) as a key seeding event in the pathogenesis of Alzheimer disease (AD), immunoneutralization strategies have been actively pursued both in AD and in models of AD as a potential means for treating AD. Both active and passive immunizations targeted at fibrillar Abeta successfully remove cerebral plaque load and attenuate Abeta-induced toxicity. Consistently with this, intracerebroventricular (ICV) passive immunization established in our laboratory using antibody against fibrillar Abeta (anti-fAbeta) reduced cerebral plaque load and reversed early synaptic deficits at pre/early plaque stage when there is an abundance of soluble dimeric/oligomeric Abeta but sparse fibrillar Abeta, indicating that anti-fAbeta-mediated partial neutralization of toxic oligomeric Abeta species might have reduced early synaptotoxicity. In the previous investigation, we found that immunoneutralization with anti-fAbeta transiently reduced cerebral Abeta and associated toxicity. The current investigation tested whether ICV im munization using antibody to conformationally changed oligomeric Abeta (anti-oligoAbeta) will overcome the transient restorative nature of anti-fAbeta and produce persistent, long-lasting preventive effects. Because oligomeric Abeta is strongly correlated with synaptotoxicity, we investigated whether immunoneutralization of oligomeric Abeta will reverse synaptic deficits by analyzing presynaptic molecular marker (SNAP-25) profile within hippocampal dendritic fields, where SNAP-25 is abundantly expressed. Results show that, in contrast to ICV anti-fAbeta antibody, ICV anti-oligoAbeta antibody significantly prevented cerebral Abeta build and almost completely restored SNAP-25 immunoreaction up to 8 weeks postinjection in TgCRND8 brain. Results show that ICV passive immunization with anti-oligoAbeta antibody might be an improved ICV immunization strategy for preventing permanent structural damage in AD.     

Beta-amyloid racemized at the Ser26 residue in the brains of patients with Alzheimer disease: implications in the pathogenesis of Alzheimer disease

Oligomeric and fibrillar beta-amyloid (Abeta) may be toxic in Alzheimer disease (AD), especially after post-translation modification cumulative over time. Racemization of Ser and Asp residues of Abeta in senile plaques (SPs) occurs as an age-dependent process in AD. We previously reported that Abeta1-40 racemized at Ser26 is soluble and susceptible to proteolysis yielding toxic [D-Ser26]Abeta25-35/40 fragments in vitro and in vivo. Here, we focus on the localization of racemized Ser26 residues in AD brains within the limbic system, the earliest site of AD histopathology. We developed antisera (20.1 and 22.7). each with epitopes within [D-Ser26]Abeta25-40. Two forms of truncated [D-Ser26]Abeta were detected either in SPs or within neurons in all 11 AD-affected brains, but not in age-matched controls. [D-Ser26]Abeta25/26-35 (detected by 20.1) was localized to plaque cores, extracellular neurofibrillary "ghost" tangles and vascular amyloid deposits. In contrast, [D-Ser26]Abeta25-40 (detected by 22.7) was observed in most neurons containing intracellular neurofibrillary tangles, but not in SPs. These results suggest [D-Ser26]Abeta]1-40, formed during aging, becomes soluble and diffuses from SPs. It is then proteolyzed to [D-Ser26]Abeta25-35/40, which is toxic and may contribute to the neurodegeneration. This hypothesis may explain the long lag between SP formation and neurofibrillary degeneration in AD brains.     

Intracellular amyloid-β accumulation in calcium-binding protein-deficient neurons leads to amyloid-β plaque formation in animal model of Alzheimer's disease

One of the major hallmarks of Alzheimer's disease (AD) is the extracellular deposition of amyloid-β (Aβ) as senile plaques in specific brain regions. Clearly, an understanding of the cellular processes underlying Aβ deposition is a crucial issue in the field of AD research. Recent studies have found that accumulation of intraneuronal Aβ (iAβ) is associated with synaptic deficits, neuronal death, and cognitive dysfunction in AD patients. In this study, we found that Aβ deposits had several shapes and sizes, and that iAβ occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice, an animal model of AD. We also observed pyroglutamate-modified Aβ (N3pE-Aβ), which has been suggested to be a seeding molecule for senile plaques, inside the Aβ plaques only after iAβ accumulation, which argues against its seeding role. In addition, we found that iAβ accumulates in calcium-binding protein (CBP)-free neurons, induces neuronal death, and then develops into senile plaques in 2-4-month-old 5XFAD mice. These findings suggest that N3pE-Aβ-independent accumulation of Aβ in CBP-free neurons might be an early process that triggers neuronal damage and senile plaque formation in AD patients. Our results provide new insights into several long-standing gaps in AD research, namely how Aβ plaques are formed, what happens to iAβ and how Aβ causes selective neuronal loss in AD patients.     

Amyloid beta protein deposition and neuron loss in osteopetrotic (op/op) mice

Formation of senile plaques (SPs) by amyloid beta (Abeta) protein is a neuropathological change which characterizes Alzheimer's disease (AD), and Abeta deposition and neuron loss are essential for the pathological cascade of the disease. Although the mechanism of Abeta deposition remains unclear, it has been suggested that clearance of Abeta protein may be impaired in the AD brain. Previous studies demonstrated that microglia were able to remove Abeta by releasing a metalloprotease or by phagocytosis, suggesting that microglia may play an important role in preventing Abeta deposition in the central nervous system (CNS). On the other hand, it was reported that the number of microglia was reduced in osteopetrotic (op/op) toothless mice resulting from the lack of functional macrophage colony-stimulating factor (M-CSF). The present study was thus designed to examine the Abeta deposition and the number of hippocampal neurons in the brain of op/op mice. A number of fibrillar plaques were detected in the cerebral cortex, hippocampus, amygdala and hypothalamus in op/op mice, however, no quantitative evidence of Abeta deposition was observed in normal mice. Moreover, the total number of pyramidal cells in the hippocampal CA1, and CA3 regions was significantly reduced in op/op mice when compared to the controls. These results demonstrate that Abeta deposition influence neuron loss and it may be suspected that expression of SPs may be in part regulated by microglia under physiological conditions.     

Deposits of fibrillar Aβ do not cause neuronal loss or ferritin expression in adult rat brain

In Alzheimer's disease (AD), senile plaques containing amyloid-beta (Abeta) are associated with neurodegeneration, yet little quantitative data are available concerning the spatiotemporal patterns of neuronal death that result from exposure to Abeta deposits. Furthermore, plaques are accompanied by ferritin-rich cells but no data exist regarding the spatiotemporal expression of ferritin in response to Abeta. The present study has obtained such data after injecting aged Abeta peptide into the parietal cortex of adult rats. Injected deposits of fibrillar Abeta (1 microliter of 1 mM in saline) were cleared within 7 days but did not cause a significant increase in ferritin expression. Counts of dying neurones showed that human Abeta1-40 killed as many neurones as control injections of saline, while human Abeta1-42 and rat Abeta1-40 killed significantly less. We conclude that the fibrillar Abeta in plaques is not likely to be directly responsible for the neurodegeneration and ferritin expression that occurs in AD.     

Modeling behavioral and neuronal symptoms of Alzheimer's disease in mice: a role for intraneuronal amyloid

The amyloid Abeta-peptide (Abeta) is suspected to play a critical role in the cascade leading to AD as the pathogen that causes neuronal and synaptic dysfunction and, eventually, cell death. Therefore, it has been the subject of a huge number of clinical and basic research studies on this disease. Abeta is typically found aggregated in extracellular amyloid plaques that occur in specific brain regions enriched in nAChRs in Alzheimer's disease (AD) and Down syndrome (DS) brains. Advances in the genetics of its familiar and sporadic forms, together with those in gene transfer technology, have provided valuable animal models that complement the traditional cholinergic approaches, although modeling the neuronal and behavioral deficits of AD in these models has been challenging. More recently, emerging evidence indicates that intraneuronal accumulation of Abeta may also contribute to the cascade of neurodegenerative events and strongly suggest that it is an early, pathological biomarker for the onset of AD and associated cognitive and other behavioral deficits. The present review covers these studies in humans, in in vitro and in transgenic models, also providing more evidence that adult 3xTg-AD mice harboring PS1M146V, APPSwe, tauP301L transgenes, and mimicking many critical hallmarks of AD, show cognitive deficits and other behavioral alterations at ages when overt neuropathology is not yet observed, but when intraneuronal Abeta, synaptic and cholinergic deficits can already be described.     

Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses

Synaptic dysfunction is increasingly viewed as an early manifestation of Alzheimer's disease (AD), but the cellular mechanism by which beta-amyloid (Abeta) may affect synapses remains unclear. Since cultured neurons derived from APP mutant transgenic mice secrete elevated levels of Abeta and parallel the subcellular Abeta accumulation seen in vivo, we asked whether alterations in synapses occur in this setting. We report that cultured Tg2576 APP mutant neurons have selective alterations in pre- and post-synaptic compartments compared to wild-type neurons. Post-synaptic compartments appear fewer in number and smaller, while active pre-synaptic compartments appear fewer in number and enlarged. Among the earliest changes in synaptic composition in APP mutant neurons were reductions in PSD-95, a protein involved in recruiting and anchoring glutamate receptor subunits to the post-synaptic density. In agreement, we observed early reductions in surface expression of glutamate receptor subunit GluR1 in APP mutant neurons. We provide evidence that Abeta is specifically involved in these alterations in synaptic biology, since alterations in PSD-95 and GluR1 are blocked by gamma-secretase inhibition, and since exogenous addition of synthetic Abeta to wild-type neurons parallels changes in synaptic PSD-95 and GluR1 observed in APP mutant neurons.     

LRP and senile plaques in Alzheimer's disease: colocalization with apolipoprotein E and with activated astrocytes

The low density lipoprotein receptor-related protein (LRP) is a multifunctional receptor which is present on senile plaques in Alzheimer's disease (AD). It is suggested to play an important role in the balance between amyloid beta (Abeta) synthesis and clearance mechanisms. One of its ligands, apolipoprotein E (apoE), is also present on senile plaques and has been implicated as a risk factor for AD, potentially affecting the deposition, fibrillogenesis and clearance of Abeta. Using immunohistochemistry we show that LRP was present only on cored, apoE-containing senile plaques, in both PDAPP transgenic mice and human AD brains. We detected strong LRP staining in neurons and in reactive astrocytes, and immunostaining of membrane-bound LRP showed colocalization with fine astrocytic processes surrounding senile plaques. LRP was not present in plaques in young transgenic mice or in plaques of APOE-knockout mice. As LRP ligands associated with Abeta deposits in AD brain may play an important role in inducing levels of LRP in both neurons and astrocytes, our findings support the idea that apoE might be involved in upregulation of LRP (present in fine astrocytic processes) and act as a local scaffolding protein for LRP and Abeta. The upregulation of LRP would allow increased clearance of LRP ligands as well as clearance of Abeta/ApoE complexes.     

Progressive age-related development of Alzheimer-like pathology in APP/PS1 mice

Increasing evidence points to synaptic plasticity impairment as one of the first events in Alzheimer's disease (AD). However, studies on synaptic dysfunction in different transgenic AD models that overexpress familial AD mutant forms of amyloid precursor protein (APP) and/or presenilin (PS) have provided conflicting results. Both long-term potentiation (LTP) and basal synaptic transmission (BST) have been found to be both unchanged and altered in different models and under differing experimental conditions. Because of their more robust amyloid-beta (Abeta) deposition, double transgenic mice currently are used by several laboratories as an AD model. Here, we report that mice overexpressing APP (K670N:M671L) together with PS1 (M146L) have abnormal LTP as early as 3 months of age. Interestingly, reduced LTP paralleled plaque appearance and increased Abeta levels and abnormal short-term memory (working memory). BST and long-term memory (reference memory) are impaired only later (approximately 6 months) as amyloid burden increases. Abeta pathology across different ages did not correlate with synaptic and cognitive deficits, suggesting that Abeta levels are not a marker of memory decline. In contrast, progression of LTP impairment correlated with the deterioration of working memory, suggesting that percentage of potentiation might be an indicator of the cognitive decline and disease progression in the APP/PS1 mice.     

Role of the blood-brain barrier in the pathogenesis of Alzheimer's disease

Cerebrovascular dysfunction contributes to the cognitive decline and dementia in Alzheimer's disease (AD), and may precede cerebral amyloid angiopathy and brain accumulation of the Alzheimer's neurotoxin, amyloid beta-peptide (Abeta). The blood-brain barrier (BBB) is critical for brain Abeta homeostasis and regulates Abeta transport via two main receptors, the low density lipoprotein receptor related protein 1 (LRP1) and the receptor for advanced glycation end products (RAGE). According to the neurovascular hypothesis of AD, faulty BBB clearance of Abeta through deregulated LRP1/RAGE-mediated transport, aberrant angiogenesis and arterial dysfunction may initiate neurovascular uncoupling, Abeta accumulation, cerebrovascular regression, brain hypoperfusion and neurovascular inflammation. Ultimately these events lead to BBB compromise and chemical imbalance in the neuronal 'milieu', and result in synaptic and neuronal dysfunction. Based on the neurovascular hypothesis, we suggest an array of new potential therapeutic approaches that could be developed for AD to reduce neuroinflammation, enhance Abeta clearance and neurovascular repair, and improve cerebral blood flow. RAGE-based and LRP1-based therapeutic strategies have potential to control brain Abeta in AD, and possibly related familial cerebrovascular beta-amyloidoses. In addition, we have identified two vascularly restricted genes, GAX (growth arrest-specific homeobox), which controls LRP1 expression in brain capillaries and brain angiogenesis, and MYOCD (myocardin), which controls contractility of cerebral arterial smooth muscle cells and influences cerebral blood flow. These findings provide insights into new pathogenic pathways for the vascular dysfunction in AD and point to new therapeutic targets for AD.     

Hemoglobin promotes Abeta oligomer formation and localizes in neurons and amyloid deposits

The objective of this study was to search for brain-specific binding proteins that participated in Abeta aggregation. Immunoprecipitation of Abeta in Alzheimer's brain homogenate revealed a major co-precipitating 16-kDa protein band, which was identified through mass spectrometry as hemoglobin (Hb) alpha and beta chains. Hemoglobin was distributed in Alzheimer's disease (AD) patients in a brain region-dependent manner, with the highest levels in the hippocampus and parietal gray (PG) matter, followed by parietal white matter (PW), and the lowest in cerebellum (Cb). AD parietal gray and white matters exhibited higher Hb levels than those in the nondemented (ND) group. Likewise, RT-PCR revealed that the Hb mRNA levels in AD inferior temporal gyri were higher than those of ND subjects. Furthermore, Hb was shown to promote Abeta oligomer formation. Immunohistochemical studies indicated that Hb was localized within the cytosol of pyramidal neurons in the hippocampus, suggesting a potential source of intracerebral Hb. Finally, double immunofluorescent assay confirmed the co-localization of Hb with senile plaques (SP) and cerebral amyloid angiopathy (CAA). We propose that an elevation in brain Hb via circulation leakage or perturbations of Hb gene regulation may participate in AD pathogenesis.     

Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by amyloid deposition in the cerebral neuropil and vasculature. These amyloid deposits comprise predominantly fragments and full-length (40 or 42 residue) forms of the amyloid beta-protein (Abeta) organized into fibrillar assemblies. Compelling evidence indicates that factors that increase overall Abeta production or the ratio of longer to shorter forms, or which facilitate deposition or inhibit elimination of amyloid deposits, cause AD or are risk factors for the disease. In vitro studies have demonstrated that fibrillar Abeta has potent neurotoxic effects on cultured neurons. In vivo experiments in non-human primates have demonstrated that Abeta fibrils directly cause pathologic changes, including tau hyperphosphorylation. In concert with histologic studies revealing a lack of tissue injury in areas of the neuropil in which non-fibrillar deposits were found, these data suggested that fibril assembly was a prerequisite for Abeta-mediated neurotoxicity in vivo. Recently, however, both in vitro and in vivo studies have revealed that soluble, oligomeric forms of Abeta also have potent neurotoxic activities, and in fact, may be the proximate effectors of the neuronal injury and death occurring in AD. A paradigm shift is thus emerging that necessitates the reevaluation of the relative importance of polymeric (fibrillar) vs. oligomeric assemblies in the pathobiology of AD. In addition to AD, an increasing number of neurodegenerative disorders, including Parkinson's disease, familial British dementia, familial amyloid polyneuropathy, amyotrophic lateral sclerosis, and prion diseases, are associated with abnormal protein assembly processes. The archetypal features of the assembly-dependent neuropathogenetic effects of Abeta may thus be of relevance not only to AD but to these other disorders as well.