Ajeet Rijal Upadhaya

Biochemical stages of amyloid-β peptide aggregation and accumulation in the human brain and their association with symptomatic and pathologically preclinical Alzheimer’s disease

Alzheimers disease is characterized by the deposition of amyloid-β peptide in the brain. N-terminal truncation resulting in the formation of AβN3pE and phosphorylation at serine 8 have been reported to modify aggregation properties of amyloid-β. Biochemically, soluble, dispersible, membrane-associated, and insoluble, plaque-associated amyloid-β aggregates have been distinguished. Soluble and dispersible amyloid-β aggregates are both in mixture with the extracellular or intracellular fluid but dispersible aggregates can be cleared from proteins in solution by ultracentrifugation. To clarify the role of phosphorylated amyloid-β and AβN3pE in soluble, dispersible, membrane-associated, and plaque-associated amyloid-β aggregates in the pathogenesis of Alzheimers disease we studied brains from 21 cases with symptomatic Alzheimers disease, 33 pathologically preclinical Alzheimers disease cases, and 20 control cases. Western blot analysis showed that soluble, dispersible, membrane-associated and plaque-associated amyloid-β aggregates in the earliest preclinical stage of Alzheimers disease did not exhibit detectable amounts of AβN3pE and phosphorylated amyloid-β. This stage was referred to as biochemical stage 1 of amyloid-β aggregation and accumulation. In biochemical amyloid-β stage 2, AβN3pE was additionally found whereas phosphorylated amyloid-β was restricted to biochemical amyloid-β stage 3, the last stage of amyloid-β aggregation. Phosphorylated amyloid-β was seen in the dispersible, membrane-associated, and plaque-associated fraction. All cases with symptomatic Alzheimers disease in our sample fulfilled biochemical amyloid-β stage 3 criteria, i.e. detection of phosphorylated amyloid-β. Most, but not all, cases with pathologically preclinical Alzheimers disease had biochemical amyloid-β stages 1 or 2. Immunohistochemistry confirmed the hierarchical occurrence of amyloid-β, AβN3pE, and phosphorylated amyloid-β in amyloid plaques. Phosphorylated amyloid-β containing plaques were, thereby, seen in all symptomatic cases with Alzheimers disease but only in a few non-demented control subjects. The biochemical amyloid-β stages correlated with the expansion of amyloid-β plaque deposition and with that of neurofibrillary tangle pathology. Taken together, we demonstrate that AβN3pE and phosphorylated amyloid-β are not only detectable in plaques, but also in soluble and dispersible amyloid-β aggregates outside of plaques. They occur in a hierarchical sequence that allows the distinction of three stages. In light of our findings, it is tempting to speculate that this hierarchical, biochemical sequence of amyloid-β aggregation and accumulation is related to disease progression and may be relevant for an increasing toxicity of amyloid-β aggregates.

Cerebral small vessel disease-induced Apolipoprotein E leakage is associated with Alzheimer disease and the accumulation of amyloid [beta]-protein in perivascular astrocytes

Apolipoprotein E (apoE) plays a role in the pathogenesis of Alzheimer disease (AD). It is involved in the receptor-mediated cellular clearance of the amyloid &bgr;-protein (A&bgr;) and in the perivascular drainage of the extracellular fluid. Microvascular changes are also associated with AD and have been discussed as a possible reason for altered perivascular drainage. To further clarify the role of apoE in the perivascular and vascular pathology in AD patients, we studied its occurrence and distribution in the perivascular space, the perivascular neuropil, and in the vessel wall of AD and control cases with and without small vessel disease (SVD). Apolipoprotein E was found in the perivascular space and in the neuropil around arteries of the basal ganglia from control and AD cases disclosing no major differences. Western blot analysis of basal ganglia tissue also revealed no significant differences pertaining to the amount of full-length and C-terminal truncated apoE in AD cases compared with controls. In contrast, A&bgr; occurred in apoE-positive perivascular astrocytes in AD cases but not in controls. In blood vessels, apoE and immunoglobulin G were detected within the SVD-altered vessel wall. The severity of SVD was associated with the occurrence of apoE in the vessel wall and with that of A&bgr; in perivascular astrocytes. These results point to an important role of apoE in the perivascular clearance of A&bgr; in the human brain. The occurrence of apoE and immunoglobulin G in SVD lesions and in the perivascular space suggests that the presence of SVD results in plasma-protein leakage into the brain. It is therefore tempting to speculate that apoE represents a pathogenetic link between SVD and AD.

High-molecular weight Aβ oligomers and protofibrils are the predominant Aβ species in the native soluble protein fraction of the AD brain

Alzheimer’s disease (AD) is characterized by the aggregation and deposition of amyloid β protein (Aβ) in the brain. Soluble Aβ oligomers are thought to be toxic. To investigate the predominant species of Aβ protein that may play a role in AD pathogenesis, we performed biochemical analysis of AD and control brains. Sucrose buffer‐soluble brain lysates were characterized in native form using blue native (BN)‐PAGE and also in denatured form using SDS‐PAGE followed by Western blot analysis. BN‐PAGE analysis revealed a high‐molecular weight smear (>1000 kD) of Aβ42‐positive material in the AD brain, whereas low‐molecular weight and monomeric Aβ species were not detected. SDS‐PAGE analysis, on the other hand, allowed the detection of prominent Aβ monomer and dimer bands in AD cases but not in controls. Immunoelectron microscopy of immunoprecipitated oligomers and protofibrils/fibrils showed spherical and protofibrillar Aβ‐positive material, thereby confirming the presence of high‐molecular weight Aβ (hiMWAβ) aggregates in the AD brain. In vitro analysis of synthetic Aβ40‐ and Aβ42 preparations revealed Aβ fibrils, protofibrils, and hiMWAβ oligomers that were detectable at the electron microscopic level and after BN‐PAGE. Further, BN‐PAGE analysis exhibited a monomer band and less prominent low‐molecular weight Aβ (loMWAβ) oligomers. In contrast, SDS‐PAGE showed large amounts of loMWAβ but no hiMWAβ40 and strikingly reduced levels of hiMWAβ42. These results indicate that hiMWAβ aggregates, particularly Aβ42 species, are most prevalent in the soluble fraction of the AD brain. Thus, soluble hiMWAβ aggregates may play an important role in the pathogenesis of AD either independently or as a reservoir for release of loMWAβ oligomers.

Transgenic Expression of Intraneuronal Aβ42 But Not Aβ40 Leads to Cellular Aβ Lesions, Degeneration, and Functional Impairment without Typical Alzheimer's Disease Pathology

An early role of amyloid-β peptide (Aβ) aggregation in Alzheimers disease pathogenesis is well established. However, the contribution of intracellular or extracellular forms of Aβ to the neurodegenerative process is a subject of considerable debate. We here describe transgenic mice expressing Aβ1–40 (APP47) and Aβ1–42 (APP48) with a cleaved signal sequence to insert both peptides during synthesis into the endoplasmic reticulum. Although lower in transgene mRNA, APP48 mice reach a higher brain Aβ concentration. The reduced solubility and increased aggregation of Aβ1–42 may impair its degradation. APP48 mice develop intracellular Aβ lesions in dendrites and lysosomes. The hippocampal neuron number is reduced already at young age. The brain weight decreases during aging in conjunction with severe white matter atrophy. The mice show a motor impairment. Only very few Aβ1–40 lesions are found in APP47 mice. Neither APP47 nor APP48 nor the bigenic mice develop extracellular amyloid plaques. While intracellular membrane expression of Aβ1–42 in APP48 mice does not lead to the AD-typical lesions, Aβ aggregates develop within cells accompanied by considerable neurodegeneration.

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 Alzheimers 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.

Amyloid-β protein modulates the perivascular clearance of neuronal apolipoprotein E in mouse models of Alzheimer’s disease

The deposition of amyloid-β protein (Aβ) in the brain is a hallmark of Alzheimer’s disease (AD). Apolipoprotein E (apoE) is involved in the clearance of Aβ from brain and the APOE ε4 allele is a major risk factor for sporadic AD. We have recently shown that apoE is drained into the perivascular space (PVS), where it co-localizes with Aβ. To further clarify the role of apoE in perivascular clearance of Aβ, we studied apoE-transgenic mice over-expressing human apoE4 either in astrocytes (GE4) or in neurons (TE4). These animals were crossbred with amyloid precursor protein (APP)-transgenic mice and with APP-presenilin-1 (APP-PS1) double transgenic mice. Using an antibody that specifically detects human apoE (h-apoE), we observed that astroglial expression of h-apoE in GE4 mice leads to its perivascular drainage, whereas neuronal expression in TE4 mice does not, indicating that neuron-derived apoE is usually not the subject of perivascular drainage. However, h-apoE was observed not only in the PVS of APP-GE4 and APP-PS1-GE4 mice, but also in that of APP-TE4 and APP-PS1-TE4 mice. In all these mouse lines, we found co-localization of neuron-derived h-apoE and Aβ in the PVS. Aβ and h-apoE were also found in the cytoplasm of perivascular astrocytes indicating that astrocytes take up the neuron-derived apoE bound to Aβ, presumably prior to its clearance into the PVS. The uptake of apoE–Aβ complexes into glial cells was further investigated in glioblastoma cells. It was mediated by α2macroglobulin receptor/low density lipoprotein receptor-related protein (LRP-1) and inhibited by adding receptor-associated protein (RAP). It results in endosomal Aβ accumulation within these cells. These results suggest that neuronal apoE–Aβ complexes, but not neuronal apoE alone, are substrates for LRP-1-mediated astroglial uptake, transcytosis, and subsequent perivascular drainage. Thus, the production of Aβ and its interaction with apoE lead to the pathological perivascular drainage of neuronal apoE and provide insight into the pathological interactions of Aβ with neuronal apoE metabolism.

The type of Aβ-related neuronal degeneration differs between amyloid precursor protein (APP23) and amyloid β-peptide (APP48) transgenic mice.

BackgroundThe deposition of the amyloid β-peptide (Aβ) in the brain is one of the hallmarks of Alzheimer’s disease (AD). It is not yet clear whether Aβ always leads to similar changes or whether it induces different features of neurodegeneration in relation to its intra- and/or extracellular localization or to its intracellular trafficking routes. To address this question, we have analyzed two transgenic mouse models: APP48 and APP23 mice. The APP48 mouse expresses Aβ1-42 with a signal sequence in neurons. These animals produce intracellular Aβ independent of amyloid precursor protein (APP) but do not develop extracellular Aβ plaques. The APP23 mouse overexpresses human APP with the Swedish mutation (KM670/671NL) in neurons and produces APP-derived extracellular Aβ plaques and intracellular Aβ aggregates.ResultsTracing of commissural neurons in layer III of the frontocentral cortex with the DiI tracer revealed no morphological signs of dendritic degeneration in APP48 mice compared to littermate controls. In contrast, the dendritic tree of highly ramified commissural frontocentral neurons was altered in 15-month-old APP23 mice. The density of asymmetric synapses in the frontocentral cortex was reduced in 3- and 15-month-old APP23 but not in 3- and 18-month-old APP48 mice. Frontocentral neurons of 18-month-old APP48 mice showed an increased proportion of altered mitochondria in the soma compared to wild type and APP23 mice. Aβ was often seen in the membrane of neuronal mitochondria in APP48 mice at the ultrastructural level.ConclusionsThese results indicate that APP-independent intracellular Aβ accumulation in APP48 mice is not associated with dendritic and neuritic degeneration but with mitochondrial alterations whereas APP-derived extra- and intracellular Aβ pathology in APP23 mice is linked to dendrite degeneration and synapse loss independent of obvious mitochondrial alterations. Thus, Aβ aggregates in APP23 and APP48 mice induce neurodegeneration presumably by different mechanisms and APP-related production of Aβ may, thereby, play a role for the degeneration of neurites and synapses.

Modified amyloid variants in pathological subgroups of β‐amyloidosis

Amyloid β (Aβ) depositions in plaques and cerebral amyloid angiopathy (CAA) represent common features of Alzheimers disease (AD). Sequential deposition of post‐translationally modified Aβ in plaques characterizes distinct biochemical stages of Aβ maturation. However, the molecular composition of vascular Aβ deposits in CAA and its relation to plaques remain enigmatic.

tau pathology in Young people and its progression Before Abeta Deposition

forms of frontotemporal dementia, establishing that tau protein dysfunction is sufficient to cause neurodegeneration and dementia. Thus, transgenic mice expressing mutant (e.g. P301S) human tau in nerve cells exhibit the essential features of tauopathies, including neurodegeneration and abundant filaments made of hyperphosphorylated tau protein. In contrast, mouse lines expressing single isoforms of wild-type human tau do not produce tau filaments or display neurodegeneration. Methods: Here we have used tau-expressing lines to investigate whether experimental tauopathy can be transmitted. Results: We show that the injection of brain extract from mutant P301S tau-expressing mice into the brain of transgenic wild-type tau-expressing animals induces the assembly of wild-type human tau into filaments and the spreading of pathology from the site of injection to neighbouring brain regions. The study will be complemented by the intracerebral injection of human tissues from various tauopathies into the brain of both transgenic wild-type tau-expressing mice and wild-type B6 mice.

Elevated levels of soluble, high molecular weight SDS-denaturable Aβ-aggregates are associated with dendritic degeneration in APP-transgenic mouse models

concern in Alzheimer’s disease (AD) patients. Interestingly, it has been suggested that the pathological mechanism(s) underlying POCDmimic AD. Indeed, anesthesia might be a risk factor for the development of neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Patients with AD are considered to be particularly at risk for some of the cognitive side effects of anesthesia, and there is also concern that general anesthesia is a risk factor for AD, with in vitro and in vivo studies suggesting that anesthetics may promote and intensify the neuropathogenesis of AD. Methods: After a short review of the clinical literature on anesthesia and AD, we will focus on describing the impact of anesthesia on the two pathological hallmarks of AD: beta-amyloid (Abeta) accumulation and aberrant tau phosphorylation and aggregation, with an emphasis on our most recent data. Results: Evidence from in vitro and animal models demonstrate that exposure to inhaled anesthetics, such as isoflurane and halothane, can increase Abeta production, enhance Abeta oligomerization, and promote plaque formation, while exposure to intravenous anesthetics, such as propofol, thiopental or chloral hydrate, has no effect. We have also demonstrated that exposure to isoflurane could lead to tau hyperphosphorylation, detachment from microtubules and enhanced aggregation in vivo, albeit indirectly, by inducing hypothermia in mouse models of tauopathies. On the other hand, some anesthetics such as propofol also have a direct effect on tau phosphorylation, as demonstrated by maintaining the animals normothermic. Conclusions:While there has been clinical interest on the relation between anesthesia and AD for at least a quarter of a century, the biochemical consequences of anesthesia on AD neuropathogenic pathways have only begun to be studied very recently. Overall, epidemiological evidence establishing a link between anesthetic exposure and the risk of AD remains controversial. On the other hand, clinical studies examining AD biomarkers, and studies exploring the impact of anesthetics on Abeta and tau, converge to indicate that anesthetics could affect AD pathogenesis, either directly or indirectly.