Intraneuronal Aβ Research Articles

REGION-SPECIFIC NEURON AND SYNAPSE LOSS IN THE HIPPOCAMPUS OF APPSL/PS1 KNOCK-IN MICE.

Transgenic mouse models with knock-in (KI) expression of human mutant amyloid precursor protein (APP) and/or human presenilin 1 (PS1) may be helpful to elucidate the cellular consequences of APP and PS1 misprocessing in the aging brain. Age-related alterations in total numbers of neurons and in numbers of synaptophysin-immunoreactive presynaptic boutons (SIPB), as well as the amyloid plaque load were analyzed in the hippocampal dentate gyrus (DG), CA3, and CA1-2 of 2- and 10-month-old APPSL/PS1 homozygous KI, APPSL (expressing human mutant APP751 carrying the Swedish [K670N/M671L] and London [V717I] mutations under Thy-1 promoter), and PS1 homozygous KI mice (expressing human PS1 mutations [M233T and L235P]). APPSL/PS1 homozygous KI mice, but neither APPSL mice nor PS1 homozygous KI mice, showed substantial age-related loss of neurons (-47.2%) and SIPB (-22.6%), specifically in CA1-2. PS1 homozygous KI mice showed an age-related increase in hippocampal granule cell numbers (+37.9%). Loss of neurons and SIPB greatly exceeded the amount of local extracellular Aβ aggregation and astrocytes, whereas region-specific accumulation of intraneuronal Aβ preceded neuron and synapse loss. An age-related increase in the ratio of SIPB to neuron numbers in CA1-2 of APPSL/PS1 homozygous KI mice was suggestive of compensatory synaptic plasticity. These findings indicate a region-selectivity in intra- and extraneuronal Aβ accumulation in connection with neuron and synapse loss in the hippocampus of APPSL/PS1 homozygous KI mice.

PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer's amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE).

BackgroundAlzheimer’s disease (AD) is intimately tied to amyloid-β (Aβ) peptide. Extraneuronal brain plaques consisting primarily of Aβ aggregates are a hallmark of AD. Intraneuronal Aβ subunits are strongly implicated in disease progression. Protein sequence mutations of the Aβ precursor protein (APP) account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP) may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or “proximal regulatory element” (PRE), at −76/−47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA) and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones.ResultsEMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2), nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF), and specificity protein 1 (SP1). These sites crossed a known single nucleotide polymorphism (SNP). EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis.ConclusionsWe propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also interacts with the integrins. These proteins are connected to vital cellular and neurological functions. In addition, the transcription factor PuF is a known inhibitor of metastasis and regulates cell growth during development. Given that APP is a known cell adhesion protein and ferroxidase, this suggests biochemical links among cell signaling, the cell cycle, iron metabolism in cancer, and AD in the context of overall aging.

APOE4-specific Changes in Aβ Accumulation in a New Transgenic Mouse Model of Alzheimer Disease

APOE4 is the greatest risk factor for Alzheimer disease (AD) and synergistic effects with amyloid-β peptide (Aβ) suggest interactions among apoE isoforms and different forms of Aβ accumulation. However, it remains unclear how the APOE genotype affects plaque morphology, intraneuronal Aβ, soluble Aβ42, and oligomeric Aβ (oAβ), particularly in vivo. As the introduction of human APOE significantly delays amyloid deposition in transgenic mice expressing familial AD (FAD) mutations (FAD-Tg), 5xFAD-Tg mice, which exhibit amyloid deposition by age 2 months, were crossed with apoE-targeted replacement mice to produce the new EFAD-Tg mice. Compared with 5xFAD mice, Aβ deposition was delayed by ∼4 months in the EFAD mice, allowing detection of early changes in Aβ accumulation from 2-6 months. Although plaque deposition is generally greater in E4FAD mice, E2/E3FAD mice have significantly more diffuse and E4FAD more compact plaques. As a first report in FAD-Tg mice, the APOE genotypes had no effect on intraneuronal Aβ accumulation in EFAD mice. In E4FAD mice, total apoE levels were lower and total Aβ levels higher than in E2FAD and E3FAD mice. Profiles from sequential three-step extractions (TBS, detergent, and formic acid) demonstrated that the lower level of total apoE4 is reflected only in the detergent-soluble fraction, indicating that less apoE4 is lipoprotein-associated, and perhaps less lipidated, compared with apoE2 and apoE3. Soluble Aβ42 and oAβ levels were highest in E4FAD mice, although soluble apoE2, apoE3, and apoE4 levels were comparable, suggesting that the differences in soluble Aβ42 and oAβ result from functional differences among the apoE isoforms. Thus, APOE differentially regulates multiple aspects of Aβ accumulation.

The β-Secretase-Derived C-Terminal Fragment of βAPP, C99, But Not Aβ, Is a Key Contributor to Early Intraneuronal Lesions in Triple-Transgenic Mouse Hippocampus

Triple-transgenic mice (3xTgAD) overexpressing Swedish-mutated β-amyloid precursor protein (βAPP(swe)), P310L-Tau (Tau(P301L)), and physiological levels of M146V-presenilin-1 (PS1(M146V)) display extracellular amyloid-β peptides (Aβ) deposits and Tau tangles. More disputed is the observation that these mice accumulate intraneuronal Aβ that has been linked to synaptic dysfunction and cognitive deficits. Here, we provide immunohistological, genetic, and pharmacological evidences for early, age-dependent, and hippocampus-specific accumulation of the β-secretase-derived βAPP fragment C99 that is observed from 3 months of age and enhanced by pharmacological blockade of γ-secretase. Notably, intracellular Aβ is only detectable several months later and appears, as is the case of C99, in enlarged cathepsin B-positive structures, while extracellular Aβ deposits are detected ~12 months of age and beyond. Early C99 production occurs mainly in the CA1/subicular interchange area of the hippocampus corresponding to the first region exhibiting plaques and tangles in old mice. Furthermore, the comparison of 3xTgAD mice with double-transgenic mice bearing the βAPP(swe) and Tau(P301L) mutations but expressing endogenous PS1 (2xTgAD) demonstrate that C99 accumulation is not accounted for by a loss of function triggered by PS1 mutation that would have prevented C99 secondary cleavage by γ-secretase. Together, our work identifies C99 as the earliest βAPP catabolite and main contributor to the intracellular βAPP-related immunoreactivity in 3xTgAD mice, suggesting its implication as an initiator of the neurodegenerative process and cognitive alterations taking place in this mouse model.

Age-dependent alterations in the presynaptic active zone in a Drosophila model of Alzheimer's Disease

The accumulation of beta amyloid (Aβ) can cause synaptic impairments, but the characteristics and mechanisms of the synaptic impairment induced by the accumulation of Aβ in Alzheimer's disease (AD) remain unclear. In identified single neurons in a newly developed Drosophila AD model, in which Aβ accumulates intraneuronally, we found an age-dependent reduction in the synaptic vesicle release probability that was associated with a decrease in the density of presynaptic calcium channel clusters and an increase in the presynaptic and postsynaptic contact length. Moreover, these alterations occurred in the absence of presynaptic bouton loss. In addition, we found that Aβ expression also produced an age-dependent decrease in the amount of Bruchpilot (Brp), which plays an important role in controlling Ca2+ channel clustering and synaptic vesicle release in the presynaptic active zone. Our study indicates that the chronic accumulation of intraneuronal Aβ can induce functional and structural changes in the presynaptic active zone prior to a loss of presynaptic buttons in the same neuron.

Early accumulation of intracellular fibrillar oligomers and late congophilic amyloid angiopathy in mice expressing the Osaka intra-Aβ APP mutation

Pathogenic amyloid-β peptide precursor (APP) mutations clustered around position 693 of APP—position 22 of the Aβ sequence—are commonly associated with congophilic amyloid angiopathy (CAA) and intracerebral hemorrhages. In contrast, the Osaka (E693Δ) intra-Aβ APP mutation shows a recessive pattern of inheritance that leads to AD-like dementia despite low brain amyloid on in vivo positron emission tomography imaging. Here, we investigated the effects of the Osaka APP mutation on Aβ accumulation and deposition in vivo using a newly generated APP transgenic mouse model (E22ΔAβ) expressing the Osaka mutation together with the Swedish (K670N/M671L) double mutation. E22ΔAβ mice exhibited reduced α-processing of APP and early accumulation of intraneuronal fibrillar Aβ oligomers associated with cognitive deficits. In line with our in vitro findings that recombinant E22Δ-mutated Aβ peptides form amyloid fibrils, aged E22ΔAβ mice showed extracellular CAA deposits in leptomeningeal cerebellar and cortical vessels. In vitro results from thioflavin T aggregation assays with recombinant Aβ peptides revealed a yet unknown antiamyloidogenic property of the E693Δ mutation in the heterozygous state and an inhibitory effect of E22Δ Aβ42 on E22Δ Aβ40 fibrillogenesis. Moreover, E22Δ Aβ42 showed a unique aggregation kinetics lacking exponential fibril growth and poor seeding effects on wild-type Aβ aggregation. These results provide a possible explanation for the recessive trait of inheritance of the Osaka APP mutation and the apparent lack of amyloid deposition in E693Δ mutation carriers.

High tryptophan diet reduces CA1 intraneuronal β‐amyloid in the triple transgenic mouse model of Alzheimer’s disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease that impairs mnesic functions. The histopathology of the disease is manifested by the accumulation of intracellular β-amyloid (Aβ) and subsequent formation of neuritic plaques as well as the presence neurofibrillary tangles in specific brain regions associated with learning and memory including the hippocampus. Here, we analysed the effect of chronic (1 month) food diets containing low (LTrP), normal (NTrP) and high tryptophan (HTrP), 0.04, 0.20 and 0.40 g/100 g, respectively, on CA1 serotonin transporter (SERT) fibre density, intraneuronal Aβ deposition and total number of serotonergic (5-HT) neurons in an AD triple transgenic (3xTg-AD) mouse model. Nontransgenic (non-Tg) animals fed with HTrP displayed increased SERT fibre density in CA1 (35%) and in stratum lacunosum moleculare (S.Mol) (48%) compared to LTrP diet. Transgenic animals showed increased SERT fibre density in CA1 S.Mol compared to diet-matched non-Tg irrespective of dietary tryptophan content (104% for LTrP, 74% for NTrP and 35% for HTrP); no differences were observed in the total number of 5-HT neurons neither in the dorsal nor in the median raphe nuclei. However, and more relevant to AD, HTrP diet reduced intraneuronal Aβ density (by a 17%) in transgenic animals compared to transgenic animals fed with NTrP diet. Our results show that increased dietary TrP intake reduces intraneuronal Aβ load in the 3xTg-AD mouse model of AD, suggesting that enhanced TrP intake and in consequence a potential increase in 5-HT neurotransmission may be effective in reducing plaque pathology in AD.

Critical role of intraneuronal Aβ in Alzheimer's disease: Technical challenges in studying intracellular Aβ

AimsMultiple lines of evidence have implicated β-amyloid (Aβ) in the pathogenesis of Alzheimer's disease (AD). However, the mechanism(s) whereby Aβ is involved in the disease process remains unclear. The dominant hypothesis in AD has been that Aβ initiates the disease via toxicity from secreted, extracellular Aβ aggregates. More recently, an alternative hypothesis has emerged focusing on a pool of Aβ that accumulates early on within AD vulnerable neurons of the brain. Although the topic of intraneuronal Aβ has been of major interest in the field, technical difficulties in detecting intraneuronal Aβ have also made this topic remarkably controversial. Here we review evidence pointing to the critical role of intraneuronal Aβ in AD and provide insights both into challenges faced in detecting intracellular Aβ and the prion-like properties of Aβ. Main methodsImmunoprecipitation and Western blot are used for Aβ detection. Key findingsWe highlight that a standard biochemical method can underestimate intraneuronal Aβ and that extracellular Aβ can up-regulate intracellular Aβ. We also show that detergent can remove intraneuronal Aβ. SignificanceThere is a growing awareness that intraneuronal Aβ is a key pathogenic pool of Aβ involved in causing synapse dysfunction. Difficulties in detecting intraneuronal Aβ are an insufficient reason for ignoring this critical pool of Aβ.

Alzheimer’s Disease Amyloid β-Protein Mutations and Deletions That Define Neuronal Binding/Internalization as Early Stage Nonfibrillar/Fibrillar Aggregates and Late Stage Fibrils

Accumulation of amyloid β-protein (Aβ) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aβ accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aβ might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aβ40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aβ40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aβ lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aβ in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aβ40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aβ seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aβ39E22Δ, Aβ41E22Δ, and Aβ42 but not Aβ40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aβ39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aβ, therefore, results from aggregate binding to neurons, which requires H13 for Aβ40 but not for E22Δ or Aβ42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aβ40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aβ40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aβ proteins and their aggregation states or fibrillar structure.

Abnormal Intracellular Accumulation and Extracellular Aβ Deposition in Idiopathic and Dup15q11.2-q13 Autism Spectrum Disorders

BackgroundIt has been shown that amyloid ß (Aβ), a product of proteolytic cleavage of the amyloid β precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type–specific amount.Methodology/Principal FindingsIn the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aβ load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aβ load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aβ was mainly N-terminally truncated. Increased intraneuronal accumulation of Aβ17–40/42 in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aβ accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aβ1–40/42 detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aβ and an extracellular deposition of full-length Aβ in nonfibrillar plaques.Conclusions/SignificanceThe higher prevalence of excessive Aβ accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links between autism, epilepsy and alterations of APP processing leading to neuronal and astrocytic Aβ accumulation and diffuse plaque formation.

Identification and Characterization of an Aβ Oligomer Precipitating Peptide That May Be Useful to Explore Gene Therapeutic Approaches to Alzheimer Disease

A key feature of Alzheimer disease (AD) is the pathologic self-association of the amyloid-β (Aβ) peptide, leading to the formation of diffusible toxic Aβ oligomers and extracellular amyloid plaques. Next to extracellular Aβ, intraneuronal Aβ has important pathological functions in AD. Agents that specifically interfere with the oligomerization processes either outside or inside of neurons are highly desired for the elucidation of the pathologic mechanisms of AD and might even pave the way for new AD gene therapeutic approaches. Here, we characterize the Aβ binding peptide L3 and its influence on Aβ oligomerization in vitro. Preliminary studies in cell culture demonstrate that stably expressed L3 reduces cell toxicity of externally added Aβ in neuroblastoma cells.

Intraneuronal Aβ detection in 5xFAD mice by a new Aβ-specific antibody

BackgroundThe form(s) of amyloid-β peptide (Aβ) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aβ accumulation is an issue of considerable controversy; even the existence of Aβ deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for Aβ. To further address this issue, an anti-Aβ antibody was developed (MOAB-2) that specifically detects Aβ, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Aβ in transgenic mice with increased levels of human Aβ in 5xFAD and 3xTg mice.ResultsMOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aβ residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Aβ residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aβ40 and Aβ42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aβ, distinct from Aβ associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues.ConclusionsBoth intraneuronal Aβ accumulation and extracellular Aβ deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular Aβ from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal Aβ with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.

AβPP Accumulation and/or Intraneuronal Amyloid-β Accumulation? The 3xTg-AD Mouse Model Revisited

The triple-transgenic Alzheimer's disease (AD) mouse model, 3xTg-AD, played an important role in supporting the intraneuronal amyloid-β (Aβ) hypothesis in AD. However, recent evidence claims that the 3xTg-AD mice accumulate amyloid-β protein precursor (AβPP) instead of Aβ within neurons. In the present report, we re-investigated the 3xTg-AD mouse model and confirmed recent findings of age-dependent AβPP accumulations. In addition, intraneuronal Aβ was detected mainly in the neocortex using conformation-specific as well as antibodies directed against Aβ neo-epitopes. In contrast to previous work, however, only minor levels of intraneuronal Aβ were detected in the CA1 region of the hippocampus in aged 3xTg-AD mice.

β‐amyloid peptide is internalized into chick retinal neurons and alters the distribution of myosin Vb

The most common neurodegenerative disorder afflicting the aging human population is Alzheimer's disease (AD). A major hallmark of AD is dementia from a loss of neuronal function, attributed to the presence and accumulation of β-amyloid (Aβ) peptide into senile plaques. Preceding senile plaque formation, abnormalities in axons can be observed as changes in morphologies and intracellular trafficking. Recently, it has been recognized that Aβ also accumulates within neurons and this intraneuronal Aβ accumulation has been reported to be critical in the disruption of synapses and cognitive function. Here, we report on the internalization of a fluorescently labeled Aβ peptide into cultured chick retinal neurons. The pattern of Aβ distribution during the time course of incubation is reminiscent of the endocytic pathway. Furthermore, the distribution of the internalized Aβ peptide converges with that of myosin Vb and both relocalize from the axon to cell body. These observations are consistent with the hypothesis that AD proceeds as a result of an imbalance between Aβ production and Aβ clearance, suggesting a role for myosin Vb in this process.

Transgenic Expression of Intraneuronal Aβ42But Not Aβ40Leads to Cellular Aβ Lesions, Degeneration, and Functional Impairment without Typical Alzheimer's Disease Pathology

An early role of amyloid-β peptide (Aβ) aggregation in Alzheimer's 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.

Hypercholesterolemia accelerates intraneuronal accumulation of Aβ oligomers resulting in memory impairment in Alzheimer's disease model mice

AimsHypercholesterolemia is known to be a risk factor for Alzheimer's disease (AD), and diet-induced hypercholesterolemia has been shown to accelerate amyloid pathology in animals. While growing evidence has shown that synaptic and cognitive dysfunction in AD is associated with intraneuronal accumulation of Aβ, the relationships between hypercholesterolemia, memory impairment, and intraneuronal Aβ remains unclear. The present study aims to clarify this association. Main methodsTransgenic mice expressing amyloid precursor protein (APP) harboring the Osaka (E693∆) mutation (APPOSK-Tg mice) were used. These mice exhibit intraneuronal Aβ oligomers and memory impairment from 8months of age. Five-month-old male APPOSK-Tg mice and non-Tg littermates were fed a high-cholesterol diet for 1month to induce hypercholesterolemia. At 6months of age, their cognitive function was evaluated by the Morris water maze. Intraneuronal Aβ, synaptic density, and tau phosphorylation were examined by immunohistochemistry. Key findingsSerum and brain cholesterol levels were significantly higher in APPOSK-Tg mice and non-Tg littermates that were fed a high-cholesterol diet than in control mice that were fed normal chow, indicating that hypercholesterolemia was successfully induced. Hypercholesterolemic APPOSK-Tg mice, but not control APPOSK-Tg mice or hypercholesterolemic non-Tg littermates, exhibited impaired spatial reference memory, which was accompanied with intraneuronal accumulation of Aβ oligomers, reduced synaptophysin immunoreactivity, and abnormal tau phosphorylation in the hippocampus. Hypercholesterolemia-accelerated accumulation of intraneuronal Aβ oligomers was also observed in another model mouse, Tg2576. SignificanceOur findings suggest that hypercholesterolemia accelerates intraneuronal accumulation of Aβ oligomers and subsequent synapse loss, resulting in memory impairment.

Impaired β-Amyloid Secretion in Alzheimer's Disease Pathogenesis

A central question in Alzheimer's disease (AD) research is what role β-amyloid peptide (Aβ) plays in synaptic dysfunction. Synaptic activity increases Aβ secretion, potentially inhibiting synapses, but also decreases intraneuronal Aβ, protecting synapses. We now show that levels of secreted Aβ fall with time in culture in neurons of AD-transgenic mice, but not wild-type mice. Moreover, the ability of synaptic activity to elevate secreted Aβ and reduce intraneuronal Aβ becomes impaired in AD-transgenic but not wild-type neurons with time in culture. We demonstrate that synaptic activity promotes an increase in the Aβ-degrading protease neprilysin at the cell surface and a concomitant increase in colocalization with Aβ42. Remarkably, AD-transgenic but not wild-type neurons show reduced levels of neprilysin with time in culture. This impaired ability to secrete Aβ and reduce intraneuronal Aβ has important implications for the pathogenesis and treatment of AD.

High-Resolution 3D Reconstruction Reveals Intra-Synaptic Amyloid Fibrils

β-Amyloid (Aβ) accumulation and aggregation are hallmarks of Alzheimer's disease (AD). High-resolution three-dimensional (HR-3D) volumetric imaging allows for better analysis of fluorescence confocal microscopy and 3D visualization of Aβ pathology in brain. Early intraneuronal Aβ pathology was studied in AD transgenic mouse brains by HR-3D volumetric imaging. To better visualize and analyze the development of Aβ pathology, thioflavin S staining and immunofluorescence using antibodies against Aβ, fibrillar Aβ, and structural and synaptic neuronal proteins were performed in the brain tissue of Tg19959, wild-type, and Tg19959-YFP mice at different ages. Images obtained by confocal microscopy were reconstructed into three-dimensional volumetric datasets. Such volumetric imaging of CA1 hippocampus of AD transgenic mice showed intraneuronal onset of Aβ42 accumulation and fibrillization within cell bodies, neurites, and synapses before plaque formation. Notably, early fibrillar Aβ was evident within individual synaptic compartments, where it was associated with abnormal morphology. In dendrites, increasing intraneuronal thioflavin S correlated with decreases in neurofilament marker SMI32. Fibrillar Aβ aggregates could be seen piercing the cell membrane. These data support that Aβ fibrillization begins within AD vulnerable neurons, leading to disruption of cytoarchitecture and degeneration of spines and neurites. Thus, HR-3D volumetric image analysis allows for better visualization of intraneuronal Aβ pathology and provides new insights into plaque formation in AD.

Allopregnanolone Promotes Regeneration and Reduces β-Amyloid Burden in a Preclinical Model of Alzheimer's Disease

Previously, we demonstrated that allopregnanolone (APα) promoted proliferation of rodent and human neural progenitor cells in vitro. Further, we demonstrated that APα promoted neurogenesis in the hippocampal subgranular zone (SGZ) and reversed learning and memory deficits in the male triple transgenic mouse model of Alzheimer's (3xTgAD). In the current study, we determined the efficacy of APα to promote the survival of newly generated neural cells while simultaneously reducing Alzheimer's disease (AD) pathology in the 3xTgAD male mouse model. Comparative analyses between three different APα treatment regimens indicated that APα administered 1/week for 6 months was maximally efficacious for simultaneous promotion of neurogenesis and survival of newly generated cells and reduction of AD pathology. We further investigated the efficacy of APα to impact Aβ burden. Treatment was initiated either prior to or post intraneuronal Aβ accumulation. Results indicated that APα administered 1/week for 6 months significantly increased survival of newly generated neurons and simultaneously reduced Aβ pathology with greatest efficacy in the pre-pathology treatment group. APα significantly reduced Aβ generation in hippocampus, cortex, and amygdala, which was paralleled by decreased expression of Aβ-binding-alcohol-dehydrogenase. In addition, APα significantly reduced microglia activation as indicated by reduced expression of OX42 while increasing CNPase, an oligodendrocyte myelin marker. Mechanistic analyses indicated that pre-pathology treatment with APα increased expression of liver-X-receptor, pregnane-X-receptor, and 3-hydroxy-3-methyl-glutaryl-CoA-reductase (HMG-CoA-R), three proteins that regulate cholesterol homeostasis and clearance from brain. Together these findings provide preclinical evidence for the optimal treatment regimen of APα to achieve efficacy as a disease modifying therapeutic to promote regeneration while simultaneously decreasing the pathology associated with Alzheimer's disease.

Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration in a mouse model of Alzheimer's disease

Increasing evidence suggests that the accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria causes synaptic mitochondrial failure and synaptic degeneration in Alzheimer's disease (AD). The purpose of this study was to better understand the effects of Aβ in mitochondrial activity and synaptic alterations in neurons from a mouse model of AD. Using primary neurons from a well-characterized Aβ precursor protein transgenic (AβPP) mouse model (Tg2576 mouse line), for the first time, we studied mitochondrial activity, including axonal transport of mitochondria, mitochondrial dynamics, morphology and function. Further, we also studied the nature of Aβ-induced synaptic alterations, and cell death in primary neurons from Tg2576 mice, and we sought to determine whether the mitochondria-targeted antioxidant SS31 could mitigate the effects of oligomeric Aβ. We found significantly decreased anterograde mitochondrial movement, increased mitochondrial fission and decreased fusion, abnormal mitochondrial and synaptic proteins and defective mitochondrial function in primary neurons from AβPP mice compared with wild-type (WT) neurons. Transmission electron microscopy revealed a large number of small mitochondria and structurally damaged mitochondria, with broken cristae in AβPP primary neurons. We also found an increased accumulation of oligomeric Aβ and increased apoptotic neuronal death in the primary neurons from the AβPP mice relative to the WT neurons. Our results revealed an accumulation of intraneuronal oligomeric Aβ, leading to mitochondrial and synaptic deficiencies, and ultimately causing neurodegeneration in AβPP cultures. However, we found that the mitochondria-targeted antioxidant SS31 restored mitochondrial transport and synaptic viability, and decreased the percentage of defective mitochondria, indicating that SS31 protects mitochondria and synapses from Aβ toxicity.