Seeded strain‐like transmission of β‐amyloid morphotypes in APP transgenic mice

The polymorphic β‐amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral β‐amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop β‐amyloid depositions that differ in morphology, binding of amyloid conformation‐sensitive dyes, and Aβ40/Aβ42 peptide ratio. To determine the nature of such β‐amyloid morphotypes, β‐amyloid‐containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced β‐amyloid deposition with the morphological, conformational, and Aβ40/Aβ42 ratio characteristics of β‐amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced β‐amyloid deposits with the characteristics of β‐amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced β‐amyloid deposits, although less prominent, and the induced deposits were similar to the β‐amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aβ in APP transgenic mice can be maintained by seeded conversion.     

Intraneuronal amyloid precursor protein (APP) and appearance of extracellular β‐amyloid peptide (aβ) in the brain of aging kokanee salmon

Antibodies to human amyloid precursor protein (APP₆₉₅) and beta‐amyloid peptide (Aβ_1‐42) were used to determine timing of amyloidosis in the brain of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages: immature (IM), maturing (MA), sexually mature (SM), and spawning (SP), representing a range of aging from somatically mature but sexually immature to spawning and somatic senescence. In IM fish, immunoreactive (ir) intracellular APP occurred in 18 of 23 brain regions. During sexual maturation and aging, the number of neurons expressing APP increased in 11 of these APP‐ir regions. Aβ‐ir was absent in IM fish, present in seven regions in MA fish, moderately abundant in 15 regions in SM fish, and was most abundant in all brain regions of SP fish exhibiting Aβ‐ir. Intracellular APP‐ir was observed in brain regions involved in sensory integration, olfaction, vision, stress responses, reproduction, and coordination. Intra‐ and extracellular Aβ_1‐42 immunoreactivity (Aβ‐ir) was present in all APP‐ir regions except the nucleus lateralis tuberis (hypothalamus) and Purkinje cells (cerebellum). APP‐ir and Aβ deposition increase during aging. APP‐ir is present in IM fish; Aβ‐ir usually appears first in MA or SM fish and increases in SM fish as does APP‐ir. Extracellular Aβ deposition dramatically increases between SM and SP stages (1–2 weeks) in all fish, indicating an extremely rapid and synchronized process. Rapid senescence observed in pacific salmon could make them a useful model to investigate timing of amyloidosis and neurodegeneration during brain aging. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 11–20, 2002     

Mutant ubiquitin decreases amyloid β plaque formation in a transgenic mouse model of Alzheimer's disease

The mutant ubiquitin UBB(+1) is a substrate as well as an inhibitor of the ubiquitin-proteasome system (UPS) and accumulates in the neuropathological hallmarks of Alzheimer's disease (AD). A role for the UPS has been suggested in the generation of amyloid β (Aβ) plaques in AD. To investigate the effect of UBB(+1) expression on amyloid pathology in vivo, we crossed UBB(+1) transgenic mice with a transgenic line expressing AD-associated mutant amyloid precursor protein (APPSwe) and mutant presenilin 1 (PS1dE9), resulting in APPPS1/UBB(+1) triple transgenic mice. In these mice, we determined the Aβ levels at 3, 6, 9 and 11months of age. Surprisingly, we found a significant decrease in Aβ deposition in amyloid plaques and levels of soluble Aβ(42) in APPPS1/UBB(+1) transgenic mice compared to APPPS1 mice at 6months of age, without alterations in UBB(+1) protein levels or proteasomal chymotrypsin activity. These lowering effects of UBB(+1) on Aβ deposition were transient, as this relative decrease in plaque load was not significant in APPPS1/UBB(+1) mice at 9 and 11months of age. We also show that APPPS1/UBB(+1) mice exhibit astrogliosis, indicating that they may not be improved functionally compared to APPPS1 mice despite the Aβ reduction. The molecular mechanism underlying this decrease in Aβ deposition in APPPS1/UBB(+1) mice is more complex than previously assumed because UBB(+1) is also ubiquitinated at K63 opening the possibility of additional effects of UBB(+1) (e.g. kinase activation).     

Beta-amyloid peptide variants in brains and cerebrospinal fluid from amyloid precursor protein (APP) transgenic mice: comparison with human Alzheimer amyloid

In this study, we report a detailed analysis of the different variants of amyloid-β (Aβ) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the Aβ peptides Aβ(1-40), -(1-42), -(1-39), -(1-38), -(1-37), -(2-40), and -(3-40) as well as minor amounts of pyroglutamate-modified Aβ (Aβ(N3pE)) and endogenous murine Aβ in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of Aβ were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of Aβ peptides, such as Aβ(N3pE), Aβ(1-42), and N-terminally truncated Aβ(2/3-42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients.     

Regional differences in the morphological and functional effects of aging on cerebral basement membranes and perivascular drainage of amyloid‐β from the mouse brain

Development of cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD) is associated with failure of elimination of amyloid‐β (Aβ) from the brain along perivascular basement membranes that form the pathways for drainage of interstitial fluid and solutes from the brain. In transgenic APP mouse models of AD, the severity of cerebral amyloid angiopathy is greater in the cerebral cortex and hippocampus, intermediate in the thalamus, and least in the striatum. In this study we test the hypothesis that age‐related regional variation in (1) vascular basement membranes and (2) perivascular drainage of Aβ contribute to the different regional patterns of CAA in the mouse brain. Quantitative electron microscopy of the brains of 2‐, 7‐, and 23‐month‐old mice revealed significant age‐related thickening of capillary basement membranes in cerebral cortex, hippocampus, and thalamus, but not in the striatum. Results from Western blotting and immunocytochemistry experiments showed a significant reduction in collagen IV in the cortex and hippocampus with age and a reduction in laminin and nidogen 2 in the cortex and striatum. Injection of soluble Aβ into the hippocampus or thalamus showed an age‐related reduction in perivascular drainage from the hippocampus but not from the thalamus. The results of the study suggest that changes in vascular basement membranes and perivascular drainage with age differ between brain regions, in the mouse, in a manner that may help to explain the differential deposition of Aβ in the brain in AD and may facilitate development of improved therapeutic strategies to remove Aβ from the brain in AD.     

Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Blocking the apolipoprotein E/Amyloid β interaction in triple transgenic mice ameliorates Alzheimer's disease related amyloid β and tau pathology

Inheritance of the apolipoprotein E4 (apoE4) genotype has been identified as the major genetic risk factor for late‐onset Alzheimer's disease (AD). Studies have shown that the binding between apoE and amyloid‐β (Aβ) peptides occurs at residues 244–272 of apoE and residues 12–28 of Aβ. ApoE4 has been implicated in promoting Aβ deposition and impairing clearance of Aβ. We hypothesized that blocking the apoE/Aβ interaction would serve as an effective new approach to AD therapy. We have previously shown that treatment with Aβ12‐28P can reduce amyloid plaques in APP/PS1 transgenic (Tg) mice and vascular amyloid in TgSwDI mice with congophilic amyloid angiopathy. In the present study, we investigated whether the Aβ12‐28P elicits a therapeutic effect on tau‐related pathology in addition to amyloid pathology using old triple transgenic AD mice (3xTg, with PS1_M146V, APP_Swe and tau_P30IL transgenes) with established pathology from the ages of 21 to 26 months. We show that treatment with Aβ12‐28P substantially reduces tau pathology both immunohistochemically and biochemically, as well as reducing the amyloid burden and suppressing the activation of astrocytes and microglia. These affects correlate with a behavioral amelioration in the treated Tg mice.

     

Dependency of Glucose Transport on d-Glucoside 3-Dehydrogenase as a Conversion System of Glucose to 3-Ketoglucose in Agrobacterium tumefaciens

Alzheimer’s disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.     

Identification of new Presenilin‐1 phosphosites: implication for γ‐secretase activity and Aβ production

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.

     

Seed‐induced Aβ deposition is modulated by microglia under environmental enrichment in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is characterized by severe neuronal loss as well as the accumulation of amyloid‐β (Aβ), which ultimately leads to plaque formation. Although there is now a general agreement that the aggregation of Aβ can be initiated by prion‐like seeding, the impact and functional consequences of induced Aβ deposits (Aβ seeding) on neurons still remain open questions. Here, we find that Aβ seeding, representing early stages of plaque formation, leads to a dramatic decrease in proliferation and neurogenesis in two APP transgenic mouse models. We further demonstrate that neuronal cell death occurs primarily in the vicinity of induced Aβ deposits culminating in electrophysiological abnormalities. Notably, environmental enrichment and voluntary exercise not only revives adult neurogenesis and reverses memory deficits but, most importantly, prevents Aβ seeding by activated, phagocytic microglia cells. Our work expands the current knowledge regarding Aβ seeding and the consequences thereof and attributes microglia an important role in diminishing Aβ seeding by environmental enrichment.     

Lactuca capensis reverses memory deficits in Aβ1‐42‐induced an animal model of Alzheimer's disease

We investigated the neuropharmacological effects of the methanolic extract from Lactuca capensis Thunb. leaves (100 and 200 mg/kg) for 21 days on memory impairment in an Alzheimer's disease (AD) rat model produced by direct intraventricular delivery of amyloid‐β1‐42 (Aβ1‐42). Behavioural assays such as Y‐maze and radial arm maze test were used for assessing memory performance. Aβ1‐42 decreased cognitive performance in the behavioural tests which were ameliorated by pre‐treatment with the methanolic extract. Acetylcholinesterase activity and oxidant–antioxidant balance in the rat hippocampus were abnormally altered by Aβ1‐42 treatment while these deficits were recovered by pre‐treatment with the methanolic extract. In addition, rats were given Aβ1‐42 exhibited in the hippocampus decreased brain‐derived neurotrophic factor (BDNF) mRNA copy number and increased IL‐1β mRNA copy number which was reversed by the methanolic extract administration. These findings suggest that the methanolic extract could be a potent neuropharmacological agent against dementia via modulating cholinergic activity, increasing of BDNF levels and promoting antioxidant action in the rat hippocampus.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Defective neurite extension is caused by a mutation in amyloid β/A4 (Aβ) protein precursor found in Familial Alzheimer's Disease

Clonal central nervous system neuronal cells, B103, do not synthesize detectable endogenous APP or APLP. B103 cells transfected with both wild-type (B103/APP) and mutant APP construct (B103/APPΔNL) secreted comparable amounts of soluble forms of APP (sAPP). B103/APP cells produced sAPP and cleaved at amyloid β/A4 (Aβ) 16, the α-secretase site, and B103/APPΔNL cells produced sAPPβ cleaved at Aβ 1, the β-secretase site. B103/APPΔNL cells developed fewer neurites than B103/APP cells in a serum-free defined medium. Neurite numbers of parent B103 cells were increased by the 50% conditioned medium (CM) from B103/APP cells but reduced by the CM from B103/APPΔNL cells. Chemically synthesized Aβ at concentration levels higher than 1 nM reduced numbers of neurites from B103 or B103/APPΔNL cells. However, Aβ at 1–100 nM could not reduce the neurite number of B103/APP cells. The protective activity against Aβ's deleterious effect to reduce neurite numbers was attributed to sAPPα in the CM. Although sAPPα could block the effect of Aβ, sAPPβ could not do so under the identical condition, suggesting the importance of the C-terminal 15-amino acid sequence in sAPPα. Nevertheless, sAPPα's protective activity required the N-terminal sequence around RERMS, previously identified to be the active domain of sAPPβ. The overall effect of APP mutation which overproduced Aβ and sAPPβ and underproduced sAPPα was a marked decline in the neurotrophic effect of APP. We suggest that the disruption of balance between the detrimental effect of Aβ and the trophic effect of sAPP may be important in the pathogenesis of AD caused by this pathogenic APP mutation © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 469–480, 1997     

Silymarin attenuated the amyloid β plaque burden and improved behavioral abnormalities in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.     

Anthocyanin-enriched bilberry and blackcurrant extracts modulate amyloid precursor protein processing and alleviate behavioral abnormalities in the APP/PS1 mouse model of Alzheimer's disease

A growing body of epidemiological evidence suggests that fruit and vegetable juices containing various phenolic compounds can reduce the risk of Alzheimer's disease (AD). As the altered amyloid precursor protein (APP) processing leading to increased β-amyloid (Aβ) production is a key pathogenic feature of AD, we elucidated the effects of different polyphenols on neuroprotection and APP processing under different in vitro stress conditions. The effects of these compounds were also investigated in transgenic AD mice (APdE9). Free radical toxicity and apoptosis were induced in human SH-SY5Y neuroblastoma cells overexpressing APP751. Menadione-induced production of reactive oxygen species was significantly decreased upon treatment with myricetin, quercetin or anthocyanin-rich extracts in a dose-dependent manner. However, these extracts did not affect caspase-3 activation, APP processing or Aβ levels upon staurosporine-induced apoptosis. APdE9 mice fed with anthocyanin-rich bilberry or blackcurrant extracts showed decreased APP C-terminal fragment levels in the cerebral cortex as compared to APdE9 mice on the control diet. Soluble Aβ40 and Aβ42 levels were significantly decreased in bilberry-fed mice as compared to blackcurrant-fed mice. Conversely, the ratio of insoluble Aβ42/40 was significantly decreased in blackcurrant-fed mice relative to bilberry-fed mice. Both berry diets alleviated the spatial working memory deficit of aged APdE9 mice as compared to mice on the control diet. There were no changes in the expression or phosphorylation status of tau in APdE9 mice with respect to diet. These data suggest that anthocyanin-rich bilberry and blackcurrant diets favorably modulate APP processing and alleviate behavioral abnormalities in a mouse model of AD.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of the interaction between Aβ 1–42 and glyceraldehyde phosphodehydrogenase

Advances in the understanding of AD pathogenesis have recently provided strong support for a modified Aβ protein cascade hypothesis, stating that several different Aβ assemblies contribute to the triggering of a complex pathological cascade leading to neurodegeneration. Both in vitro and in vivo, Aβ rapidly forms fibrils (fAβ), which are able to interact with various molecular partners, including proteins, lipids and proteoglycans. In a previous study aimed to identify some of these molecular partners of fAβ, we demonstrated that the GAPDH was specifically coprecipitated with fAβ. The aim of this study was to characterize this interaction. First, it was shown by TEM that synthetic GAPDH directly binds fAβ 1–42. Then rat synaptosomal proteins were purified and incubated with different forms of Aβ in various conditions, and the presence of GAPDH among the proteins coprecipitated with Aβ was studied by western blotting. Results showed that the interaction between GAPDH and fAβ 1–42 is nonionic, as is not impaired by increasing salt concentrations. GAPDH is coprecipitated not only by fAβ, but also by nonfibrillar forms of Aβ 1–42. The 41–42 Aβ sequence seems to be important in the interaction of GAPDH and Aβ, as more GAPDH was coprecipitated with fAβ 1–42 than with fAβ 1–40. GAPDH extracted from various subcellular fractions including mitochondria, was shown to interact with fAβ. Our data demonstrate a direct interaction between Aβ and GAPDH and support the possibility that this interaction has an important pathogenic role in AD. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38) occurs as Aβ(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.     

Mitofusin‐2 knockdown increases ER–mitochondria contact and decreases amyloid β‐peptide production

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria‐associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM‐associated proteins and enhanced ER to mitochondria Ca²⁺ transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β‐peptide (Aβ)‐related neuronal models. Here, we report that siRNA knockdown of mitofusin‐2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca²⁺ transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra‐ and extracellular Aβ₄₀ and Aβ₄₂. Analysis of γ‐secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ‐secretase complex function. Amyloid‐β precursor protein (APP), β‐site APP‐cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER–mitochondria contact affects γ‐secretase activity and Aβ generation. Increased ER–mitochondria contact results in lower γ‐secretase activity suggesting a new mechanism by which Aβ generation can be controlled.