Systemic immune‐checkpoint blockade with anti‐PD1 antibodies does not alter cerebral amyloid‐β burden in several amyloid transgenic mouse models

Chronic inflammation represents a central component in the pathogenesis of Alzheimer's disease (AD). Recent work suggests that breaking immune tolerance by Programmed cell Death‐1 (PD1) checkpoint inhibition produces an IFN‐γ‐dependent systemic immune response, with infiltration of the brain by peripheral myeloid cells and neuropathological as well as functional improvements even in mice with advanced amyloid pathology (Baruch et al., ( 2016 ): Nature Medicine, 22:135–137). Immune checkpoint inhibition was therefore suggested as potential treatment for neurodegenerative disorders when activation of the immune system is appropriate. Because a xenogeneic rat antibody (mAb) was used in the study, whether the effect was specific to PD1 target engagement was uncertain. In the present study we examined whether PD1 immunotherapy can lower amyloid‐β pathology in a range of different amyloid transgenic models performed at three pharmaceutical companies with the exact same anti‐PD1 isotype and two mouse chimeric variants. Although PD1 immunotherapy stimulated systemic activation of the peripheral immune system, monocyte‐derived macrophage infiltration into the brain was not detected, and progression of brain amyloid pathology was not altered. Similar negative results of the effect of PD1 immunotherapy on amyloid brain pathology were obtained in two additional models in two separate institutions. These results show that inhibition of PD1 checkpoint signaling by itself is not sufficient to reduce amyloid pathology and that additional factors might have contributed to previously published results (Baruch et al., ( 2016 ): Nature Medicine, 22:135–137). Until such factors are elucidated, animal model data do not support further evaluation of PD1 checkpoint inhibition as a therapeutic modality for Alzheimer's disease.     

The N‐terminal fragment of the β‐amyloid precursor protein of Alzheimer's disease (N‐APP) binds to phosphoinositide‐rich domains on the surface of hippocampal neurons

The function of the β‐amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPPα) can be readily cleaved to produce a small N‐terminal fragment (N‐APP) that contains heparin‐binding and metal‐binding domains and that has been found to have biological activity. In the present study, we examined whether N‐APP can bind to lipids. We found that N‐APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂)‐rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N‐APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N‐APP to neurons increased the level of cell‐surface PI(4,5)P₂ and phosphatidylinositol 3,4,5‐trisphosphate. However, PIPs were not the principal cell‐surface binding site for N‐APP, because N‐APP binding to neurons was not inhibited by a short‐acyl‐chain PIP analogue, and N‐APP did not bind to glial cells which also possessed PI(4,5)P₂ on the cell surface. The data are explained by a model in which N‐APP binds to two distinct components on neurons, one of which is an unidentified receptor and the second of which is a PIP lipid, which binds more weakly to a distinct site within N‐APP. Our data provide further support for the idea that N‐APP may be an important mediator of APP's biological activity. © 2014 Wiley Periodicals, Inc.     

β‐amyloid in lewy body disease is related to Alzheimer's disease‐like atrophy

The aim of this study was to investigate whether amyloid deposition is associated with Alzheimer's disease (AD)‐like cortical atrophy in Lewy body (LB) disease (LBD). Participants included 15 LBD with dementia patients (8 with dementia with Lewy bodies [DLB] and 7 with Parkinson's disease [PD] with dementia [PDD]), 13 AD patients, and 17 healthy controls. Age, gender, and Mini–Mental State Examination scores were matched between patient groups. All subjects underwent PET scans with [¹¹C]Pittsburgh Compound B to measure brain amyloid deposition as well as three‐dimensional T1‐weighted MRI. Gray‐matter volumes (GMVs) were estimated by voxel‐based morphometry. Volumes‐of‐interest analyses were also performed. Forty percent of the 15 DLB/PDD patients were amyloid positive, whereas all AD patients and none of the healthy controls were amyloid positive. Amyloid‐positive DLB/PDD and AD patients showed very similar patterns of cortical atrophy in the parahippocampal area and lateral temporal and parietal cortices, with 95.2% of cortical atrophy distribution being overlapped. In contrast, amyloid‐negative DLB/PDD patients had no significant cortical atrophy. Compared to healthy controls, parahippocampal GMVs were reduced by 26% in both the amyloid‐positive DLB/PDD and AD groups and by 10% in the amyloid‐negative DLB/PDD group. The results suggest that amyloid deposition is associated with AD‐like atrophy in DLB/PDD patients. Early intervention against amyloid may prevent or delay AD‐like atrophy in DLB/PDD patients with amyloid deposition. © 2012 Movement Disorder Society     

Dual pathways mediate β‐amyloid stimulated glutathione release from astrocytes

Oxidative stress plays an important role in the progression of Alzheimer's disease (AD) and other neurodegenerative conditions. Glutathione (GSH), the major antioxidant in the central nervous system, is primarily synthesized and released by astrocytes. We determined if β‐amyloid (Aβ42), crucially involved in Alzheimer's disease, affected GSH release. Monomeric Aβ (mAβ) stimulated GSH release from cultured cortical astrocytes more effectively than oligomeric Aβ (oAβ) or fibrillary Aβ (fAβ). Monomeric Aβ increased the expression of the transporter ABCC1 (also referred to as MRP1) that is the main pathway for GSH release. GSH release from astrocytes, with or without mAβ stimulation, was reduced by pharmacological inhibition of ABCC1. Astrocytes robustly express connexin proteins, especially connexin43 (Cx43), and mAβ also stimulated Cx43 hemichannel‐mediated glutamate and GSH release. Aβ‐stimulation facilitated hemichannel opening in the presence of normal extracellular calcium by reducing astrocyte cholesterol level. Aβ treatment did not alter the intracellular concentration of reduced or oxidized glutathione. Using a mouse model of AD with early onset Aβ deposition (5xFAD), we found that cortical ABCC1 was significantly increased in temporal register with the surge of Aβ levels in these mice. ABCC1 levels remained elevated from 1.5 to 3.5 months of age in 5xFAD mice, before plunging to subcontrol levels when amyloid plaques appeared. Similarly, in cultured astrocytes, prolonged incubation with aggregated Aβ, but not mAβ, reduced induction of ABCC1 expression. These results support the hypothesis that in the early stage of AD pathogenesis, less aggregated Aβ increases GSH release from astrocytes (via ABCC1 transporters and Cx43 hemichannels) providing temporary protection from oxidative stress which promotes AD development. GLIA 2015;63:2208–2219     

Secreted amyloid precursor protein and holo‐APP bind amyloid β through distinct domains eliciting different toxic responses on hippocampal neurons

Amyloid β (Aβ) is a metabolic product of Aβ precursor protein (APP). Deposition of Aβ in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Aβ induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Increasing evidence implicates APP as a receptor‐like protein for Aβ fibrils (fAβ). In this study, we present further experimental support for the direct interaction of APP with fAβ and for its involvement in Aβ neurotoxicity. Using recombinant purified holo‐APP (h‐APP), we have shown that it directly binds fAβ. Employing deletion mutant forms of APP, we show that two different sequences are involved in the binding of APP to fAβ. One sequence in the n‐terminus of APP is required for binding of fAβ to secreted APP (s‐APP) but not to h‐APP. In addition, the extracellular juxtamembrane Aβ‐sequence mediates binding of fAβ to h‐APP but not to s‐APP. Deletion of the extracellular juxtamembrane Aβ sequence abolishes abnormal h‐APP accumulation and toxicity induced by fAβ deposition, whereas deletions in the n‐terminus of APP do not affect Aβ toxicity. These experiments show that interaction of toxic Aβ species with its membrane‐anchored parental protein promotes toxicity in hippocampal neurons, adding further support to an Aβ‐receptor‐like function of APP directly implicated in neuronal degeneration in AD. © 2010 Wiley‐Liss, Inc.     

The E693Δ (Osaka) mutation in amyloid precursor protein potentiates cholesterol‐mediated intracellular amyloid β toxicity via its impaired cholesterol efflux

It has been shown that amyloid β (Aβ) secretion regulates cholesterol efflux from cells and that the E693Δ (Osaka) mutation in amyloid precursor protein (APP) promotes intracellular accumulation of Aβ and thus reduces its secretion. These findings led us to speculate that APP with the Osaka mutation (APP_OSK) might have a defect in cholesterol efflux and thus cause cellular malfunction. We therefore examined the effects of this mutation on intracellular cholesterol transport and efflux in cultured cells. Upon cholesterol loading, APP_OSK‐expressing cells exhibited higher levels of cellular cholesterol than wild‐type APP‐expressing cells, suggesting impaired cholesterol efflux. It is known that, after its internalization, cholesterol is transported from the endosomes to the endoplasmic reticulum (ER) and Golgi apparatus and then to the plasma membrane. In APP_OSK‐expressing cells, cholesterol accumulated with Aβ in the ER and Golgi apparatus and alone in endosomes/lysosomes. These results imply that the mutation‐induced disturbance of Aβ trafficking from the ER to the plasma membrane affects cholesterol transport to cause cholesterol accumulation in the ER and Golgi apparatus and, consequently, in endosomes. Furthermore, we detected an enhanced mitochondrial accumulation of Aβ and cholesterol in APP_OSK‐expressing cells, and this was accompanied by an increase in the generation of reactive oxygen species (ROS). The present findings suggest that Aβ trafficking is important for intracellular cholesterol transport and efflux and that the Osaka mutation potentiates cholesterol‐dependent exacerbation of intracellular Aβ toxicity, i.e. Aβ‐induced ROS generation, by disturbing Aβ‐mediated cholesterol efflux from the cell. © 2013 Wiley Periodicals, Inc.     

Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Aβ1-42 pathogenesis

Previous studies have implicated the failure to degrade aggregated Aβ1-42 in late endosomes or secondary lysosomes as a mechanism for the accumulation of β-amyloid in Alzheimer's disease. We examined the consequences of intracellular accumulation of Aβ1-42 on the integrity of the endosomal/lysosomal compartment by monitoring Lucifer Yellow fluorescence and the release of lysosomal hydrolases into the soluble, cytosolic fraction. In control cells, the Lucifer Yellow fluorescence is observed as punctate staining in a perinuclear distribution with no apparent cytoplasmic fluorescence, consistent with its localization in late endosomes or secondary lysosomes. After incubation with Aβ1-42 for 6 hr, a loss of lysosomal membrane impermeability is observed as evidenced by redistribution of the fluorescence to a diffuse, cytoplasmic pattern. The loss of lysosomal membrane impermeability is correlated with Aβ1-42 accumulation, since incubation of the cells with the nonaccumulating isoform of amyloid, Aβ1-40, does not induce leakage. The same results were obtained using the release of soluble lysosomal hydrolases, cathepsin D and β-hexosaminidase, into the cytosol as an assay for the leakage of lysosomal contents. Together, our results suggest that the loss of lysosomal membrane impermeability may be an early event in Aβ pathogenesis, and provide an explanation for the miscompartmentalization of extracellular and cytoplasmic components observed in Alzheimer's disease (AD). The release of hydrolases may further cause the breakdown of the cytoskeleton and the blebbing of the plasma membrane, and the leakage of heparan sulfate glycosaminoglycans from the lysosome may ultimately promote the assembly of tau into neurofibrillary tangles (NFT). J. Neurosci. Res. 52:691–698, 1998. © 1998 Wiley-Liss, Inc.     

Oxidative damage and amyloid‐β metabolism in brain regions of the longest‐lived rodents

Naked mole rats (NMRs) are the longest‐lived rodents, with young individuals having high levels of Aβ in their brains. The purpose of this study was twofold: to assess the distribution of Aβ in key regions of NMR brains (cortex, hippocampus, cerebellum) and to understand whether the accumulation of Aβ is due to enhanced production or decreased degradation. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Amyloid precursor protein processing, lipid peroxidation, Hsps, redox status, and protein degradation processes were therefore assessed in key NMR brain regions. NMR brains had high levels of lipid peroxidation compared with mice, and the NMR hippocampus had the highest levels of the most toxic moiety of Aβ (soluble Aβ_1–42). This was due not to increased Aβ production but rather to low antioxidant potential, which was associated with low induction of Hsp70 and heme oxygenase‐1 as well as low ubiquitin‐proteasome activity. NMRs may therefore serve as natural models for understanding the relationship between oxidative stress and Aβ levels and its effects on the brain. © 2013 Wiley Periodicals, Inc.     

TGF‐β2 and TGF‐β3 from cultured β‐amyloid‐treated or 3xTg‐AD‐derived astrocytes may mediate astrocyte–neuron communication

Astrocytes participate in the development and resolution of neuroinflammation in numerous ways, including the release of cytokines and growth factors. Among many, astrocytes release transforming growth factors beta (TGF‐β) TGF‐β1, TGF‐β2 and TGF‐β3. TGF‐β1 is the most studied isoform, while production and release of TGF‐β2 and TGF‐β3 by astrocytes have been poorly characterized. Here, we report that purified cultures of hippocampal astrocytes produce mainly TGF‐β3 followed by TGF‐β2 and TGF‐β1. Furthermore, astrocytes release principally the active form of TGF‐β3 over the other two. Changes in release of TGF‐β were sensitive to the calcineurin (CaN) inhibitor FK506. Starvation had no effect on TGF‐β1 and TGF‐β3 while TGF‐β2 mRNA was significantly up‐regulated in a CaN‐dependent manner. We further investigated production and release of astroglial TGF‐β in Alzheimer's disease‐related conditions. Oligomeric β‐amyloid (Aβ) down‐regulated TGF‐β1, while up‐regulating TGF‐β2 and TGF‐β3, in a CaN‐dependent manner. In cultured hippocampal astrocytes from 3xTg‐AD mice, TGF‐β2 and TGF‐β3, but not TGF‐β1, were up‐regulated, and this was CaN‐independent. In hippocampal tissues from symptomatic 3xTg‐AD mice, TGF‐β2 was up‐regulated with respect to control mice. Finally, treatment with recombinant TGF‐βs showed that TGF‐β2 and TGF‐β3 significantly reduced PSD95 protein in cultured hippocampal neurons, and this effect was paralleled by conditioned media from Aβ‐treated astrocytes or from astrocytes from 3xTg‐AD mice. Taken together, our data suggest that TGF‐β2 and TGF‐β3 are produced by astrocytes in a CaN‐dependent manner and should be investigated further in the context of astrocyte‐mediated neurodegeneration.     

Cilostazol suppresses β‐amyloid production by activating a disintegrin and metalloproteinase 10 via the upregulation of SIRT1‐coupled retinoic acid receptor‐β

The accumulation of plaques of β‐amyloid (Aβ) peptides, a hallmark of Alzheimer's disease, results from the sequential cleavage of amyloid precursor protein (APP) by activation of β‐ and γ‐secretases. However, the production of Aβ can be avoided by alternate cleavage of APP by α‐and γ‐secretases. We hypothesized that cilostazol attenuates Aβ production by increasing a disintegrin and metalloproteinase 10 (ADAM10)/α‐secretase activity via SIRT1‐coupled retinoic acid receptor‐β (RARβ) activation in N2a cells expressing human APP Swedish mutation (N2aSwe). To evoke endogenous Aβ overproduction, the culture medium was switched from medium containing 10% fetal bovine serum (FBS) to medium containing 1% FBS, and cells were cultured for 3～24 hr. After depletion of FBS in media, N2aSwe cells showed increased accumulations of full‐length APP (FL‐APP) and Aβ in a time‐dependent manner (3–24 hr) in association with decreased ADAM10 protein expression. When pretreated with cilostazol (10–30 μM), FL‐APP and Aβ levels were significantly reduced, and ADAM10 and α‐secretase activities were restored. Furthermore, the effect of cilostazol on ADAM10 expression was antagonized by pretreating Rp‐cAMPS and sirtinol and by SIRT1‐gene silencing. In the N2aSwe cells overexpressing the SIRT1 gene, ADAM10, and sAPPα levels were significantly elevated. In addition, like all‐trans retinoic acid, cilostazol enhanced the protein expressions of RARβ and ADAM10, and the cilostazol‐stimulated ADAM10 elevation was significantly attenuated by LE135 (a RARβ inhibitor), sirtinol, and RARβ‐gene silencing. In conclusion, cilostazol suppresses the accumulations of FL‐APP and Aβ by activating ADAM10 via the upregulation of SIRT1‐coupled RARβ. © 2014 Wiley Periodicals, Inc.     

Increased frequency of the α‐1‐antichymotrypsin T allele in cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) is a process of unknown etiology characterized by amyloid deposition in the wall of small cerebral and meningeal blood vessels. CAA is also a feature of Alzheimer's disease (AD) and of a subgroup of elderly people. α‐1‐Antichymotrypsin (ACT) is a serum glycoprotein frequently associated with vascular and senile plaque amyloid. The ACT gene is known to have a bi‐allele polymorphism that causes a simple amino acid substitution. In an attempt to clarify the possible role of ACT polymorphism in AD and in cases of CAA, the ACT genotype was investigated in AD, CAA, and intellectually intact controls. Representative brain areas (cerebral cortex, hippocampus, putamen, white matter, and gyrus cinguli) from all cases were studied using classical histologic staining techniques (hematoxylin–eosin (HE), Mallory's thrichromic or alkaline congo red stain), and immunohistochemistry for tau and β‐amyloid proteins. There was a significantly increased T allele and TT genotype frequency in the CAA group, but not in the AD group, suggesting a role for the ACT genotype in the development of vascular lesions. The presence of the apolipoprotein E4 allele (ApoE4) did not correlate with the ACT‐A allele, as previously reported, and appeared to be independent of the risk for developing AD.     

Serum amyloid β protein in young and elderly depression: a pilot study

Background: Depression may increase the risk of developing Alzheimer's disease (AD). Recent large cohort studies have also shown that a low plasma amyloid β (Aβ)‐42 level combined with a high Aβ40 level increases the risk of developing AD, suggesting plasma Aβ42/40 ratio as useful for identifying risk of developing mild cognitive impairment and AD. Although several studies have examined Aβ levels in the peripheral blood of elderly individuals with depression, results have been inconsistent. Furthermore, no results have been described for younger depression. Methods: Serum Aβ40, Aβ42 level and Aβ40/42 ratio were evaluated using enzyme‐linked immunosorbent assay in 60 patients with major depressive disorder (MDD) and 60 healthy controls. The results were analyzed in two age groups (young, <60 years; elderly, ≥60 years). Results: Serum Aβ40 level was significantly higher in young MDD patients compared to young controls (P < 0.001), but it was not significantly deferent in the elderly group. Serum Aβ42 level did not differ significantly in both young and elderly groups. Aβ40/42 ratio was significantly higher in both young (P < 0.001) and elderly (P < 0.001) patients with MDD compared to controls. Conclusions: Serum Aβ40/42 ratio was significantly higher in MDD patients than in controls, and this difference was seen for both elderly and young subjects. This may suggest that even young subjects with MDD undergo pathological changes in the very early stage of amyloid deposition.     

Relative efficacies of amyloid β peptide (Aβ) binding proteins in Aβ aggregation

The aggregation of amyloid β peptide (Aβ) into its fibrillar, cross β-pleated configuration is generally viewed as a critical event in the pathophysiology of Alzheimer's disease (AD). A diverse group of molecules, the Aβ binding proteins, has been evaluated for their effects on this process. However, most of these studies have used micromolar or greater reagent concentrations, and their different methods have not permitted quantitative comparisons of the efficacy of different Aβ binding proteins in augmenting or inhibiting aggregation. In the present work we have undertaken a coherent analysis using fluorimetry of thioflavin T-stained experimental solutions. The complement protein C1q, serum amyloid P, and transthyretin significantly enhanced the formation of precipitable, cross β-pleated aggregates in solutions of 800 nM Aβ1–42. Under these same experimental conditions, α₁-antichymotrypsin had no significant effect on the aggregation process, and both the E3 and E4 isoforms of apolipoprotein E were significant inhibitors. There was a non-significant trend toward the E3 isoform exhibiting greater inhibition than the E4 isoform. Of the aggregation-facilitating molecules, C1q was substantially and significantly the most potent. © 1996 Wiley-Liss, Inc.     

Baicalein reduces β‐amyloid and promotes nonamyloidogenic amyloid precursor protein processing in an Alzheimer's disease transgenic mouse model

Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ‐aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABA_A modulation for Alzheimer's disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild‐type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of β‐amyloid (Aβ) by increasing APP α‐processing. These effects were blocked by the GABA_A antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α‐secretase processing, reduced Aβ production, and reduced AD‐like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aβ production and improving cognitive performance, by activating GABA_A receptors. © 2013 Wiley Periodicals, Inc.     

Radiosynthesis of the candidate β‐amyloid radioligand [11C]AZD2184: Positron emission tomography examination and metabolite analysis in cynomolgus monkeys

β‐Amyloid accumulation is associated with the pathogenesis of Alzheimer's disease (AD). AZD2184, a new radioligand for high‐contrast positron emission tomography (PET) imaging of Aβ‐deposits, has recently been developed and characterized in vitro and in rodents ex vivo. The objective of this study was to label AZD2184 with carbon‐11, to perform in vivo characterization of [¹¹C]AZD2184 ([¹¹C] 5 ) in the cynomolgus monkey brain as well as whole‐body dosimetry, and to examine the metabolism of the labeled radioligand. [¹¹C] 5 was prepared by a two‐step radiosynthesis starting with the reaction of 5‐(6‐(tert‐butyldimethylsilyloxy)benzo[d]thiazol‐2‐yl)pyridin‐2‐amine with [¹¹C]methyl iodide followed by deprotection using water. Four brain PET measurements in two cynomolgus monkeys and one whole‐body PET measurement were performed with [¹¹C] 5 . There was a high and rapid brain uptake (2.2–3.4% of injected dose at 2 min). The distribution of brain radioactivity was fairly uniform, with early to late‐brain concentration ratios (peak vs. 60 min) higher for [¹¹C] 5 than ratios previously reported for [¹¹C]PIB (8.2 and 4.6, respectively). Based on the whole‐body data, it was estimated that an effective dose in an adult male would be 6.2 μSv/MBq and thus would be safe from a radiation point of view for multiple scans within the same year. [¹¹C] 5 shows binding characteristics, suggesting low levels of white‐matter retention, and may thus provide improved contrast when compared with currently used PET radioligands for visualization of Aβ‐deposits. On the basis of the labeling chemistry and the results of the biological evaluation, we conclude that [¹¹C] 5 should be useful for routine clinical studies. Synapse 64:733–741, 2010. © 2010 Wiley‐Liss, Inc.