Costimulatory Effects of Interferon-γ and Interleukin-1β or Tumor Necrosis Factor α on the Synthesis of Aβ1-40 and Aβ1-42 by Human Astrocytes

Chronic inflammation and astrocytosis are characteristic histopathological features of Alzheimer's Disease (AD). Astrocytes are one of the predominant cell types in the brain. In AD they are activated and produce inflammatory components such as complement components, acute phase proteins, and cytokines. In this study we analyzed the effect of cytokines on the production of amyloid β (Aβ) in the astrocytoma cell line U373 and in primary human astrocytes isolated postmortem from healthy aged persons as well as from patients with AD. Astrocytes did not produce Aβ in the absence of stimuli or following stimulation with IL-1β, TNFα, IL-6, and TGF-β1. Neither did combinations of TNFα and IL-1β, IL-6 or TGF-β1, or the coadministration of IFNγ and IL-6 or TGF-β1 induce Aβ production. In contrast, pronounced production of Aβ1-40 and Aβ1-42 was observed when primary astrocytes or astrocytoma cells were stimulated with combinations of IFNγ and TNFα or IFNγ and IL-1β. Induction of Aβ production was accompanied by decreased glycosylation of APP as well as by increased secretion of APPsβ. Our results suggest that astrocytes may be an important source of Aβ in the presence of certain combinations of inflammatory cytokines. IFNγ in combination with TNFα or IL-1β seems to trigger Aβ production by supporting β-secretase cleavage of the immature APP molecule.     

Immunodominant epitope and properties of pyroglutamate-modified Aβ-specific antibodies produced in rabbits

N-truncated and N-modified forms of amyloid beta (Aβ) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Aβ is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 3 (AβN3(pE)). We demonstrated that AβN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AβN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AβN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AβN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides.     

Long‐term oral intake of aluminium or zinc does not accelerate Alzheimer pathology in AβPP and AβPP/tau transgenic mice

Whether or not the oral intake of metals such as aluminium (Al) and zinc (Zn) is a risk for Alzheimer's disease (AD) has been a matter of controversy. Lack of AD pathology in patients with Al encephalopathy indicates Al does not cause AD. On the other hand, some epidemiological studies have suggested high Al increases the occurrence of AD. Our purpose is to test if high Al in drinking water is a risk factor for AD. We administered Al and Zn in drinking water to Tg2576, a transgenic mouse model for amyloid β‐protein (Aβ) deposition with the Aβ precursor protein (AβPP) mutations (K670N/M671L), and Tg2576/tau(P301L), a model for Aβ and tau deposition. Deionized water was given to the control Tg2576 and Tg2576/tau. After administration for 4–10 months of approximately 100 mg/kg body weight Al or Zn per day, we were not able to find by quantitative immunohistochemical analyses differences in the deposition of Aβ and tau between the treated and untreated groups. Nor did the Al or Zn treatment affect the amount of soluble Aβ and Aβ*56, an Aβ oligomer, measured by ELISA or immunoblot. The oral intake of excess Al or Zn does not accelerate AD pathology in the transgenic mouse models for Aβ and tau accumulation. Such results do not seem to support the notion that excessive oral intake of Al or Zn is a risk factor for AD.     

γ‐Secretase binding sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a putative origin of CSF Aβ

Deposition of β ‐amyloid (Aβ) peptides, cleavage products of β‐amyloid precursor protein (APP) by β‐secretase‐1 (BACE1) and γ‐secretase, is a neuropathological hallmark of Alzheimer's disease (AD). γ‐Secretase inhibition is a therapeutical anti‐Aβ approach, although changes in the enzyme's activity in AD brain are unclear. Cerebrospinal fluid (CSF) Aβ peptides are thought to derive from brain parenchyma and thus may serve as biomarkers for assessing cerebral amyloidosis and anti‐Aβ efficacy. The present study compared active γ‐secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored the possibility of Aβ production and secretion by the choroid plexus (CP). The specific binding density of [³H]‐L‐685,458, a radiolabeled high‐affinity γ‐secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with similar ages and post‐mortem delays. The CP in post‐mortem samples exhibited exceptionally high [³H]‐L‐685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin‐1 immunoreactivity, and β‐site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins and released Aβ40 and Aβ42 into the medium. Overall, our results suggest that γ‐secretase activity appears unaltered in the cerebrum in AD and is not correlated with regional amyloid plaque pathology. The CP appears to be a previously unrecognised non‐neuronal contributor to CSF Aβ, probably at reduced levels in AD.     

Aβ-fiber intensity stimulation of chronically constricted median nerve induces c-fos expression in thalamic projection neurons of the cuneate nucleus in rats with behavioral signs of neuropathic pain

This study was aimed to investigate the possible involvement of neurons in the cuneate nucleus (CN) in the processing of Aβ afferent inputs evoked by electrical stimulation of constricted median nerve in rats with behavioral signs of neuropathic pain. Immunohistochemical localization of Fos protein was used to examine the neuronal activation, and the combination of Fos immunohistochemistry with the retrograde labeling of Fluoro-Gold (FG) injected into the ventrobasal complex of the thalamus was used to characterize the activated neurons. Two weeks after unilateral median nerve constriction injury, the rats exhibited behavioral signs of neuropathic pain in the affected forepaws. In rats after nerve injury but without electrical stimulation, some Fos-like immunoreactive (Fos-LI) neurons were detected in the dorsal horn of the seventh cervical segment (C7) but none was found in the CN. Similar features were also noted when the stimulation of the intact median nerve served as an additional control. After Aβ-fiber intensity stimulation of the previously constricted median nerve, an increase in number of Fos-LI neurons occurred in the medial half of the ipsilateral C7 dorsal horn as well as in the ipsilateral CN. In the latter, the Fos-LI neurons were located in the median nerve projection territory throughout the nucleus. Most of the Fos-LI neurons were distributed in the middle region of the CN, with about 78% of them emitting FG fluorescence indicating that they were cuneothalamic projection neurons. The results of this study suggest that the dorsal column-medial lemniscal system may contribute to the transmission and modulation of Aβ-fiber mediated neuropathic pain signals.     

Neurotrophin-3 antisense oligonucleotide attenuates nerve injury-induced Aβ-fibre sprouting

It is proposed that following peripheral nerve injury abnormal sprouting of Aβ-fibre primary afferent neurons in the spinal cord contributes to the allodynia that often occurs with such injury. Allodynia is characterized as pain due to a stimulus which is normally non-noxious. Our recent in vivo experiments show that intrathecal administration of neurotrophin-3 (NT-3), in normal animals, induces allodynia and sprouting of Aβ-fibres. In this study, we examine whether intrathecal administration of NT-3 antisense oligonucleotides (50 μM), via an osmotic pump for 14 days, attenuates nerve injury-induced sprouting and allodynia. The oligonucleotides used in this study were phosphorothioate modified and control experiments, using an ELISA, confirm that intrathecal administration of the antisense induces a significant decrease in NT-3 levels in the spinal cord. All surgery was conducted on anaesthetized Wistar rats (sodium pentobarbitone, i.p. 50 mg/kg). Consistent with previous studies, transganglionic labelling of Aβ-fibres with choleragenoid-horseradish peroxidase (C-HRP) shows that complete transection of the sciatic nerve induces an expansion of C-HRP label into lamina II of the spinal dorsal horn. Using image analysis, we find that intrathecal administration of NT-3 antisense attenuates the density of C-HRP labelling in lamina II in nerve injured animals. A NT-3 sense oligonucleotide (50 μM) has no effect. To test the effect of NT-3 antisense on allodynia, the nociceptive flexion reflex is examined, using an Ugo Basile Analgesymeter, in animals with partial sciatic nerve ligation. Intrathecal administration of 50 μM NT-3 antisense significantly attenuates nerve injury-induced allodynia, whereas the sense oligonucleotide has no effect. These results provide further evidence that endogenous NT-3 contributes to both nerve injury-induced Aβ-fibre sprouting and allodynia and demonstrates the potential of neurotrophin-3 antisense oligonucleotides as therapeutic agents for neuropathic pain.     

Amyloid-β: a chameleon walking in two worlds: a review of the trophic and toxic properties of amyloid-β

Although much maligned, the amyloid-β (Aβ) protein has been shown to possess a number of trophic properties that emanate from the protein’s ability to bind Cu, Fe and Zn. Aβ belongs to a group of proteins that capture redox metal ions (even under mildly acidotic conditions), thereby preventing them from participating in redox cycling with other ligands. The coordination of Cu appears to be crucial for Aβ’s own antioxidant activity that has been demonstrated both in vitro as well as in the brain, cerebrospinal fluid and plasma. The chelation of Cu by Aβ would therefore be predicted to dampen oxidative stress in the mildly acidotic and oxidative environment that accompanies acute brain trauma and Alzheimer’s disease (AD). Given that oxidative stress promotes Aβ generation, the formation of diffuse amyloid plaques is likely to be a compensatory response to remove reactive oxygen species. This review weighs up the evidence supporting both the trophic and toxic properties of Aβ, and while evidence for direct Aβ neurotoxicity in vivo is scarce, we postulate that the product of Aβ’s antioxidant activity, hydrogen peroxide (H₂O₂), is likely to mediate toxicity as the levels of this oxidant rise with the accumulation of Aβ in the AD brain. We propose that metal ion chelators, antioxidants, antiinflammatories and amyloid-lowering drugs that target the reduction of H₂O₂ and/or Aβ generation may be efficacious in decreasing neurotoxicity. However, given the antioxidant activity of Aβ, we suggest that the excessive removal of Aβ may prevent adequate chelation of metal ions and removal of O₂⁻, leading to enhanced, rather than reduced, neuronal oxidative stress.     

Intracellular sAPP retention in response to Aβ is mapped to cytoskeleton‐associated structures

Amyloid β (Aβ) contributes to neurodegeneration in Alzheimer's disease and provides a close association between molecular events and pathology, although the underlying molecular mechanisms are unclear. In the work described here, Aβ did not induce amyloid precursor protein (APP) expression, but APP processing/trafficking was markedly affected. In COS‐7 cells, Aβ provokes retention of intracellular sAPPα (isAPPα). Intracellular holo‐APP levels remain unchanged, and extracellular total sAPP increases, although extracellular sAPPα alone was not altered significantly. In primary neuronal cultures and PC12 cells, isAPP also increased, but this was mirrored by a decrease in extracellular total sAPP. The isAPP retention was particularly associated with the cytoskeletal fraction. The retention “per se” occurred in vesicular‐like densities, negative for a C‐terminal antibody and strongly positive for the 6E10 antibody, clearly showing abnormal intracellular accumulation of sAPPα in response to Aβ. Our data support a dynamic model for intracellular retention of sAPPα as an early response to Aβ exposure. Particularly noteworthy was the observation that removal of Aβ reversed the isAPP accumulation. Mechanistically, these findings disclose an attractive physiological response, revealing the capacity of cells to deal with adverse effects induced by Aβ. © 2008 Wiley‐Liss, Inc.     

Immunohistochemical localization of neprilysin in the human cerebral cortex: inverse association with vulnerability to amyloid β-protein (Aβ) deposition

We investigated the immunohistochemical localization of neprilysin, a putative amyloid β-protein (Aβ)-degrading enzyme, in postmortem human brain tissues. In the cerebral cortex, neprilysin immunoreactivity was weak, but relatively dense distribution was found in the primary somatosensory and visual cortices compared with the hippocampus and association cortices. In Alzheimer brain, neprilysin-positive dystrophic neurites occurred in senile plaques in the primary cortices, an observation that supports the relative abundance of neprilysin-positive neuronal processes. A paucity of neprilysin in the hippocampus and association cortices may contribute to the vulnerability of these areas to Aβ deposition.     

Sex Hormone Decline and Amyloid β Synthesis,Transport and Clearance in the Brain

Sex hormones (SH) are essential regulators of the central nervous system. The decline in SH levels along with ageing may contribute to compromised neuroprotection and set the grounds for neurodegeneration and cognitive impairments. In Alzheimer's disease, besides other pathological features, there is an imbalance between amyloid β (Aβ) production and clearance, leading to its accumulation in the brain of older subjects. Aβ accumulation is a primary cause for brain inflammation and degeneration, as well as concomitant cognitive decline. There is mounting evidence that SH modulate Aβ production, transport and clearance. Importantly, SH regulate most of the molecules involved in the amyloidogenic pathway, their transport across brain barriers for elimination, and their degradation in the brain interstitial fluid. This review brings together data on the regulation of Aβ production, metabolism, degradation and clearance by SH.     

A critical role of TRPM2 channel in Aβ42‐induced microglial activation and generation of tumor necrosis factor‐α

Amyloid β (Aβ)‐induced neuroinflammation plays an important part in Alzheimer's disease (AD). Emerging evidence supports a role for the transient receptor potential melastatin‐related 2 (TRPM2) channel in Aβ‐induced neuroinflammation, but how Aβ induces TRPM2 channel activation and this relates to neuroinflammation remained poorly understood. We investigated the mechanisms by which Aβ₄₂ activates the TRPM2 channel in microglial cells and the relationships to microglial activation and generation of tumor necrosis factor‐α (TNF‐α), a key cytokine implicated in AD. Exposure to 10–300 nM Aβ₄₂ induced concentration‐dependent microglial activation and generation of TNF‐α that were ablated by genetically deleting (TRPM2 knockout ;TRPM2‐KO) or pharmacologically inhibiting the TRPM2 channel, revealing a critical role of this channel in Aβ₄₂‐induced microglial activation and generation of TNF‐α. Mechanistically, Aβ₄₂ activated the TRPM2 channel via stimulating generation of reactive oxygen species (ROS) and activation of poly(ADPR) polymerase‐1 (PARP‐1). Aβ₄₂‐induced generation of ROS and activation of PARP‐1 and TRPM2 channel were suppressed by inhibiting protein kinase C (PKC) and NADPH oxidases (NOX). Aβ₄₂‐induced activation of PARP‐1 and TRPM2 channel was also reduced by inhibiting PYK2 and MEK/ERK. Aβ₄₂‐induced activation of PARP‐1 was attenuated by TRPM2‐KO and moreover, the remaining PARP‐1 activity was eliminated by inhibiting PKC and NOX, but not PYK2 and MEK/ERK. Collectively, our results suggest that PKC/NOX‐mediated generation of ROS and subsequent activation of PARP‐1 play a role in Aβ₄₂‐induced TRPM2 channel activation and TRPM2‐dependent activation of the PYK2/MEK/ERK signalling pathway acts as a positive feedback to further facilitate activation of PARP‐1 and TRPM2 channel. These findings provide novel insights into the mechanisms underlying Aβ‐induced AD‐related neuroinflammation.     

Small Aβ1–42 oligomer‐induced membrane depolarization of neuronal and microglial cells: Role of N‐methyl‐D‐aspartate receptors

Although it is well documented that soluble beta amyloid (Aβ) oligomers are critical factors in the pathogenesis of Alzheimer's disease (AD) by causing synaptic dysfunction and neuronal death, the primary mechanisms by which Aβ oligomers trigger neurodegeneration are not entirely understood. We sought to investigate whether toxic small Aβ_1–42 oligomers induce changes in plasma membrane potential of cultured neurons and glial cells in rat cerebellar granule cell cultures leading to neuronal death and whether these effects are sensitive to the N‐methyl‐D‐aspartate receptor (NMDA‐R) antagonist MK801. We found that small Aβ_1–42 oligomers induced rapid, protracted membrane depolarization of both neurons and microglia, whereas there was no change in membrane potential of astrocytes. MK801 did not modulate Aβ‐induced neuronal depolarization. In contrast, Aβ_1?42 oligomer‐induced decrease in plasma membrane potential of microglia was prevented by MK801. Small Aβ_1–42 oligomers significantly elevated extracellular glutamate and caused neuronal necrosis, and both were prevented by MK801. Also, small Aβ_1–42 oligomers decreased resistance of isolated brain mitochondria to calcium‐induced opening of mitochondrial permeability transition pore. In conclusion, the results suggest that the primary effect of toxic small Aβ oligomers on neurons is rapid, NMDA‐R‐independent plasma membrane depolarization, which leads to neuronal death. Aβ oligomers‐induced depolarization of microglial cells is NMDA‐R dependent. © 2014 Wiley Periodicals, Inc.     

CSF Aβ42 and tau in Parkinson's disease with cognitive impairment

We tested the hypothesis that the CSF biomarker signature associated with Alzheimer's disease (AD) is present in a subset of individuals with Parkinson's disease and Dementia (PD‐D) or with PD and Cognitive Impairment, Not Dementia (PD‐CIND). We quantified CSF Aβ₄₂, total tau (T‐tau), and phospho‐tau (P181‐tau) using commercially available kits. Samples were from 345 individuals in seven groups (n): Controls ≤50 years (35), Controls >50 years (115), amnestic Mild Cognitive Impairment (aMCI) (24), AD (49), PD (49), PD‐CIND (62), and PD‐D (11). We observed expected changes in AD or aMCI compared with age‐matched or younger controls. CSF Aβ₄₂ was reduced in PD‐CIND (P < 0.05) and PD‐D (P < 0.01), whereas average CSF T‐tau and P181‐tau were unchanged or decreased. One‐third of PD‐CIND and one‐half of PD‐D patients had the biomarker signature of AD. Abnormal metabolism of Aβ₄₂ may be a common feature of PD‐CIND and PD‐D. © 2010 Movement Disorder Society     

The Levels of Soluble versus Insoluble Brain Aβ Distinguish Alzheimer's Disease from Normal and Pathologic Aging

The abundance and solubility of Aβ peptides are critical determinants of amyloidosis in Alzheimer's disease (AD). Hence, we compared levels of total soluble, insoluble, and total Aβ1-40 and Aβ1-42 in AD brains with those in age-matched normal and pathologic aging brains using a sandwich enzyme-linked immunosorbent assay (ELISA). Since the measurement of Aβ1-40 and Aβ1-42 depends critically on the specificity of the monoclonal antibodies used in the sandwich ELISA, we first demonstrated that each assay is specific for Aβ1-40 or Aβ1-42 and the levels of these peptides are not affected by the amyloid precursor protein in the brain extracts. Thus, this sandwich ELISA enabled us to show that the average levels of total cortical soluble and insoluble Aβ1-40 and Aβ1-42 were highest in AD, lowest in normal aging, and intermediate in pathologic aging. Remarkably, the average levels of insoluble Aβ1-40 were increased 20-fold while the average levels of insoluble Aβ1-42 were increased only 2-fold in the AD brains compared to pathologic aging brains. Further, the soluble pools of Aβ1-40 and Aβ1-42 were the largest fractions of total Aβ in the normal brain (i.e., 50 and 23%, respectively), but they were the smallest in the AD brain (i.e., 2.7 and 0.7%, respectively) and intermediate (i.e., 8 and 0.8%, respectively) in pathologic aging brains. Thus, our data suggest that pathologic aging is a transition state between normal aging and AD. More importantly, our findings imply that a progressive shift of brain Aβ1-40 and Aβ1-42 from soluble to insoluble pools and a profound increase in the levels of insoluble Aβ1-40 plays mechanistic roles in the onset and/or progression of AD.     

X11α haploinsufficiency enhances Aβ amyloid deposition in Alzheimer's disease transgenic mice

The neuronal adaptor protein X11α/mint-1/APBA-1 binds to the cytoplasmic domain of the amyloid precursor protein (APP) to modulate its trafficking and metabolism. We investigated the consequences of reducing X11α in a mouse model of Alzheimer's disease (AD). We crossed hAPPswe/PS-1ΔE9 transgenic (AD tg) mice with X11α heterozygous knockout mice in which X11α expression is reduced by approximately 50%. The APP C-terminal fragments C99 and C83, as well as soluble Aβ40 and Aβ42, were increased significantly in brain of X11α haploinsufficient mice. Aβ/amyloid plaque burden also increased significantly in the hippocampus and cortex of one year old AD tg/X11α (+/−) mice compared to AD tg mice. In contrast, the levels of sAPPα and sAPPβ were not altered significantly in AD tg/X11α (+/−) mice. The increased neuropathological indices of AD in mice expressing reduced X11α suggest a normal suppressor role for X11α on CNS Aβ/amyloid deposition.     

Change in excitability and phenotype of substance P and its receptor in cat Aβ sensory neurons following peripheral inflammation

The effect of peripheral inflammation on spontaneous firing and level of substance P (SP) and its receptor in electrophysiologically identified cat Aβ neurons of dorsal root ganglion (DRG) was studied in vivo using a combination of intracellular recording, dye injection and immunohistochemical techniques. Following injection of carrageenan (Carg) into cat hindpaw, the number of Aβ neurons with spontaneous firing was enhanced significantly (42.9%, n=182) in comparison with control (16.8%, n=149, P<0.01). DRG Aβ neurons became less depolarized 2–4 h following Carg injection. After identifying the cell properties, Lucifer Yellow was injected and SP-like immunoreactivity (SP-LI) was then detected. A total of 17% of Aβ sensory neurons exhibited SP-LI in inflammatory cat. We also found in rat DRGs that the number of SP-LI positive large cells (>35 μm) was also significantly increased in Carg-treated DRG (11.8±1.2, n=8) compared with untreated DRG (1.8±0.8, n=8, P<0.01). In control cat, the topical use of SP in DRG did not induce any response of Aβ neurons. However, in Carg-treated cat, SP depolarized the membrane potential in most Aβ neurons (68.2%, n=22). L668,169, an antagonist of SP receptor, completely blocked the SP-induced responses. Furthermore, repeated application of SP did not induce obvious desensitization of Aβ neurons. These data suggest that peripheral inflammation increased the excitability, SP level and sensitivity of SP receptor of Aβ neurons. Therefore, we concluded that Aβ sensory neurons appear to contribute to inflammatory allodynia.