Novel demonstration of amyloid-β oligomers in sporadic inclusion-body myositis muscle fibers

Accumulation of amyloid-β (Aβ) within muscle fibers has been considered an upstream step in the development of the s-IBM pathologic phenotype. Aβ42, which is considered more cytotoxic than Aβ40 and has a higher propensity to oligomerize, is preferentially increased in s-IBM muscle fibers. In Alzheimer disease (AD), low-molecular weight Aβ oligomers and toxic oligomers, also referred to as “Aβ-Derived Diffusible Ligands” (ADDLs), are considered strongly cytotoxic and proposed to play an important pathogenic role. ADDLs have been shown to be increased in AD brain. We now report for the first time that in s-IBM muscle biopsies Aβ-dimer, -trimer, and -tetramer are identifiable by immunoblots. While all the s-IBM samples we studied had Aβ-oligomers, their molecular weights and intensity varied between the patient samples. None of the control muscle biopsies had Aβ oligomers. Dot-immunoblots using highly specific anti-ADDL monoclonal antibodies also showed highly increased ADDLs in all s-IBM biopsies studied, while controls were negative. By immunofluorescence, in some of the abnormal s-IBM muscle fibers ADDLs were accumulated in the form of plaque-like inclusions, and were often increased diffusely in very small fibers. Normal and disease-controls were negative. By gold-immuno-electron microscopy, ADDL-immunoreactivities were in close proximity to 6–10 nm amyloid-like fibrils, and also were immunodecorating amorphous and floccular material. In cultured human muscle fibers, we found that inhibition of autophagy led to the accumulation of Aβ oligomers. This novel demonstration of Aβ42 oligomers in s-IBM muscle biopsy provides additional evidence that intra-muscle fiber accumulation of Aβ42 oligomers in s-IBM may contribute importantly to s-IBM pathogenic cascade.     

Contrasting protective effects of cannabinoids against oxidative stress and amyloid-β evoked neurotoxicity in vitro

Cannabinoids have been widely reported to have neuroprotective properties in vitro and in vivo. In this study we compared the effects of CB1 and CB2 receptor-selective ligands, the endocannabinoid anandamide and the phytocannabinoid cannabidiol, against oxidative stress and the toxic hallmark Alzheimer's protein, β-amyloid (Aβ) in neuronal cell lines. PC12 or SH-SY5Y cells were selectively exposed to either hydrogen peroxide, tert-butyl hydroperoxide or Aβ, alone or in the presence of the CB1 specific agonist arachidonyl-2'-chloroethylamide (ACEA), CB2 specific agonist JWH-015, anandamide or cannabidiol. Cannabidiol improved cell viability in response to tert-butyl hydroperoxide in PC12 and SH-SY5Y cells, while hydrogen peroxide-mediated toxicity was unaffected by cannabidiol pretreatment. Aβ exposure evoked a loss of cell viability in PC12 cells. Of the cannabinoids tested, only anandamide was able to inhibit Aβ-evoked neurotoxicity. ACEA had no effect on Aβ-evoked neurotoxicity, suggesting a CB1 receptor-independent effect of anandamide. JWH-015 pretreatment was also without protective influence on PC12 cells from either pro-oxidant or Aβ exposure. None of the cannabinoids directly inhibited or disrupted preformed Aβ fibrils and aggregates. In conclusion, the endocannabinoid anandamide protects neuronal cells from Aβ exposure via a pathway unrelated to CB1 or CB2 receptor activation. The protective effect of cannabidiol against oxidative stress does not confer protection against Aβ exposure, suggesting divergent pathways for neuroprotection of these two cannabinoids.     

Florbetaben to trace amyloid-β in the Alzheimer brain by means of PET

PET imaging of amyloid-β has recently emerged as a valuable biomarker to support the in vivo diagnosis of Alzheimer's disease (AD). So far, however, no tracer is available suitable for general clinical routine application. Florbetaben is a promising 18F-labeled amyloid-β-targeted PET tracer currently in Phase 2/3 clinical development. This review provides an overview on the current knowledge and future research activities on florbetaben. Recently, the first worldwide multi-center trial to test the diagnostic performance of amyloid-β PET in AD was conducted with this tracer. From this trial, a sensitivity and specificity of 80 and 91% in the discrimination between patients with probable AD and age-matched healthy controls was reported. Ongoing florbetaben PET trials deal with correlating the in vivo PET signal to post mortem histopathology evaluation, and with investigating the value of the tracer to predict progression to AD at the stage of mild cognitive impairment. The preclinical and clinical data currently available verify florbetaben as a safe and efficacious PET tracer suitable for detection of amyloid-β deposition in the brain. The results of the ongoing trials will contribute to current knowledge on the characteristics of florbetaben, and will help to determine the future potential of florbetaben PET imaging as a visual adjunct to supplement the routine clinical "AD diagnostic toolbox".     

Proteome-based plasma markers of brain amyloid-β deposition in non-demented older individuals

Blood-based markers reflecting core pathological features of Alzheimer's disease (AD) in pre-symptomatic individuals are likely to accelerate the development of disease-modifying treatments. Our aim was to discover plasma proteins associated with brain amyloid-β (Aβ) burden in non-demented older individuals. We performed discovery-phase experiments using two dimensional gel electrophoresis (2DGE) and mass spectrometry-based proteomic analysis of plasma in combination with 11C-PiB PET imaging of the brain in samples collected 10 years prior to the PET scans. Confirmatory studies used ELISA assays in a separate set of blood samples obtained within a year of the PET scans. We observed that a panel of 18 2DGE plasma protein spots effectively discriminated between individuals with high and low brain Aβ. Mass spectrometry identified these proteins, many of which have established roles in Aβ clearance, including a strong signal from apolipoprotein-E (ApoE). In validation-phase studies, we observed a strong association between plasma ApoE concentration and Aβ burden in the medial temporal lobe. Targeted voxel-based analysis localized this association to the hippocampus and entorhinal cortex. APOE ε4 carriers also showed greater Aβ levels in several brain regions relative to ε4 non-carriers. These results suggest that both peripheral concentration of ApoE protein and APOE genotype are related to early neuropathological changes in brain regions vulnerable to AD pathology even in the non-demented elderly. Our strategy combining proteomics with in vivo brain amyloid imaging holds promise for the discovery of biologically relevant peripheral markers in those at risk for AD.     

Overexpression of amyloid-β protein precursor induces mitochondrial oxidative stress and activates the intrinsic apoptotic cascade

Aberrant processing of amyloid-β protein precursor (AβPP) into amyloid-β (Aβ) fragments underlies the formation of senile plaques in Alzheimer's disease (AD). Moreover, Aβ fragments, particularly Aβ(42), exert direct toxic effects within neurons including the induction of mitochondrial oxidative stress (MOS). Interestingly, individuals with Down syndrome (DS) frequently develop early onset AD as a major co-morbid phenotype. One hypothesis for AD associated with DS involves the overexpression of wild type (WT) AβPP protein, due to its location on chromosome 21. However, the mechanism by which the overexpression of WT AβPP might trigger MOS and induce cell death is presently unclear. Here we show that transient overexpression of DsRed2-tagged AβPP (WT) in CHO cells induces caspase-3 activation and nuclear fragmentation indicative of apoptosis. AβPP localizes to the mitochondrial fraction of transfected CHO cells and induces glutathione-sensitive opening of the mitochondrial permeability transition pore (mPTP) and cytochrome c release. MOS and intrinsic apoptosis induced by AβPP are significantly inhibited by co-expression of Bcl-2 or treatment with either glutathione or a pan-caspase inhibitor. The mPTP inhibitor, cyclosporin A, also significantly attenuates AβPP-induced apoptosis. AβPP-induced apoptosis is unaffected by a β-secretase inhibitor and is independent of detectable Aβ(42); however, a γ-secretase inhibitor significantly protects against AβPP overexpression, suggesting a possible role of the AβPP intracellular domain in cell death. These data indicate that overexpression of WT AβPP is sufficient to induce MOS and intrinsic apoptosis, suggesting a novel pro-oxidant role for AβPP at mitochondria which may be relevant in AD and DS disease pathologies.     

Overexpression of human wild-type amyloid-β protein precursor decreases the iron content and increases the oxidative stress of neuroblastoma SH-SY5Y cells

The accumulation of amyloid-β protein precursor (AβPP) is related to the pathogenesis of Alzheimer's disease (AD); however, the underlying mechanism is still unclear. The abnormal interactions of AβPP with metal ions such as iron are implicated in the process of oxidative stress in AD brains. In this study, we found that the overexpression of wild-type human AβPP695 decreased the iron content and increased the oxidative stress in neuroblastoma SH-SY5Y cells. The catalase activity of stably transfected cells overexpressing wild-type AβPP695 (AβPP cells) was significantly lower than that of the control cells. Intracellular reactive oxygen species (ROS) generation and calcium levels significantly increased in AβPP cells compared to control cells. The mitochondrial membrane potential of AβPP cells was significantly lower than that of the control cells. Moreover, iron treatment decreased ROS and calcium levels and increased cell viability of AβPP cells. The iron deficiency in AβPP cells may contribute to the pathogenesis of AD.     

Intracellular amyloid-β accumulation in calcium-binding protein-deficient neurons leads to amyloid-β plaque formation in animal model of Alzheimer's disease

One of the major hallmarks of Alzheimer's disease (AD) is the extracellular deposition of amyloid-β (Aβ) as senile plaques in specific brain regions. Clearly, an understanding of the cellular processes underlying Aβ deposition is a crucial issue in the field of AD research. Recent studies have found that accumulation of intraneuronal Aβ (iAβ) is associated with synaptic deficits, neuronal death, and cognitive dysfunction in AD patients. In this study, we found that Aβ deposits had several shapes and sizes, and that iAβ occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice, an animal model of AD. We also observed pyroglutamate-modified Aβ (N3pE-Aβ), which has been suggested to be a seeding molecule for senile plaques, inside the Aβ plaques only after iAβ accumulation, which argues against its seeding role. In addition, we found that iAβ accumulates in calcium-binding protein (CBP)-free neurons, induces neuronal death, and then develops into senile plaques in 2-4-month-old 5XFAD mice. These findings suggest that N3pE-Aβ-independent accumulation of Aβ in CBP-free neurons might be an early process that triggers neuronal damage and senile plaque formation in AD patients. Our results provide new insights into several long-standing gaps in AD research, namely how Aβ plaques are formed, what happens to iAβ and how Aβ causes selective neuronal loss in AD patients.     

Heparanase overexpression impairs inflammatory response and macrophage-mediated clearance of amyloid-β in murine brain

Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimer's disease, as well as after traumatic injury and pathogen infection. Resident immune cells, microglia and astrocytes, are activated and joined by blood-borne monocytes that traverse the blood-brain barrier and convert into activated macrophages. The activated cells express various cytokines, chemokines and proteolytic enzymes. To study the role of heparan sulfate proteoglycans in neuroinflammation, we employed a transgenic mouse overexpressing heparanase, an endoglucuronidase that specifically degrades heparan sulfate side chains. Neuroinflammation was induced by systemic challenge with lipopolysaccharide, or by localized cerebral microinjection of aggregated amyloid-β peptide, implicated in Alzheimer's disease. Lipopolysaccharide-treated control mice showed massive activation of resident microglia as well as recruitment of monocyte-derived macrophages into the brain parenchyma. Microinjection of aggregated amyloid-β elicited a similar inflammatory response, albeit restricted to the injection site, which led to dispersion and clearance of the amyloid. In the heparanase-overexpressing mice, all aspects of immune cell recruitment and activation were significantly attenuated in both inflammation models, as was amyloid dispersion. Accordingly, an in vitro blood-brain barrier model constructed from heparanase-overexpressing cerebral vascular cells showed impaired transmigration of monocytes compared to a corresponding assembly of control cells. Our data indicate that intact heparan sulfate chains are required at multiple sites to mediate neuroinflammatory responses, and further point to heparanase as a modulator of this process, with potential implications for Alzheimer's disease.     

Context-dependent toxicity of amyloid-β peptides on mouse cerebellar cells

Alzheimer's disease (AD) is the major cause of dementia in old people. AD pathology is characterized by amyloid-β (Aβ) deposits in several regions of the brain, and links have been hypothesized between Aβ toxicity and apoptosis. Cerebellar granule cells (CGCs) have been widely used as in vitro tools for molecular studies correlating apoptosis with AD, although the cerebellum is a relatively spared area of the brain in vivo. We have used mixed neuronal-glial cerebellar cultures (NGCCs) and organotypic cerebellar cultures (OCCs) obtained from postnatal mice to assess the toxic effect of the Aβ oligomer 1-40 (Aβ1-40) after propidium iodide uptake in vitro. Our results demonstrate that NGCCs, which are primarily composed of CGCs, are resistant to Aβ1-40 challenge (5-10 μM) when cultured in physiological (5 mM) extracellular KCl ([K+]e) concentrations, i.e., in a condition in which CGCs undergo full maturation. Conversely, when 10 μM Aβ1-40 is given to NGCCs cultured in elevated (25 mM) [K+]e (and thus maintained in an immature state), there is a statistically significant increase in cell death. Cell death is by apoptosis, as demonstrated by ultrastructural examination. OCCs are resistant to Aβ challenge in any of the conditions tested (variation of [K+]e, presence or absence of serum, or addition of the neprilysin blocker phosphoramidon). Altogether these observations lead us to conclude that cerebellar cells in an organotypic context may be less susceptible to damage by Aβ, raising the question whether isolated CGCs are a reliable assay in drug discovery studies of AD.     

Bidirectional relationship between functional connectivity and amyloid-β deposition in mouse brain

Brain region-specific deposition of extracellular amyloid plaques principally composed of aggregated amyloid-β (Aβ) peptide is a pathological signature of Alzheimer's disease (AD). Recent human neuroimaging data suggest that resting-state functional connectivity strength is reduced in patients with AD, cognitively normal elderly harboring elevated amyloid burden, and in advanced aging. Interestingly, there exists a striking spatial correlation between functional connectivity strength in cognitively normal adults and the location of Aβ plaque deposition in AD. However, technical limitations have heretofore precluded examination of the relationship between functional connectivity, Aβ deposition, and normal aging in mouse models. Using a novel functional connectivity optical intrinsic signal (fcOIS) imaging technique, we demonstrate that Aβ deposition is associated with significantly reduced bilateral functional connectivity in multiple brain regions of older APP/PS1 transgenic mice. The amount of Aβ deposition in each brain region was associated with the degree of local, age-related bilateral functional connectivity decline. Normal aging was associated with reduced bilateral functional connectivity specifically in retrosplenial cortex. Furthermore, we found that the magnitude of regional bilateral functional correlation in young APP/PS1 mice before Aβ plaque formation was proportional to the amount of region-specific plaque deposition seen later in older APP/PS1 mice. Together, these findings suggest that Aβ deposition and normal aging are associated with region-specific disruption of functional connectivity and that the magnitude of local bilateral functional connectivity predicts regional vulnerability to subsequent Aβ deposition in mouse brain.     

Cold stress induced morphological microglial activation and increased IL-1β expression in astroglial cells in rat brain

The present study investigated the possible impact of cold stress on the immune functions of the brain. Wistar rats were exposed to 4°C for 2h prior to analysis of immunohistochemical analysis of OX-42 and IL-1β, which are markers of microglia and inflammation, respectively. Exposure to cold stress induced morphological microglial activation in as early as 30 min, and the activation lasted up to 2h following the stress. In addition, increased IL-1β-immunoreactivity was detected in the hippocampus and hypothalamus. However, IL-1β was not co-localized with microglia, and was predominantly expressed in astroglia. The present study provides the first evidence that cold stress contributes to neuro-immunomodulation in the brain through microglial activation and expression of IL-1β in astroglia.     

Hemoglobin-induced oxidative stress contributes to matrix metalloproteinase activation and blood–brain barrier dysfunction in vivo

Hemoglobin (Hb) released from extravasated erythrocytes is implicated in brain edema after intracerebral hemorrhage (ICH). Hemoglobin is a major component of blood and a potent mediator of oxidative stress after ICH. Oxidative stress and matrix metalloproteinases (MMPs) are associated with blood–brain barrier (BBB) dysfunction. This study was designed to elucidate whether Hb-induced oxidative stress contributes to MMP-9 activation and BBB dysfunction in vivo. An intracerebral injection of Hb into rat striata induced increased hydroethidine (HEt) signals in parallel with MMP-9 levels. In situ gelatinolytic activity colocalized with oxidized HEt signals in vessel walls, accompanied by immunoglobulin G leakage and a decrease in immunoactivity of endothelial barrier antigen, a marker of endothelial integrity. Administration of a nonselective MMP inhibitor prevented MMP-9 levels and albumin leakage in injured striata. Moreover, reduction in oxidative stress by copper/zinc-superoxide dismutase (SOD1) overexpression reduced oxidative stress, MMP-9 levels, albumin leakage, and subsequent apoptosis compared with wild-type littermates. We speculate that Hb-induced oxidative stress may contribute to early BBB dysfunction and subsequent apoptosis, partly through MMP activation, and that SOD1 overexpression may reduce Hb-induced oxidative stress, BBB dysfunction, and apoptotic cell death.     

Cell-derived soluble oligomers of human amyloid-β peptides disturb cellular homeostasis and induce apoptosis in primary hippocampal neurons

The concentrations of soluble β-amyloid (Aβ) oligomers paralleled with the extent of synaptic loss and severity of cognitive impairment in Alzheimer patients. However, the neurotoxicity of the naturally generated Aβ species remains unknown. This study was designed to examine the effects of naturally generated Aβ oligomers, secreted from amyloid precursor protein-expressing cells, on the homeostasis and viability of primary hippocampal neurons. Our results showed that primary hippocampal neurons incubated with condition media containing cell-secreted soluble Aβ had higher levels of heat-shock protein (HSP)27, HSP60 and HSP70, and lower levels of HSP32 than those of the control neurons. The cell-secreted soluble Aβ caused mitochondria dysfunction in hippocampal neurons as demonstrated by depolarized membrane potential and decreased cytochrome c oxidase activity and ATP levels. The levels of pro-apoptotic proteins, Bid, Bax and cytochrome C, were elevated; whereas anti-apoptotic Bcl-2 protein was reduced in the soluble Aβ-cultured neurons. Apoptosis was also evident in these soluble Aβ-cultured neurons. These results indicate that naturally secreted Aβ induces neuronal injury/death by activating an apoptotic pathway involving impaired mitochondria function and cellular homeostasis.     

Meta-analysis of plasma amyloid-β levels in Alzheimer's disease

Plasma amyloid-β (Aβ) levels have been proposed as biomarkers of Alzheimer's disease (AD), but studies have produced inconsistent results. We present a meta-analytic review of cross-sectional studies that examined plasma Aβ levels in AD and cognitively normal subjects, and longitudinal studies that used baseline plasma Aβ levels to predict conversion from normal cognition to AD. Medline and EMBASE databases were searched to generate an initial list of relevant studies, and selected authors approached for additional data. Twelve cross- sectional studies (n = 1483) and seven longitudinal (n = 3920) met the inclusion criteria for meta-analysis. Random effects model was used to calculate the weighted mean difference (WMD) by Review Manager Version 4.2. In longitudinal studies, cognitively normal individuals who converted to AD had higher baseline Aβ1-40 and Aβ1-42 levels (WMD: 10.29, z = 3.80, p = 0.0001 and WMD: 8.01, z = 2.76, p = 0.006, respectively), and non-significantly increased Aβ1-42/Aβ1-40 ratio (WMD: 0.03, z = 1.65, p = 0.10). In cross sectional studies, compared to cognitively normal individuals, AD patients had marginally but non-significantly lower Aβ1-42 levels (WMD:-2.84, z = 1.73, p = 0.08), but Aβ1-40 levels were not significantly different (WMD: 3.43, z = 0.40, p = 0.69). Our systematic review suggests a model of differential longitudinal changes in plasma Aβ levels in cognitively stable individuals versus those who go on to develop AD dementia. Baseline Aβ1-40 and Aβ1-42 levels in cognitively normal elderly individuals might be predictors of higher rates of progression to AD, and should be further explored as potential biomarkers.     

Do gap junctions couple interstitial cells of Cajal pacing and neurotransmission to gastrointestinal smooth muscle?

Interstitial cells of Cajal (ICC) pace gastrointestinal phasic activity and transmit nerve activity. Gap junctions may couple these cells to smooth muscle, but no functional evidence exists. The objective of this study was to use uncouplers of gap junctions, 18 alpha-glycyrrhetenic acid and its water-soluble analogue carbenoxolone, to evaluate if gap junctions function in pacing and neurotransmission. After inhibition of nerve function with tetrodotoxin (TTX) and N(G)-nitro-L-arginine (L-NOARG), ionomycin- or carbachol-initiated regular phasic activities of circular muscle strips from canine colon and ileum. In some cases, the primary ICC network responsible for pacing was removed. The effects of inhibitors of gap junction conductance (10(-5)-10(-4) mol L(-1)) on frequencies and amplitudes of contraction were compared to appropriate time controls. Lower oesophageal sphincter (LOS) relaxations to nerve stimulation were studied before and after inhibition of gap junction functions. No major changes in LOS relaxations or frequencies of colonic or ileal contractions occurred, but amplitudes of contractions decreased from these agents. Similar results were obtained when the myenteric plexus-ICC network of ileum was removed. Regular phasic activity was not obtained after removal of the colon submuscular plexus ICC. These findings suggest that mechanisms other than gap junctions couple gut pacemaking activity and nerve transmission.     

Femtomole immunodetection of synthetic and endogenous amyloid-β oligomers and its application to Alzheimer’s disease drug candidate screening

Alzheimer’s disease (AD) is a fatal, progressive dementia for which there is no cure and for which a molecular basis has yet to be established. However, considerable evidence suggests that AD is linked to neurotoxic assemblies of the 42-amino-acid peptide amyloid β (Aβ). There is now a clear body of evidence that shows this neurotoxicity resides not only in insoluble fibrils of Aβ but also in soluble Aβ ADDLs (Aβ-derived diffusible ligands) and larger protofibrils. Further, anti-Aβ antibodies have been reported to reverse memory failure in human amyloid precursor protein (hAPP)-expressed transgenic mice in a manner that suggests symptom reversal is attributable to targeting of ADDLs. Clearly, a search for drugs targeting the assembly of these soluble Aβ species represents a new and potentially important approach to the treatment of AD. In this work we describe the development of a dot-blot immunoassay to measure ADDL at the femtomole level, its use in defining the time course of ADDL formation, and its use in determining the presence of ADDLs in the hAPP transgenic mouse brain. Discussion of a protocol to screen agents for inhibition of neurotoxic ADDL formation both in vivo and in vitro is also presented. The methods are suitable for screening combinatorial libraries and, importantly, provide the potential for simultaneous information on candidate transport across the blood-brain barrier.     

Matrix metalloproteinase-9 participates in NGF-induced α-secretase cleavage of amyloid-β protein precursor in PC12 cells

Amyloid-β protein precursor (AβPP) is a ubiquitously expressed glycoprotein, which under physiological conditions can be cleaved following two alternative routes; the non-amyloidogenic and the amyloidogenic pathway. Shift of AβPP processing in favor of the amyloidogenic pathway is a key event in the pathogenesis of Alzheimer's disease (AD). Among the factors that regulate AβPP processing, nerve growth factor (NGF) appears to play an important role; abnormal NGF signaling has been implicated in the onset of AD. In the present study, we used PC12 cells to study the effects of NGF on AβPP processing and provide evidence that NGF, through binding to its high affinity receptor, TrkA moderately down-regulates the expression of the β-secretase β-site AβPP cleaving enzyme-1 and, most importantly, upregulates the expression of two enzymes with α-secretase activity, a disintegrin and metalloprotease-17 and to a greater extent matrix metalloproteinase-9 (MMP9) in a phosphoinositide kinase-3 dependent manner. Finally, we demonstrate that MMP9 actively participates in NGF-induced α-secretase cleavage of AβPP, thus it contributes to the shift of AβPP processing towards the non-amyloidogenic pathway precluding the formation of neurotoxic Aβ peptides.