β-amyloid controls altered Reelin expression and processing in Alzheimer's disease

Reelin is a glycoprotein that modulates synaptic function and plasticity in the mature brain, thereby favouring memory formation. We recently reported altered cerebral Reelin expression in Alzheimer's disease (AD). Here we demonstrate pronounced Reelin changes at protein and mRNA levels in the frontal cortex in adult Down's syndrome (DS), where the extra copy of chromosome 21 leads to overexpression of β-amyloid. In cortical extracts of fetal DS samples we detected increased levels of the full-length Reelin and the 310-kDa fragment. Overexpression of mutant human amyloid precursor protein also led to an increase in levels of Reelin fragments in Tg2576 transgenic mice for human β-amyloid. Finally, in vitro Aβ42 treatment of SH-SY5Y neuroblastoma cells led to increased Reelin levels. An altered pattern of Reelin glycosylation was detected in extracts from the frontal cortex of AD patients and in Aβ42-treated SH-SY5Y cells, supporting the notion that β-amyloid triggers altered Reelin processing. These results provide evidence that Reelin expression and processing is altered in several amyloid conditions.     

Peripheral reduction of β-amyloid is sufficient to reduce brain β-amyloid: implications for Alzheimer's disease

Three loci that modify β-amyloid (Aβ) accumulation and deposition in the brains of a mouse model of Alzheimer's disease have been previously described. One encompasses the Psen2 gene encoding presenilin 2, a component of the γ-secretase activity responsible for generating Aβ by proteolysis. We show that the activity of mouse Psen2, as measured by levels of mRNA accumulation, unexpectedly is heritable in the liver but not the brain, suggesting liver as the origin of brain Aβ deposits. Administration of STI571, a cancer therapeutic that does not cross the blood-brain barrier, reduced accumulation of Aβ in both the blood and the brain, confirming brain Aβ's peripheral origin and suggesting that STI571 and related compounds might have therapeutic/prophylactic value in human Alzheimer's disease. The genes Cib1 and Zfhx1b reside within the other modifier loci and also exhibit heritable expression in the liver, suggesting that they too contribute to Aβ accumulation.     

Alzheimer's disease: β-amyloid plaque formation in human brain

Although the precise cause of Alzheimer's disease is not known, the β-amyloid peptide chains of 40-42 amino acids are suspected to contribute to the disease. The β-amyloid precursor protein is found on many types of cell membranes, and the action of secretases (β and γ) on this precursor protein normally releases the β-amyloids at a high rate into the plasma and the cerebrospinal fluid. However, the concentrations of the β-amyloids in the plasma and the spinal fluid vary considerably between laboratories. The β-amyloids adsorb in the nanomolar concentration range to receptors on neuronal and glial cells. The β-amyloids are internalized, become folded in the β-folded or β-pleated shape, and then stack on each other to form long fibrils and aggregates known as plaques. The β-amyloids likely act as monomers, dimers, or multimers on cell membranes to interfere with neurotransmission and memory before the plaques build up. Treatment strategies include inhibitors of β- and γ-secretase, as well as drugs and physiological compounds to prevent aggregation of the amyloids. Several immune approaches and a cholesterol-lowering strategy are also being tested to remove the β-amyloids.     

Impaired β-amyloid secretion in Alzheimer's disease pathogenesis

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

Short amyloid-β immunogens with spacer-enhanced immunogenicity without junctional epitopes for Alzheimer's disease immunotherapy

Induction of an immune response to amyloid-β (Aβ) protein is effective in treating animal models of Alzheimer's disease. The Aβ1-15 sequence contains the antibody epitope(s), but lacks the T-cell reactive sites of full-length Aβ1-42. We tested two alternative peptide immunogens encompassing either a tandem repeat of GPGPG-linked Aβ1-15 sequences (2Aβ15-linker) or a tandem repeat Aβ1-15 without the spacer sequence (2Aβ15). Titers of the immunized sera were measured by indirect ELISA. We analyzed the production of interferon-γ and interleukin-4 cytokine by lymphocytes and CD4 T-cells using ELISPOT and FACS assays; we then measured CD4 T-cell proliferation using a CFSE-based lymphoproliferation assay. Immunization with 2Aβ15-linker resulted in a high anti-Aβ titer of the noninflammatory T-helper 2 isotype, a lack of lymphocyte proliferation against the spacer part peptide. We observed much lower titers against the Aβ protein after immunization with 2Aβ15. Restimulation of lymphocytes with the corresponding immunogens resulted in proliferative responses, which showed that the sequential arrangement of the epitopes created junctional epitopes. The disruption of junctional epitopes through the introduction of a GPGPG spacer restored the immunogenicity against all the epitopes. Our novel immunogen with spacer may be a safer alternative to a peptide-based vaccine.     

Spatial patterns of β-amyloid (Aβ) deposits in familial and sporadic Alzheimer's disease

The spatial patterns of the diffuse, primitive, and classic β -amyloid (Aβ) deposits were compared in cortical regions in early-onset familial Alzheimer's disease (EO-FAD) linked to mutations of the amyloid precursor protein APP) or presenilin 1 (PSEN1) genes, late-onset familial AD (LO-FAD), and sporadic AD (SAD). The objective was to determine whether genetic factors influenced the spatial patterns of the A β deposits. A β deposits were distributed either in clusters which were regularly distributed parallel to the pia mater or in larger, non-regularly distributed clusters. There were no significant differences in spatial pattern of the diffuse deposits between patient groups but mean cluster size of the diffuse deposits was larger in FAD compared with SAD. Primitive A β deposits were more frequently distributed in regular clusters and less frequently distributed in large clusters in FAD compared with SAD. Classic A β deposits were more frequently distributed in regularly spaced clusters and less frequently distributed in large clusters in LO-FAD compared with EO-FAD. There were no significant differences in the spatial patterns or cluster sizes of A β deposits in cases classified according to apolipoprotein E (APOE) genotype. These results suggest (1) greater deposition of A β in the form of clusters of diffuse deposits in FAD, (2) a greater proportion of diffuse deposits may be converted to primitive deposits in SAD, (3) classic deposits are more widely distributed in EO-FAD, and (4) the presence of APOE allele ε4 has little effect on the spatial patterns of A β deposits.     

Neuropathology after active Aβ42 immunotherapy: implications for Alzheimer’s disease pathogenesis

The amyloid cascade hypothesis of Alzheimer’s disease (AD) is testable: it implies that interference with Aβ aggregation and plaque formation may be therapeutically useful. Aβ42 immunisation of amyloid precursor protein (APP) transgenic mice prevented plaque formation and caused removal of existing plaques. The first clinical studies of Aβ immunisation in AD patients (AN1792, Elan Pharmaceuticals) were halted when some patients suffered side effects. Since our confirmation that Aβ immunisation can prompt plaque removal in human AD, we have performed a clinical and neuropathological follow up of AD patients in the initial Elan Aβ immunisation trial. In immunised AD patients, we found: a lower Aβ load, with evidence that plaques had been removed; a reduced tau load in neuronal processes, but not in cell bodies; and no evidence of a beneficial effect on synapses. There were pathological “side effects” including: increased microglial activation; increased cerebral amyloid angiopathy; and there is some evidence for increased soluble/oligomeric Aβ. A pathophysiological mechanism involving effects on the cerebral vasculature is proposed for the clinical side effects observed with some active and passive vaccine protocols. Our current knowledge of the effects of Aβ immunotherapy is based on functional information from the early clinical trials and a few post mortem cases. Several further clinical studies are underway using a variety of protocols and important clinical, imaging and neuropathological data will become available in the near future. The information obtained will be important in helping to understand the pathogenesis not only of AD but also of other neurodegenerative disorders associated with protein aggregation.     

Aβ immunotherapy for Alzheimer’s disease: effects on apoE and cerebral vasculopathy

Aβ immunotherapy for Alzheimer’s disease (AD) results in the removal of Aβ plaques and increased cerebral amyloid angiopathy (CAA). In current clinical trials, amyloid-related imaging abnormalities (ARIAs), putatively due to exacerbation of CAA, are concerning side effects. We aimed to assess the role of the Aβ transporter apolipoprotein E (apoE) in the exacerbation of CAA and development of CAA-associated vasculopathy after Aβ immunotherapy. 12 Aβ42-immunized AD (iAD; AN1792, Elan Pharmaceuticals) cases were compared with 28 unimmunized AD (cAD) cases. Immunohistochemistry was quantified for Aβ42, apoE, apoE E4 and smooth muscle actin, and CAA-associated vasculopathy was analyzed. Aβ immunotherapy was associated with redistribution of apoE from cortical plaques to cerebral vessel walls, mirroring the altered distribution of Aβ42. Concentric vessel wall splitting was increased threefold in leptomeningeal vessels after immunotherapy (cAD 6.3 vs iAD 20.6 %, P < 0.001), but smooth muscle cell abnormalities did not differ. The findings suggest that apoE is involved in the removal of plaques and transport of Aβ to the cerebral vasculature induced by Aβ immunotherapy. Immunotherapy was not associated with CAA-related vascular smooth muscle damage, but was accompanied by increased splitting of the vessel wall, perhaps reflecting enhanced deposition and subsequent removal of Aβ. ARIA occurring in some current trials of Aβ immunotherapy may reflect an extreme form of these vascular changes.     

Future directions for immune modulation in neurodegenerative disorders: focus on Parkinson’s disease

One common feature of neurodegenerative diseases is neuroinflammation. In the case of Parkinson’s disease (PD), neuroinflammation appears early and persists throughout the disease course. The principal cellular mediator of brain inflammation is the resident microglia which share many features with related hematopoietically derived macrophages. Microglia can become activated by misfolded proteins including the PD relevant example, α-synuclein, a presynaptic protein. When activated, microglia release pro-inflammatory diffusible mediators that promote dysfunction and contribute to the death of the PD vulnerable dopaminergic neurons in the midbrain. Recently, the orphan nuclear receptor Nurr1, well known as a critical determinant in dopaminergic neuron maturation, has been ascribed two new properties. First, it promotes the production and release of the neuropeptide vasoactive intestinal peptide that functions both to stimulate dopaminergic neuron survival and inhibit neuroinflammation. Second, Nurr1 suppresses the expression and release of pro-inflammatory cytokines in glial cells. Herein, we discuss these new findings in context of strategies to attenuate neuroinflammation in PD.     

Exosomes: a novel therapeutic target for Alzheimer's disease?

Extracellular exosomes are formed inside the cytoplasm of cells in compartments known as multivesicular bodies. Thus, exosomes contain cytoplasmic content. Multivesicular bodies fuse with the plasma membrane and release exosomes into the extracellular environment. Comprehensive research suggests that exosomes act as both inflammatory intermediaries and critical inducers of oxidative stress to drive progression of Alzheimer's disease. An important role of exosomes in Alzheimer's disease includes the formation of neurofibrillary tangles and beta-amyloid production, clearance, and accumulation. In addition, exosomes are involved in neuroinflammation and oxidative stress, which both act as triggers for beta-amyloid pathogenesis and tau hyperphosphorylation. Further, it has been shown that exosomes are strongly associated with beta-amyloid clearance. Thus, effective measures for regulating exosome metabolism may be novel drug targets for Alzheimer's disease.     

Young blood products:emerging treatment for Alzheimer's disease?

Alzheimer's disease is the most common neurodegenerative disorder and no disease-modifying treatment is currently available.Research has shown that while brain neurogenesis continues in adult life,it declines with age.Using parabiosis,plasma transfusions and direct administration of neural growth factors,animal studies have demonstrated the positive impact of exposure to young blood products on neurogenesis and synaptic plasticity in an aging brain.The hippocampus and the sub-ventricular zones were identified as the main regions affected.Promising findings have prompted researchers to experiment their effects in subjects with an established neurocognitive disorder,such as Alzheimer's disease.They argued that modification of brain vasculature,reactivation of adult neural stem cells,and remodeling of their synaptic activity/plasticity may lead to cognitive enhancement and increased neurogenesis.One pilot human study found that young donor plasma infusion protocols for adults with Alzheimer's disease were safe and feasible;however,no statistically significant improvements in cognition were detected.There is a need to conduct additional placebo-controlled human studies in larger samples.Future studies should focus on identifying an optimal age at which an intervention in humans may yield significant cognitive enhancement,as well as determining the types of transfusions with the best efficacy and tolerability profiles.     

Statin therapy for Alzheimer's disease: will it work?

Disease-modifying therapies are being developed for Alzheimer's disease (AD). These are expected to slow the clinical progression of the disease or delay its onset. Cerebral accumulation of amyloid beta (A beta) peptides is an early and perhaps necessary event for establishing AD pathology. Consequently therapies aimed at attenuating brain amyloidosis are expected to be disease modifying. Based on the epidemiological evidence pointing to a link between cholesterol metabolism and AD and the numerous laboratory studies implicating cholesterol in the process of A beta production and accumulation, it is now believed that cholesterol-lowering therapies will be of value as disease modifying agents. Several epidemiological studies revealed that statin use for the treatment of coronary arterial disease is associated with a decreased prevalence or a decreased risk of developing AD. These observations require both preclinical and clinical validation. The former involves testing statins in one or more animal models of AD in order to establish which disease features are affected by statin treatment, the relative efficacy with which different statins modify these features and the mechanism(s) by which statins affect AD phenotypes. The latter requires prospective, randomized, placebo controlled trials to evaluate the effect of statin treatment on cognitive and AD biomarker outcomes. We have initiated a study aimed at determining the effects of atorvastatin (Lipitor), a statin with the largest US market share, on brain A beta deposition in the PSAPP transgenic mouse model of Alzheimer's amyloidosis. Our results indicate that Lipitor treatment markedly attenuates A beta deposition in this animal model.     

Monitoring pathological assembly of tau and β-amyloid proteins in Alzheimer's disease

This double-labelling confocal microscopy study of the neuropathology of Alzheimer's disease (AD) reports the use of a fluorescent dye, thiazin red, which has staining properties similar to thioflavin-S. Thiazin red fluorescence can be visualised selectively in the red channel, and we have used this property to compare it with the labelling seen using monoclonal antibody (mAb) 423, which detects tau protein C-terminally truncated at Glu-391, and mAb 4G8, which detects -amyloid protein. Thiazin red is shown to recognized the typical histopathological deposits associated with both proteins. However, not all deposits containing these proteins are stained. Specifically, diffuse -amyloid plaques and severely degraded extracellular tangles are unlabelled. Likewise a characteristic mAb 423-reactive granular plaque-like structure, typically present in cases with abundant extracellular tangels, is unlabelled by thiazin red. Such plaques can be shown to be continuous with the basal dendrites of degraded tanglebearing pyramidal cells. These findings suggest that paired helical filaments (PHFs) continue to undergo degradation in the extracellular space, which is associated with loss of thiazin red binding sites, but preservation of mAb 423 immunoreactivity. This epitope appears to be characteristic of a stable core element of the PHF which is highly resistant to proteolysis. Compounds such as thiazin red with high affinity for -pleated protein structures can be used to monitor the state of pathological assembly of amyloidogenic protein species found in AD.Supported in part by CONACyT grant #1624-N9208 (to R.M.), the Medical Research Council (U.K.), Zeneca Pharmaceuticals and the Alzheimer Disease Research Fund and the Leopold Muller Estate