What determines the molecular composition of abnormal protein aggregates in neurodegenerative disease?

Abnormal protein aggregates, in the form of either extracellular plaques or intracellular inclusions, are an important pathological feature of the majority of neurodegenerative disorders. The major molecular constituents of these lesions, viz., beta-amyloid (Abeta), tau, and alpha-synuclein, have played a defining role in the diagnosis and classification of disease and in studies of pathogenesis. The molecular composition of a protein aggregate, however, is often complex and could be the direct or indirect consequence of a pathogenic gene mutation, be the result of cell degeneration, or reflect the acquisition of new substances by diffusion and molecular binding to existing proteins. This review examines the molecular composition of the major protein aggregates found in the neurodegenerative diseases including the Abeta and prion protein (PrP) plaques found in Alzheimer's disease (AD) and prion disease, respectively, and the cellular inclusions found in the tauopathies and synucleinopathies. The data suggest that the molecular constituents of a protein aggregate do not directly cause cell death but are largely the consequence of cell degeneration or are acquired during the disease process. These findings are discussed in relation to diagnosis and to studies of to disease pathogenesis.

Aluminium and Alzheimer's disease: review of possible pathogenic mechanisms

Chronic exposure to aluminium (Al) remains a controversial possible cause of sporadic forms of Alzheimer's disease (AD). This article reviews the evidence that once Al enters the brain and individual brain cells, it may be involved in three pathological processes: (1) the production of abnormal forms of tau leading to the formation of cellular neurofibrillary tangles and neuropil threads; (2) the processing of the amyloid precursor protein, resulting in the formation of beta-amyloid deposits and senile plaques, and (3) that via the mutual histocompatibility system, Al could be involved in the initiation of the immune response observed in AD patients. Despite recent evidence that Al could be involved in these processes, a conclusive case that exposure to Al initiates the primary pathological process in sporadic AD remains to be established.

Hypothesis: is Alzheimer's disease a metal-induced immune disorder?

A hypothesis that a metal-induced immune disorder may be involved in the pathogenesis of some forms of Alzheimer's disease (AD) is presented. The classical complement pathway is activated in AD and T cells and reactive microglia appear in the brain. Studies of metal induced autoimmunity and the use of compounds containing aluminium as vaccine adjuvants suggest that metals can activate complement and can be taken up by antigen presenting cells. The consequent immune response could contribute to neuronal damage, beta-amyloid deposition and cell death. The strengths and weaknesses of this hypothesis are discussed and tests of some aspects are proposed.

The levels of neopterin, biopterin and the neopterin/biopterin ratio in urine from control subjects and patients with Alzheimer’s disease and Down’s syndrome

The levels of neopterin, biopterin and the neopterin/biopterin ratio (N/B) were measured in urine samples taken from normal young and elderly control subjects, exceptionally healthy elderly control subjects classified according to the ‘Senieur’ protocol and patients with Down’s syndrome (DS) or Alzheimer’s disease (AD). The N/B ratio was approximately unity in control groups with the exception of the normal elderly controls. The levels of neopterin and biopterin declined with age in the exceptionally healthy ‘Senieur’ control group. The N/B ratio was elevated in young and old DS patients as a result of the significant increase in neopterin. Neopterin levels were significantly elevated in AD patients compared with the healthy elderly controls, but this did not result in a significant increase in the N/B ratio in these patients. The N/B ratio increased with age in AD patients as a result of a decline in biopterin. These results suggested that there is a cellular immune reponse in DS and AD patients which in DS, may precede the formation of beta-amyloid deposits in the brain. In addition, there may be a deficiency in tetrahydrobiopterin biosynthesis in AD which becomes more marked with age.

The molecular biology of senile plaques and neurofibrillary tangles in Alzheimer’s disease

Since the earliest descriptions of the disease, senile plaques (SP) and neurofibrillary tangles (NFT) have been regarded as the pathological 'hallmarks' of Alzheimer's disease (AD). Whether or not SP and NFT are sufficient cause to explain the neurodegeneration of AD is controversial. The major molecular constituents of these lesions, viz., beta-amyloid (Ass) and tau, have played a defining role both in the diagnosis of the disease and in studies of pathogenesis. The molecular biology of SP and NFT, however, is complex with many chemical constituents. An individual constituent could be the residue of a pathogenic gene mutation, result from cellular degeneration, or reflect the acquisition of new proteins by diffusion and molecular binding. This review proposes that the molecular composition of SP and NFT is largely a consequence of cell degeneration and the later acquisition of proteins. Such a conclusion has implications both for the diagnosis of AD and in studies of disease pathogenesis.

Is glutathione an important neuroprotective effector molecule against amyloid beta toxicity?

Cellular thiols are critical moieties in signal transduction, regulation of gene expression, and ultimately are determinants of specific protein activity. Whilst protein bound thiols are the critical effector molecules, low molecular weight thiols, such as glutathione, play a central role in cytoprotection through (1) direct consumption of oxidants, (2) regeneration of protein thiols and (3) export of glutathione containing mixed disulphides. The brain is particularly vulnerable to oxidative stress, as it consumes 20% of oxygen load, contains high concentrations of polyunsaturated fatty acids and iron in certain regions, and expresses low concentrations of enzymic antioxidants. There is substantial evidence for a role for oxidative stress in neurodegenerative disease, where excitotoxic, redox cycling and mitochondrial dysfunction have been postulated to contribute to the enhanced oxidative load. Others have suggested that loss of important trophic factors may underlie neurodegeneration. However, the two are not mutually exclusive; using cell based model systems, low molecular weight antioxidants have been shown to play an important neuroprotective role in vitro, where neurotrophic factors have been suggested to modulate glutathione levels. Glutathione levels are regulated by substrate availability, synthetic enzyme and metabolic enzyme activity, and by the presence of other antioxidants, which according to the redox potential, consume or regenerate GSH from its oxidised partner. Therefore we have investigated the hypothesis that amyloid beta neurotoxicity is mediated by reactive oxygen species, where trophic factor cytoprotection against oxidative stress is achieved through regulation of glutathione levels. Using PC12 cells as a model system, amyloid beta 25-35 caused a shift in DCF fluorescence after four hours in culture. This fluorescence shift was attenuated by both desferioxamine and NGF. After four hours, cellular glutathione levels were depleted by as much as 75%, however, 24 hours following oxidant exposure, glutathione concentration was restored to twice the concentration seen in controls. NGF prevented both the loss of viability seen after 24 hours amyloid beta treatment and also protected glutathione levels. NGF decreased the total cellular glutathione concentration but did not affect expression of GCS. In conclusion, loss of glutathione precedes cell death in PC12 cells. However, at sublethal doses the surviving fraction respond to oxidative stress by increasing glutathione levels, where this is achieved, at least in part, at the gene level through upregulation of GCS. Whilst NGF does protect against oxidative toxicity, this is not achieved through upregulation of GCS or glutathione.

Spatial distribution of diffuse, primitive, and classic amyloid-beta deposits and blood vessels in the upper laminae of the frontal cortex in Alzheimer disease

The spatial distribution of the diffuse, primitive, and classic amyloid-beta deposits was studied in the upper laminae of the superior frontal gyrus in cases of sporadic Alzheimer disease (AD). Amyloid-beta-stained tissue was counterstained with collagen IV to determine whether the spatial distribution of the amyloid-beta deposits along the cortex was related to blood vessels. In all patients, amyloid-beta deposits and blood vessels were aggregated into distinct clusters and in many patients, the clusters were distributed with a regular periodicity along the cortex. The clusters of diffuse and primitive deposits did not coincide with the clusters of blood vessels in most patients. However, the clusters of classic amyloid-beta deposits coincided with those of the large diameter (>10 microm) blood vessels in all patients and with clusters of small-diameter (< 10 microm) blood vessels in four patients. The data suggest that, of the amyloid-beta subtypes, the clusters of classic amyloid-beta deposits appear to be the most closely related to blood vessels and especially to the larger-diameter, vertically penetrating arterioles in the upper cortical laminae.

Laminar distribution of amyloid-beta (Abeta) deposits in dementia with Lewy bodies: comparison with Alzheimer's disease

Significant amyloid-beta (Abeta) deposition in cases of dementia with Lewy bodies (DLB) may represent concurrent Alzheimer's disease (AD). To test this hypothesis, the laminar distribution of the diffuse, primitive, and classic Abeta deposits was studied in the frontal and temporal cortex in cases of DLB and were compared with AD. In DLB, the diffuse and primitive deposits exhibited two common patterns of distribution; either maximum density occurred in the upper cortical laminae or a bimodal distribution was present with density peaks in the upper and lower laminae. In addition, a bimodal distribution of the classic deposits was observed in approximately half of the cortical areas analysed. A number of differences in the laminar distributions of Abeta deposits were observed in DLB and AD. First, the proportion of the primitive relative to the diffuse and classic deposits present was lower in DLB compared with AD. Second, the primitive deposits were more frequently bimodally distributed in DLB. Third, the density of the diffuse deposits reached a maximum lower in the cortical profile in AD. These data suggest differences in the pattern of cortical degeneration in the two disorders and therefore, DLB cases with significant Abeta pathology may not represent the coexistence of DLB and AD.

Oxidised LDL-lipids alter redox ratio, lipid raft formation and increase amyloid beta production by SHSY-5Y cells

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

Amyloid β 1-42 induces hypometabolism in human stem cell-derived neuron and astrocyte networks

Alzheimer's disease (AD) is the most common form of dementia, affecting more than 35 million people worldwide. Brain hypometabolism is a major feature of AD, appearing decades before cognitive decline and pathologic lesions. To date, the majority of studies on hypometabolism in AD have used transgenic animal models or imaging studies of the human brain. As it is almost impossible to validate these findings using human tissue, alternative models are required. In this study, we show that human stem cell-derived neuron and astrocyte cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose, pyruvate, lactate, and glutamate. In addition, a significant increase in the glycogen content of cells was also observed. These changes were accompanied by changes in NAD+ /NADH, ATP, and glutathione levels, suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. Further research using this model may elucidate the mechanisms associated with Aβ-induced hypometabolism.