Cora O'Neill

Defects in IGF-1 receptor, insulin receptor and IRS-1/2 in Alzheimer's disease indicate possible resistance to IGF-1 and insulin signalling

Insulin like growth factor-1 receptor (IGF-1R) and insulin receptor (IR) signalling control vital growth, survival and metabolic functions in the brain. Here we describe specific and significant alterations in IGF-1R, IR, and their key substrate adaptor proteins IRS-1 and IRS-2 in Alzheimers disease (AD). Western immunoblot analysis detected increased IGF-1R levels, and decreased levels of IGF-1-binding protein-2 (IGFBP-2), a major IGF-1-binding protein, in AD temporal cortex. Increased IGF-1R was observed surrounding and within amyloid-beta (Abeta)-containing plaques, also evident in an animal model of AD, and in astrocytes in AD. However, despite the overall increase in IGF-1R levels, a significantly lower number of neurons expressed IGF-1R in AD, and IGF-1R was aberrantly distributed in AD neurons especially evident in those with neurofibrillary tangles (NFTs). IR protein levels were similar in AD and control cases, however, the IR was concentrated intracellularly in AD neurons, unlike its distribution throughout the neuronal cell soma and in dendrites in control brain. Significant decreases in IRS-1 and IRS-2 levels were identified in AD neurons, in association with increased levels of inactivated phospho(Ser312)IRS-1 and phospho(Ser616)IRS-1, where increased levels of these phosphoserine epitopes colocalised strongly with NFTs. Our results show that IGF-1R and IR signalling is compromised in AD neurons and suggest that neurons that degenerate in AD may be resistant to IGF-1R/IR signalling.

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology

Studies suggest that activation of phosphoinositide 3‐kinase‐Akt may protect against neuronal cell death in Alzheimers disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho‐Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated‐Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated‐Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3βSer9, tauSer214, mTORSer2448, and decreased levels of the Akt target, p27kip1, were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated‐Akt and PTEN‐containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.

Oxidative stress and apoptosis in neurodegeneration

The pathogenesis of neurodegenerative diseases such as Parkinsons diseases, amyotrophic lateral sclerosis and Alzheimers disease is unknown. These diseases are characterized by a slow, progressive loss of particular subsets of neurons. Much evidence has accumulated which supports the hypothesis that oxidative stress and damage by free radicals may play an important part in these diseases. In particular recent studies with the inherited form of amyotrophic lateral sclerosis have revealed mutations in the superoxide dismutase gene, which is one of the cells main defence mechanisms against oxidative stress. These findings suggest a direct link between oxidative stress and the development of a neurodegenerative disease.

Alterations in the ryanodine receptor calcium release channel correlate with Alzheimer's disease neurofibrillary and β-amyloid pathologies

Investigation of the integrity of the ryanodine receptor in Alzheimers disease is important because it plays a critical role in the regulation of calcium release from the endoplasmic reticulum in brain, impairment of which is believed to contribute to the pathogenesis of Alzheimers disease. The present study compared ryanodine receptor levels and their functional modulation in particulate fractions from control and Alzheimers disease temporal cortex, occipital cortex and putamen. Relationships between ryanodine receptor changes and the progression of Alzheimers disease pathology were determined by examining autoradiographic [3H]ryanodine binding in entorhinal cortex/anterior hippocampus sections from 22 cases that had been staged for neurofibrillary changes and beta-amyloid deposition. A significant (P < 0.02) 40% decrease in the Bmax for [3H]ryanodine binding and significantly higher IC50 values for both magnesium and Ruthenium Red inhibition of [3H]ryanodine binding were detected in Alzheimers disease temporal cortex particulate fractions compared to controls. Immunoblot analyses showed Type 2 ryanodine receptor holoprotein levels to be decreased by 20% (P < 0.05) in these Alzheimers disease cases compared to controls. No significant differences were detected in [3H]ryanodine binding comparing control and Alzheimers disease occipital cortex or putamen samples. The autoradiography study detected increased [3H]ryanodine binding in the subiculum, CA2 and CA1 regions in cases with early (stage I-II) neurofibrillary pathology when compared to Stage 0 cases. Analysis of variance of data with respect to the different stages of neurofibrillary pathology revealed significant stage-related declines of [3H]ryanodine binding in the subiculum (P < 0.02) with trends towards significant decreases in CA1, CA2 and CA4. Post-hoc testing with Fishers PLSD showed significant reductions (74-94%) of [3H]ryanodine binding in the subiculum, and CA1-CA4 regions of the late isocortical stage (V-VI) cases compared to the early entorhinal stage I-II cases. [3H]Ryanodine binding also showed significant declines with staging for beta-amyloid deposition in the entorhinal cortex (P < 0.01) and CA4 (P < 0.05) with trends towards a significant decrease in the dentate gyrus. We conclude that alterations in ryanodine receptor binding and function are very early events in the pathogenesis of Alzheimers disease, and may be fundamental to the progression of both neurofibrillary and beta-amyloid pathologies.

Increased β-amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimer's disease APP670/671 mutation

Cell lines transfected with the Swedish Alzheimers disease amyloid precursor protein APP670/671 mutation release significantly more β‐amyloid than wild‐type cells. Citron et al. [Proc. Natl. Acad. Sci. USA (1994) in press] have recently shown that fibroblasts carrying the APP670/671 mutation also release more β‐amyloid than control cells [1]. The present study confirms a ca. threefold increase in β‐amyloid release from mutation‐bearing fibroblasts. APP mRNA levels did not differ between mutation‐bearing and control cells, although mutation‐bearing fibroblasts contained significantly more APP751/770 than controls. Mild stress decreased β‐amyloid secretion and increased APP751/770 levels in all cell lines. In conclusion, the proportion of APP committed to amyloidogenic processing is increased in fibroblasts from family members with the APP670/671 mutation, and this mutation may also compromise the APP stress response.

Insulin and IGF-1 signalling: longevity, protein homoeostasis and Alzheimer's disease.

The quality control of protein homoeostasis deteriorates with aging, causing the accumulation of misfolded proteins and neurodegeneration. Thus, in AD (Alzheimers disease), soluble oligomers, protofibrils and fibrils of the Aβ (amyloid β-peptide) and tau protein accumulate in specific brain regions. This is associated with the progressive destruction of synaptic circuits controlling memory and higher mental function. The primary signalling mechanisms that (i) become defective in AD to alter the normal proteostasis of Aβ and tau, and (ii) initiate a pathophysiological response to cause cognitive decline, are unclear. The IIS [insulin/IGF-1 (insulin-like growth factor 1)-like signalling] pathway is mechanistically linked to longevity, protein homoeostasis, learning and memory, and is emerging to be central to both (i) and (ii). This pathway is aberrantly overactivated in AD brain at the level of increased activation of the serine/threonine kinase Akt and the phosphorylation of its downstream targets, including mTOR (mammalian target of rapamycin). Feedback inhibition of normal insulin/IGF activation of the pathway also occurs in AD due to inactivation of IRS-1 (insulin receptor substrate 1) and decreased IRS-1/2 levels. Pathogenic forms of Aβ may induce aberrant sustained activation of the PI3K (phosphoinositide 3-kinase)/Akt signal in AD, also causing non-responsive insulin and IGF-1 receptor, and altered tau phosphorylation, conformation and function. Reducing IIS activity in animal models by decreasing IGF-1R levels or inhibiting mTOR activity alters Aβ and tau protein homoeostasis towards less toxic protein conformations, improves cognitive function and extends healthy lifespan. Thus normalizing IIS dysfunction may be therapeutically relevant in abrogating Aβ and tau proteotoxicity, synaptic dysfunction and cognitive decline in AD.

Adenylyl Cyclase Activity in Postmortem Human Brain: Evidence of Altered G Protein Mediation in Alzheimer's Disease

Abstract: The effects of agonal status, postmortem delay, and age on human brain adenylyl cyclase activity were determined in membrane preparations of frontal cortex from a series of 18 nondemented subjects who had died with no history of neurological or psychiatric disease. Basal and guanosine 5′‐O‐(3‐thiotriphosphate)‐, aluminum fluoride‐, and forskolin‐stimulated enzyme activities were not significantly reduced over an interval from death to postmortem of between 3 and 37 h and were also not significantly different between individuals dying with a long terminal phase of an illness and those dying suddenly. Basal and aluminum fluoride‐stimulated enzyme activities showed a negative correlation with increasing age of the individual. In subsequent experiments, basal and guanosine 5′‐O‐(3‐thiotriphosphate)‐, aluminum fluoride‐, and forskolin‐stimulated enzyme activities were compared in five brain regions from a series of eight Alzheimers disease and seven matched nondemented control subjects. No significant differences were observed between the groups for either basal activity or activities in response to forskolin stimulation of the catalytic subunit of the enzyme. In contrast, enzyme activities in response to stimulation with guanosine 5′‐O‐(3‐thiotriphosphate) and aluminum fluoride were significantly reduced in preparations of neocortex and cerebellum from the Alzheimers disease cases compared with the nondemented controls. Lower guanosine 5′‐O‐(3‐thiotriphosphate)‐, but not aluminum fluoride‐, stimulated activity was also observed in preparations of frontal cortex from a group of four disease controls compared with nondemented control values. The disease control group, which contained Parkinsons disease and progressive supranuclear palsy patients, showed increased forskolin‐stimulated activity compared with both the nondemented control and the Alzheimers disease groups. These findings indicate a widespread impairment of G protein‐stimulated adenylyl cyclase activity in Alzheimers disease brain, which occurs in the absence of altered enzyme catalytic activity and which is unlikely to be the result of non‐disease‐related factors associated with the nature of terminal illness of individuals.

Regionally selective alterations in G protein subunit levels in the Alzheimer's disease brain

In the present study the relative densities of a number of G protein subunits were quantified in membranes prepared from the hippocampus, temporal cortex and angular gyrus of Alzheimers disease and control post-mortem brain by immunoblotting with specific polyclonal antisera against Gs alpha, Gi alpha, Gi alpha-1, G(o) alpha and G beta protein subunits. In addition, basal, Gs-stimulated and Gi-inhibited adenylyl cyclase activities were measured in the same hippocampal membrane samples. Densitometric analysis of the immunoblot data revealed a 58% reduction in the levels of Gi alpha, and a 75% reduction in the levels of Gi alpha-1, in the Alzheimers disease temporal cortex. Gi alpha levels were reduced, by 37% in the angular gyrus of the Alzheimers disease cases. The ratio of large to small molecular weight isoforms of the Gs alpha subunit was significantly increased in both the hippocampus and the angular gyrus of the Alzheimers disease samples when compared to control values, although the difference in individual Gs alpha isoform levels did not attain statistical significance when comparing groups. No statistically significant differences were observed in G(o) alpha or G beta levels when comparing control and Alzheimers disease cases. Gs-stimulated adenylyl cyclase activity was significantly reduced in the Alzheimers disease samples compared to controls, whereas Gi-inhibited adenylyl cyclase activity was unchanged. No significant differences were observed between the control and Alzheimers disease samples for either basal or forskolin stimulated adenylyl cyclase activity. The ratio of hippocampal Gs-stimulated to basal adenylyl cyclase activity correlated significantly with the large to small Gs alpha subunit ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

FKBP12 associates tightly with the skeletal muscle type 1 ryanodine receptor, but not with other intracellular calcium release channels

This study compared the relative levels of ryanodine receptor (RyR) isoforms, inositol 1,4,5‐trisphosphate receptor (IP3R) isoforms, and calcineurin, plus their association with FKBP12 in brain, skeletal and cardiac tissue. FKBP12 demonstrated a very tight, high affinity association with skeletal muscle microsomes, which was displaced by FK506. In contrast, FKBP12 was not tightly associated with brain or cardiac microsomes and did not require FK506 for removal from these organelles. Furthermore, of the proteins solubilised from skeletal muscle, cardiac muscle and brain microsomes, only skeletal muscle RyR1 bound to an FKBP12–glutathione‐S‐transferase fusion protein, in a high affinity FK506 displaceable manner. These results suggest that RyR1 has distinctive FKBP12 binding properties when compared to RyR2, RyR3, all IP3R isoforms and calcineurin.

The proteins BACE1 and BACE2 and β-secretase activity in normal and Alzheimer's disease brain

The insidious progression of AD (Alzheimers disease) is believed to be linked closely to the production, accumulation and aggregation of the approximately 4.5 kDa protein fragment called Abeta (amyloid beta-peptide). Abeta is produced by sequential cleavage of the amyloid precursor protein by two enzymes referred to as beta- and gamma-secretase. beta-Secretase is of central importance, as it catalyses the rate-limiting step in the production of Abeta and was identified 7 years ago as BACE1 (beta-site APP-cleaving enzyme 1). Soon afterwards, its homologue BACE2 was discovered, and both proteins represent a new subclass of the aspartyl protease family. Studies examining the regulation and function of beta-secretase in the normal and AD brain are central to the understanding of excessive production of Abeta in AD, and in targeting and normalizing this beta-secretase process if it has gone awry in the disease. Several reports indicate this, showing increased beta-secretase activity in AD, with recent findings by our group showing changes in beta-secretase enzyme kinetics in AD brain caused by an increased V(max). This article gives a brief review of studies which have examined BACE1 protein levels and beta-secretase activity in control and AD brain, considering further the expression of BACE2 in the human brain.