Aileen M. Moloney

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.

Akt signal transduction dysfunction in Parkinson's disease.

Significant attention has been drawn to the potential role of defective PI3-kinase-Akt (PKB) signalling in Parkinsons disease (PD) neurodegeneration and to the possibility that activation of Akt may provide neuroprotection in PD. However, little knowledge exists on the integrity of the Akt system in PD. Results of the present study show diminished levels of both total and active phospho(Ser473)-Akt in the brain in PD. This was evident by western blot analysis of midbrain fractions from PD compared to non-PD control brain, but more specifically by immunofluorescence microscopy of the substantia nigra pars compacta (SNpc). Here, double immunofluorescence microscopy found Akt and phospho(Ser473)-Akt to be expressed at high levels in tyrosine hydroxylase (TH) immunopositive dopaminergic neurons in control human brain. Selective loss of these neurons was accompanied by a marked decrease of Akt and phospho(Ser473)-Akt expression in the PD brain, however Akt and active phospho(Ser473)-Akt are still evident in degenerating dopaminergic neurons in the disease. This suggests that it may be possible to target neuronal Akt in advanced PD. Converse to the marked loss of neuronal Akt in PD, increased Akt and phospho(Ser473)-Akt levels were observed in small non-TH positive cells in PD SNpc, whose increased number and small nuclear size indicate they are glia. These findings implicate defective Akt as a putative signalling pathway linked to loss of dopaminergic neurons in PD.

O3-02-02

tion of pro-death signaling. Recent studies of human postmortem brains linked the molecular and pathological lesions in AD to major impairments in: 1) insulin and insulin-like growth factor (IGF) gene expression; 2) expression and function of the insulin and IGF receptors; 3) neuronal survival signaling; and 4) acetylcholine homeostasis, and showed each of these abnormalities increases with progression of AD. The co-existence of insulin/IGF deficiency and insulin/IGF resistance suggests that AD represents a brain-specific form of diabetes, i.e. Type 3 diabetes. We generated an experimental animal model in which intracerebral Streptozotocin (icSTZ) was used to deplete brain and not pancreatic insulin/IGF, and produce neurodegeneration that is similar to human AD. The ic-STZ-injected rats did not have hyperglycemia, and pancreatic architecture and insulin immunoreactivity were similar to control, yet their brains were reduced in size and exhibited neurodegeneration with cell loss, gliosis, and increased immunoreactivity for p53, activated glycogen synthase kinase 3 , phospho-tau, ubiquitin, and amyloid. Real time quantitative RT-PCR studies demonstrated that the ic-STZ-treated brains had significantly reduced expression of genes corresponding to neurons, oligodendroglia, and choline acetyltransferase, and increased expression of genes encoding glial fibrillary acidic protein, microglia-specific proteins, acetylcholinesterase, tau, and amyloid precursor protein. These abnormalities were associated with reduced expression of genes encoding insulin, IGF-II, insulin receptor, IGF-I receptor, and insulin receptor substrate-1, and reduced ligand binding to the insulin and IGF-II receptors. Further studies showed that treatment with peroxisome-proliferator activated receptor agonists effectively prevented the ic-STZ-induced Type 3 diabetes and preserved learning and memory. These results demonstrate that many of the characteristic features of AD-type neurodegeneration can be produced experimentally by selectively impairing insulin/IGF functions together with increasing oxidative stress, and support our hypothesis that AD represents a neuro-endocrine disorder associated with brain-specific perturbations in insulin and IGF signaling mechanisms, i.e. Type 3 diabetes. The results also suggest that early treatment with insulin sensitizer agent may prevent or reduce the severity of AD.

An RNAI screen for genes that modify human amyloid induced paralysis in caenorhabditis elegans facilitates functional analysis of candidate risk factors for Alzheimer's disease

promoting neurofibrillary pathology. Methods: In order to investigate the neuroprotective effects of Cerebrolysin on TAU pathology, a new model for neurofibrillary alterations was developed using somatic gene transfer with AAV2-mutTAU (P301L). The Thy1-mutant APP tg mice (3 m/o) received bilateral injections of AAV2-mutTAU or AAV-GFP, into the hippocampus. Results: After 3 months, compared to non-tg controls, in APP tg mice intra-hippocampal injections with AAV2-mutTAU resulted in localized increased accumulation of phosphorylated TAU and neurodegeneration. Compared with vehicle-treated controls, treatment with Cerebrolysin (3 months at 5ml/kg) resulted in a significant decrease in the levels of TAU phosphorylation at critical sites dependent on GSK3b and CDK5 activity in APP tg injected with AAV2-mutTAU. This was accompanied by amelioration of the neurodegenerative alterations in the hippocampus. Conclusions: This study supports the concept that Cerebrolysin neuroprotective effects might evolve reducing the phosphorylation of TAU at critical sites.

P4-251: Changes in Akt and IGF-1 receptor signaling relate to developing Abeta and tangle pathology in the triple transgenic model of Alzheimer's disease

attention on the effect of the APP670/671 so called Swedish mutation (APPswe) on spontaneous calcium oscillations in embryonic hippocampal neurons derived from an APPswe transgenic rat. Methods: Intracellular free calcium levels were imaged by confocal microscopy using the fluorescent dye Fluo-3AM. Results: APPswe neurons showed increased frequency and unaltered amplitude of spontaneous calcium oscillations as compared to wild-type neurons. When exposed to a hyperosmotic external buffer (50 mM sucrose) the frequency of oscillations was suppressed to a similar extent in both wild-type and transgenic neurons. However whereas the amplitude of the oscillations was also decreased in wild-type neurons, it was slightly increased in the APPswe transgenic neurons. After a 26-hour exposure to 50 mM sucrose the neurons were still oscillating. Hyperosmotic shrinkage, which can occur in a variety of pathophysiological conditions, has been shown to induce multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. Loss in cell viability after 26-hour exposure to the hyperosmotic medium, as shown by the MTT assay, was comparable in both sets of cultures. Conclusions: Our results suggest alterations in calcium signaling in APPswe hippocampal transgenic neurons, and different cellular adaptation mechanism to hyperosmolality as compared to wild-type neurons. We are continuing our analysis of other perturbations in calcium signaling caused by the APPswe mutation.

P1-421: Impaired IGF-1 receptor and insulin receptor signal transduction in Alzheimer’s disease

network, the specific modulation of which may have a functional significance for the development of AD. The C-terminal region of APP and in particular the motif 682YENPTY687 is a docking site for the PTB domain of Fe65, X11, JIP1 and JIP2, mDab1, Numb, ShcA and Grb2. The signaling activity underlined by these interactions is unknown. Objectives and Methods: We have previously shown that among all these interacting proteins Grb2 requires the specific tyrosine phosphorylation of tyr-682 of APP and that the amount of ShcA is significantly increased in AD brain as compared to control, thus suggesting a pathogenic correlation. To study in detail the interaction between APP and Grb2, and to analyze the signaling beneath this interaction we transfected APP/APLPs null MEF cells, and human H4 cells with vectors encoding for human APP695 wild type or mutated in each tyrosine (Y653F, Y682F, Y687A and double mutant YY682/687FA). Results: Here we show that APP695 and its C-terminal fragments (CTFs) interact with Grb2, that this interaction is dependent on the phosphorylation of tyr-682, and that the activity of MAP Kinase ERK1/2 is modulated by APP695. Evaluating the phosphorylation status of ERK1/2 in cells transfected either with APP wt or mutated in each tyrosine residue we observed that the overexpression of APP correlates with the activation of ERK1/2 and that tyr-682 mutation, which is essential for the interaction between APP and Grb2, diminishes ERK1/2 activation while tyr-653 or tyr-687 mutations do not influence ERK1/2 phosphorylation. Conclusions: In summary, our data suggest that APP may play a role in cell signaling, similarly to that previously reported for other tyrosine kinase receptors (TRKs), through a tightly regulated coupling with intracellular adaptors such as ShcA and Grb2 targeted to the phosphorylation of ERK1/2.

P4-153: Characterization of the AKT/PTEN signalling pathway, and related proteins DJ1 and PINK-1, in sporadic Parkinson’s disease and in Alzheimer’s disease

GSS multicentric plaques. As control, specimens obtained during neurosurgical procedures at the periphery of brain tumors were used. Conclusions: In numerous specimens of the GSS case we found a large number of enlarged multivesicular bodies, several times larger than those typically encountered in human brains. We conclude that such widespread presence of MVB may suggest involvement of a microautophagy process in this disorder.