Cora O’Neill

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.

PI3-kinase/Akt/mTOR signaling: Impaired on/off switches in aging, cognitive decline and Alzheimer's disease

The normal on and off switching of the PI3-K (phosphoinositide 3-kinase)/Akt pathway, particularly by its major activators insulin and IGF-1 (insulin-like growth factor-1), is a powerful integrator of physiological responses rudimentary to successful aging. This is highlighted by extensive studies showing that reducing, but not obliterating, activation of the PI3-K/Akt/mTOR signal, at several levels, can extend healthy lifespan in organisms from yeast to mammals. Moreover, aberrant control of the PI3-K/Akt axis is emerging to be a primary causative node in all major diseases of aging: cancer, type 2 diabetes mellitus (T2DM), heart disease and neurodegeneration. Aging is the major risk factor for AD, the most common dementia disorder. The integrated coordination of neuronal responses through the PI3-K/Akt pathway has significant functional impact on key events that go awry in Alzheimers disease (AD), including: synaptic plasticity, neuronal polarity, neurotransmission, proteostasis, use-dependent translation, metabolic control and stress responses including DNA repair. Investigation of the status of the PI3-K/Akt system in brains of individuals who have had AD shows aberrant and sustained activation of neuronal PI3-K/Akt/mTOR signaling to be an early feature of the disease. This is mechanistically linked to progressive desensitization of normal brain insulin and IGF-1 responses, aberrant proteostasis of Aβ and tau, synaptic loss and cognitive decline in the disease. Notably, concomitantly with feedback inhibition of insulin and IGF-1 responses, increased activation of the neuronal PI3-K/Akt/mTOR axis is a major candidate effector system for transmission of pathophysiological signals from Aβ to tau in the context of defects in synaptic transmission that lead to cognitive decline. Therapeutic approaches targeted at normalizing signaling through either the neuronal PI3-kinase/Akt/mTOR pathway or its activation by insulin and IGF-1 have been shown to be protective against the development of AD pathology and cognitive decline in animal models of AD and some of these therapies are entering clinical trials in patients with the disease.The normal on and off switching of the PI3-K (phosphoinositide 3-kinase)/Akt pathway, particularly by its major activators insulin and IGF-1 (insulin-like growth factor-1), is a powerful integrator of physiological responses rudimentary to successful aging. This is highlighted by extensive studies showing that reducing, but not obliterating, activation of the PI3-K/Akt/mTOR signal, at several levels, can extend healthy lifespan in organisms from yeast to mammals. Moreover, aberrant control of the PI3-K/Akt axis is emerging to be a primary causative node in all major diseases of aging: cancer, type 2 diabetes mellitus (T2DM), heart disease and neurodegeneration. Aging is the major risk factor for AD, the most common dementia disorder. The integrated coordination of neuronal responses through the PI3-K/Akt pathway has significant functional impact on key events that go awry in Alzheimers disease (AD), including: synaptic plasticity, neuronal polarity, neurotransmission, proteostasis, use-dependent translation, metabolic control and stress responses including DNA repair. Investigation of the status of the PI3-K/Akt system in brains of individuals who have had AD shows aberrant and sustained activation of neuronal PI3-K/Akt/mTOR signaling to be an early feature of the disease. This is mechanistically linked to progressive desensitization of normal brain insulin and IGF-1 responses, aberrant proteostasis of Aβ and tau, synaptic loss and cognitive decline in the disease. Notably, concomitantly with feedback inhibition of insulin and IGF-1 responses, increased activation of the neuronal PI3-K/Akt/mTOR axis is a major candidate effector system for transmission of pathophysiological signals from Aβ to tau in the context of defects in synaptic transmission that lead to cognitive decline. Therapeutic approaches targeted at normalizing signaling through either the neuronal PI3-kinase/Akt/mTOR pathway or its activation by insulin and IGF-1 have been shown to be protective against the development of AD pathology and cognitive decline in animal models of AD and some of these therapies are entering clinical trials in patients with the disease.

Altered β-secretase enzyme kinetics and levels of both BACE1 and BACE2 in the Alzheimer's disease brain

β‐Secretase is the rate limiting enzymatic activity in the production of amyloid‐β peptide, the primary component of senile plaque pathology in Alzheimers disease (AD). This study performed the first comparative analysis of β‐secretase enzyme kinetics in AD and control brain tissue. Results found V max values for β‐secretase to be significantly increased, and K m values unchanged in AD temporal cortex compared to matched control temporal cortex. The increased V max in AD cases, did not correlate with levels of BACE1, and decreased BACE1 and BACE2 levels correlated with the severity of neurofibrillary pathology (I–VI), and synaptic loss in AD. These results indicate that increased V max for β‐secretase is a feature of AD pathogenesis and this increase does not correlate directly with levels of BACE1, the principal β‐secretase in brain.

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.

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.