Amyloid Beta Mediated Alterations to the Microtubule Associated Protein Tau

Distinguishing hallmarks of Alzheimer’s Disease include the accumulation of bothamyloid beta (Aβ) plaques and neurofibrillary tangles (NFT’s) in specific regions ofaffected brains. The aggregation of Aβ peptides into insoluble fibrils, the principalcomponents of plaques, involves the production of Aβ soluble oligomerintermediates. NFT’s are composed of insoluble aggregates of the microtubuleassociated protein tau. The amyloid cascade hypothesis suggests that Aβ solubleoligomers alter neuronal metabolism resulting in aberrant tau phosphorylation andtruncation, ultimately leading to the production of NFT’s and eventual neuronaldeath. In order to investigate specific effects of Aβ oligomers on tau, cultured rathippocampal neurons were treated with oligomerized Aβ and a time course ofbiochemical samples were harvested prior to, and during, the period of cell death.Multiple phosphorylation epitopes on tau were analyzed for altered abundance viawestern blotting using site and phospho-specific antibodies. Surprisingly, none ofthe 10 phospho epitopes analyzed revealed increased levels relative to controls eventhough altered signaling pathways suggest elevated cdk5 and GSK3β activity withinthe neurons. On the other hand, a prominent alteration to tau observed throughoutthe time course of Aβ treatment was fragmentation, as seen by the loss of the full-length tau and accumulation of lower molecular weight fragments. These fragmentsremained stable at later time points when significant levels of neuronal cell deathwas observed, as assayed by both metabolic function and membrane integrity.Fragmentation of tau was preceded by increased calpain and caspase proteaseactivity consistent with proteolytic action on tau. Taken together, these data indicatethat increased tau phosphorylation at the sites we analyzed does not correlate withneuronal cell death whereas there is good temporal correlation between thegeneration of tau fragments and decreased neuronal viability. Overall, tau proteolysismay be a key functional step in Aβ mediated cell death.

ABSTRACT:

Jack Reifert, DeeAnn Hartung, Stuart Feinstein

Neuroscience Research Institute and Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara CA 93106

Sustained Increases in Tau Phosphorylation is NotInduced by Aβ Oligomers

Aβ Oligomers Rapidly Increase Caspase 3/7and Calpain Activities

1) Within 30 minutes of Aβ oligomer treatment, hippocampal neurons display activated proteases such as caspase 3/7 and calpain. Calpain has been shown to cleave P35 into P25 as observed bywestern blotting after Aβ oligomer treatment in our experiments (Kusakawa et al. 2000).

2) Aβ oligomers dysregulates signaling cascades resulting in increased activity of tau targeting kinases within 1 hour. Amyloid beta treatment causes decreased levels of GSK3β phosphorylated at serine9 (an inactivating phosphorylation) suggesting increased GSK3β activity. P35 cleavage producing P25 suggests increased CDK5 activity with Aβ oligomer treatment (Cruz et. al. 2003). Together,these data imply Aβ oligomers increase activity of two tau targeting kinases.

3) Despite increased kinase activity, increased levels of tau phosphorylation is not observed in the time-course of Aβ oligomer treatment analyzed. Instead, tau degradation into low molecular weightfragments is the prominent tau alteration observed. Interestingly, no site and phospho-specific tau antibodies used detect these small tau fragments.

4) All the above events precede cell death as detected by either release of LDH into the media through a compromised membrane, or levels of ATP, and indicator of live healthy cells.

Taken together, these results highlight tau degradation by proteaes such as calpain and caspases as a prominent effect of amyloid beta treatment. Although tau targeting kinases demonstrate increasedactivity, tau phosphorylation is not dramatically increased on either the full length or the low molecular weight fragments. Overall, tau phosphorylation does not correlate well with cell death whereas taufragmentation strongly correlates to neuronal demise. Indeed, all of the phosho specific tau antibodies used give a good signal in healthy untreated controls. These data support the suggestion of atoxic tau fragment as a potential mediator of Aβ oligomer induced cell death (Cruz et. al. 2005).

Figure 3: Rapid protease activation upon treatment with Aβ oliogomers may be the principal mechanism leadingto tau degradation (figure 4). 15 DIV rat hippocampal neurons were treated with 10µM oligomerized Aβ for theindicated times, with and without a 1 hour pre-treatment with the respective protease inhibitor. Activity wasmeasured by the release of a luminescent signal upon substrate cleavage by the proteases. Relativeluminescence units (RLU’s) were normalized between live healthy cells (100%) and media alone (0%). Tauhas potential cleavage sites for both caspase 3/7 and calpain, suggesting a mechanism by which activatedproteases cleave tau into small fragments (as seen in figure 4). Graphs represent data from at least 2independent triplicate experiments. Error bars indicate SEM.

Aβ Oligomers Induce Rapid Tau Degradationand Production of Stable Tau Fragments

Supported by grants from the NIH (RO1 NS035010), Santa Barbara Cottage Hospital, and California Department of Public Health – Alzheimer’s Research Foundation (07-65802).

Program # 923

Aβ Oligomers Increase the Activity ofGSK3β and CDK5

Time Course With Aβ Oligomers @ 10µM Time Course + Aβ Dose Titration

Removal of Inactivating Phosphorylation on GSK3β Production of CDK5 Activating Subunit - P25

Figure 1: Oligomerized Aβ treatment rapidly activates GSK3β andCDK5 as indicated by reduced levels of GSK3β with inhibitingphosphorylation at serine 9 and the production of P25, anactivating subunit for CDK5. 15 DIV hippocampal neurons weretreated with 10µM oligomerized Aβ for the indicated times, andprotein was harvested and separated by SDS-PAGE. Afterprobing for GSK3β and phospho- GSK3β, or P35 and itscleavage product P25, results were normalized to GAPDHcontrol signals. Blots show representative data (n=3). The insertgraph on the left indicates the suggested relative activity ofGSK3β and CDK5 along with two survival kinases AKT andERK which were also analyzed by probing for activatingphosphorylations (data not shown).

Figure 2: Aβ oliogmers did not produce a sustained increase in tau phosphorylation at any of the 10 phospho-epitopesanalyzed. 15 DIV hippocampal neurons were treated with 10µM oligomerized Aβ for the indicated times, and protein washarvested and separated by SDS-PAGE. After probing for and site specific phospho-tau, or non-phospho specific controltau antibodies such as tau-5 or pantau, results were normalized to GAPDH signals. Other housekeeping signals measuredinclude tubulin (DM1A) and βIII tubulin. Blots show representative data for select phosphorylation sites on tau and thegraph indicates the fold expression for all the phospo-epitopes analyzed after normalization to GAPDH (n=3). Specificity ofall antibodies is demonstrated by lack of signal in samples prepared from tau knockout (KO) mouse brains (a kind gift fromHana Dawson). Untreated (UN) samples are indicated showing robust detection of phospho-tau in healthy neurons.

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Figure 4: Rapid tau degradation is a prominent feature following treatment with Aβ oligomers producing lowmolecular weight tau fragments. 15 DIV hippocampal neurons were treated with 10µM oligomerizedamyloid beta for the indicated times, and protein was harvested and separated by SDS-PAGE. Afterprobing for tau antibodies tau-5, tau-1 and pantau, results were normalized to GAPDH control signals.Blots show representative data and the graph indicates the fold expression for the pantau signalintensity (n=3). Note the tau-1 immunoblot shows increased signal through the 1 hour time-point, andaccumulation of low molecular weight fragments that give a dramatic signal after 24 hours of treatment.

No Significant Cell Death Occurs Until 24Hours of Oligomeric Aβ Treatment

Figure 4:. The CytoTox-ONE™ assay was performed using media supernatant removed just prior to lysis of the 15DIVhippocampal neurons for harvesting protein. In this manner, the same cell treatments used for western blots can beanalyzed for cell viability providing a direct comparison of protein level observations and respective membraneintegrity. Results shown are relative luminescent units (RLU’s), with media alone and healthy cell levels indicated.An increase in signal indicates a compromised membrane, and release of LDH into the media. The CellTiter-Glow® assay was performed on 15DIV hippocampal neurons seeded in 96-well plates and treated with a dosetitration of amyloid beta for various times. This assay measures ATP content, and results are normalized between acell death control (100%) and healthy cells (0%). 24 hours elicits approximately 50% cell death with 10µM Aβoligomers

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References:- Cruz et al., (2003) Neuron, 40, 471-483. - Kusakawa et al., (2000) J. Biol. Chem., 22, 17166-17172.- Park et al., (2005) J. Neurosci., 22, 5365-5375.

Special Thanks:Hana Dawson for generously providingthe tau knockout brain for our controllysates.

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