Modulating Protein Phosphatase 2A rescues disease phenotype in

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Modulating Protein Phosphatase 2A rescues disease phenotype in neurodegenerative tauopathies Simon McKenzie-Nickson, Jacky Chan, Keyla Perez, Lin W. Hung, Lesley Cheng, Amelia Sedjahtera, Lydia Gunawan, Paul A Adlard, David J Hayne, Lachlan E Mcinnes, Paul S. Donnelly, David I. Finkelstein, Professor Andrew Hill, and Kevin J Barnham ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00161 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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ACS Chemical Neuroscience

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Modulating Protein Phosphatase 2A rescues disease phenotype in

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neurodegenerative tauopathies

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Simon McKenzie-Nickson 1,2a,, Jacky Chan 1, Keyla Perez 1, Lin W. Hung 1, Lesley Cheng 3,

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Amelia Sedjahtera 1, Lydia Gunawan 1, Paul A. Adlard 1, David J. Hayne

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McInnes 2b,

Paul S.

2b

, Lachlan E.

Donnelly 2b, David I. Finkelstein 1, Andrew F. Hill 3, Kevin J. Barnham 1,2a

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8

1

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Australia

Florey Institute of Neuroscience and Mental Health, Parkville, Melbourne, VIC 3052,

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2 a

Department of Pharmacology and Therapeutics, b School of Chemistry; Bio21 Molecular

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and Biotechnology Institute, Parkville, Melbourne, VIC 3052, Australia

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3

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Trobe University, Melbourne, VIC 3086, Australia

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La

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Correspondence

to

K.J.B

([email protected])

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[email protected]).

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Author contributions: SMN, KJB, LWH, AFH, DIF designed experiments. SMN, JC, KP, LWH, LC,

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AS, LG, PAA, DJH, LEM, PSD conducted research. SMN, KP, LWH, LC, PAA analysed research.

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SMN and KJB wrote the manuscript.

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The authors declare no competing financial interests.

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ACS Paragon Plus Environment

and

S.M.N

(simon.mckenzie-

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Alzheimer’s disease (AD) is the leading cause of dementia worldwide accounting around

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70% of all cases. There is currently no treatment for AD beyond symptom management and

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attempts at developing disease-modifying therapies have yielded very little. These strategies

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have traditionally targeted the peptide Aβ which is thought to drive pathology. However, the

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lack of clinical translation of these Aβ-centric strategies underscores the need for diverse

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treatment strategies targeting other aspects of the disease. Metal dyshomeostasis is a common

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feature of several neurodegenerative diseases such as AD, Parkinson’s disease, and

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Frontotemporal dementia and manipulation of metal homeostasis has been explored as a

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potential therapeutic avenue for these diseases. The copper ionophore glyoxalbis-[N4-

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methylthiosemicarbazonato]Cu(II) (CuII(gtsm)) has previously been shown to improve the

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cognitive deficits seen in an AD animal model, however the molecular mechanism remained

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unclear. Here we report that the treatment of two animal tauopathy models (APP/PS1 and

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rTg4510) with CuII(gtsm) recovers the cognitive deficits seen in both neurodegenerative

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models. In both models, markers of tau pathology were significantly reduced with CuII(gtsm)

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treatment and in the APP/PS1 model the levels of Aβ remained unchanged. Analysis of tau

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kinases (GSK3β and CDK5) revealed no drug induced changes however both models

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exhibited a significant increase in the levels of the structural subunit of the tau phosphatase,

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PP2A. These findings suggest that targeting the tau phosphatase PP2A has therapeutic

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potential for preventing memory impairments and reducing the tau pathology seen in AD and

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other tauopathies.

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Keywords: Alzheimer’s, tau, phosphatase, copper, neurodegeneration, metals

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Funding: This work was funded by NHMRC grants APP1031193; APP1002373; 628946

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Introduction


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The growing epidemic of Alzheimer’s disease (AD) is one of the greatest health crises

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currently facing the global community. As of 2015 1, AD is reported to cost the global

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economy US$818 billion per year and with an ageing population, both the economic and

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social cost of this disease are only set to rise. Early onset or familial AD is caused by a

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mutation in one or more of the key pathological proteins such as amyloid precursor protein,

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or presenilin 1. However, by far the majority of AD cases are considered to be sporadic or

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late onset. These cases are not considered to be genetically determined 2; however, a growing

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number of polymorphisms have been shown to increase risk of the disease 3. The

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neuropathology of AD manifests as the presence of extracellular deposits, or plaques,

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consisting primarily of the amyloid-β (Aβ) peptide. This peptide is proteolytically derived

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from the amyloid precursor protein (APP). Another hallmark pathology of AD are the

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intracellular ‘tangles’ of the tau protein 4.

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These features form the basis of the amyloid cascade hypothesis which posits that an

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accumulation of Aβ (either through overproduction or through lack of clearance from the

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brain) drives the pathology as the disease progresses

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development of multiple therapeutics targeting Aβ biology; however, such compounds have

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had very little success to date 6. Therapies targeting tau have received comparatively little

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attention, despite a growing body of research demonstrating the crucial role tau plays in the

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development of disease and its potential as a therapeutic target 7–9.

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Tau is a cytoskeletal protein with complex regulation and a wide variety of roles

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function can be regulated through post-translational modifications such as acetylation 11, and,

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of particular relevance to neurodegenerative diseases, phosphorylation 12. Tau can bind to and

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stabilise microtubules, however, this process is regulated by phosphorylation at various

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epitopes

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brain tissue, tau is frequently seen with a level of tau phosphorylation higher than in non-

13

5555554

. This has resulted in the

10

. Tau

and as such the phosphorylation of tau can impact microtubule stability. .In AD



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diseased controls, both in animal models

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enters a hyperphosphorylated state there is both a toxic gain-of-function (increased

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propensity to aggregate

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Alterations in the biology of the enzymes controlling tau phosphorylation is thought to

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underlie the increase in tau phosphorylation observed in AD 19,20.

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The phosphorylation state of tau is the result of a complex interplay between kinase and

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phosphatase activity. The phosphorylation of tau is mediated primarily by the kinases

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glycogen synthase kinase 3β (GSK3β) and cyclin dependent kinase 5 (CDK5)

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ongoing interest in developing specific inhibitors for these enzymes however interest in this

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area has been dampened with the failure of lithium in clinical trials

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dephosphorylation occurs through the action of phosphatases, with protein phosphatase 2A

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(PP2A) responsible for around 70% of tau dephosphorylation

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consists of three subunits, a structural subunit (designated the A subunit), a regulatory ‘B’

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subunit, and the ‘C’ subunit which is responsible for the enzymatic activity of PP2A. The B

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subunit also provides substrate specificity 23. Impairments in PP2A are thought to contribute

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to the hyperphosphorylation observed in AD

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decreased in the brain tissue of AD patients

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Additionally,

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hyperphosphorylation and cognitive deficits in wildtype rats

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other pacific islands there is a familial parkinsonism-dementia that has strong tau pathology

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and decreased PP2A activity

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and/or activity could be an attractive therapeutic option for AD and other tauopathies.

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However to date, there has been limited activity in this field as PP2A’s complex biology and

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regulation has made this problematic 28.

inhibition

and AD patients

15,16

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. It is thought that when tau

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) and a loss-of-function (loss of microtubule stability

of

PP2A

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using

18

).

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. There is

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. Alternatively, tau

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. The PP2A holoenzyme

19

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. PP2A activity has been shown to be

alongside decreased PP2A expression okadaic

acid

treatment

induces

25

.

tau

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. Furthermore, in Guam and

. These examples may suggest that increasing PP2A levels


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Copper(II) complexes of bis(thiosemicarbazone) ligands have shown great promise in

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treating multiple animal models of neurodegenerative diseases

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utilising the APP/PS1 mouse model of AD found that treatment with glyoxalbis-[N4-

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methylthiosemicarbazonato]Cu(II) (CuII(gtsm)) improved the spatial memory deficits seen in

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the model while a related compound, CuII(atsm), that does not release bioavailable copper did

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not rescue the phenotype in this model suggesting the liberation of copper is important.

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Further, biochemical analysis of treated animals revealed a reduction in the levels of

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phosphorylated tau and a decrease in the levels of an Aβ oligomer

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treatment with CuII(gtsm) not only improves the behavioural phenotype of APP/PS1 mouse

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model of AD, but also the rTg4510 mice which overexpresses a mutant form of tau

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associated with frontotemporal dementia. Furthermore, treatment reduced measures of tau

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pathology in both models and produced an increase in the levels of the PP2A structural

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subunit.

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. A preliminary study

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. Here, we show that


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Results and discussion

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CuII(gtsm) rescues the spatial memory deficit in APP/PS1 and rTg4510 mice

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In agreement with previous studies, here we showed that the performance of both APP/PS1 31

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(Figure 1) and rTg4510

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maze and the Y-maze was impaired. In the probe trial for the Morris water maze (as

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measured by the length of time spent in the platform quadrant during the probe trial) both the

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APP/PS1 model (9 months of age (Figure 1, panel B, P = 0.0099) and the rTg4510 model

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(12 months of age; Figure 2, Panel B, P = 0.017) were impaired as compared to wildtype

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controls. We found that treatment with CuII(gtsm) significantly improved of the performance

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of both APP/PS1 (P = 0.0021 compared to vehicle treated transgenic performance) mice and

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rTg4510 mice (P = 0.018 compared to vehicle treated transgenic performance) such that they

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performed similarly to wildtype animals when assessing time spent in the platform quadrant

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(however, drug treatment didn’t influence the learning curve (Figure 1A and 2A

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respectively). When tested in the Y-maze, vehicle treated APP/PS1 and rTg4510 both

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performed significantly worse than wildtype mice (Figure 1, panel C, P = 0.0083 and Figure

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2, panel C, P = 0.0002 respectively). Treatment of APP/PS1 mice with CuII(gtsm) yielded a

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highly significant improvement in Y-maze performance (P = 0.0005 compared to vehicle

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treated transgenic performance) while treatment of rTg4510 mice did not significantly alter

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the performance (P = 0.14). Further analysis utilising a 1-sample T-test against chance

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reveals that in both APP/PS1 and rTg4510 transgenic mice performance did not differ from

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chance (P = 0.13 and P = 0.97 respectively). CuII(gtsm) treated mice in both APP/PS1 and

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rTg4510 models did in fact perform better than chance (P = 0.0062 and P = 0.02 respectively)

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revealing a preference for the novel arm over and above the chance value of 33.3%.

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(Figure 2) mouse models spatial memory in the Morris water

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CuII(gtsm) treatment rescues the severe hyperactivity phenotype of rTg4510 mice

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In an open field test, rTg4510 mice showed a marked hyperactivity phenotype as measured

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by total distance moved (P < 0.0001 compared to wildtype; Figure 3, panel A) and by the

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low total amount of time spent resting (Figure 3, panel B, P < 0.0001 compared to wildtype).

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Treatment of these mice with CuII(gtsm) significantly reduced the total distance moved (P =

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0.0003 compared to vehicle treated transgenic mice) and improved the time spent resting (P

Modulating Protein Phosphatase 2A rescues disease phenotype in

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