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Activation of the PI3K Pathway during Axonal Transport Defects can lead to Oxidative Stress-Induced NeurodegenerationClaire Thant, Megan Lamb, Timothy Hansen, Shermali GunawardenaUniversity at Buffalo Department of Biological Sciences

SummaryHigh levels of oxidative stress can be detected in neurons affected by neurodegenerative diseases such as Parkinson’s (PD), Huntington’s (HD), and Alzheimer’s diseases (AD). In addition to oxidative stress, axonal transport defects and neuronal cell death are also seen in these diseases. Here, we test the hypothesis that axonal transport defects instigates oxidative stress causing neuronal cell death. We found that Paraquat (a known inducer of oxidative stress) ingested larvae exhibits axonal blocks and neuronal cell death. Interestingly, expression of active phosphatidylinositol 3-kinase (PI3K) (a kinase in the pro cell survival pathway) suppresses Paraquat-mediated cell death but not axonal blocks. Expression of active PI3K suppresses neuronal cell death induced by expansion of polyQ repeats, but does not affect axonal transport defects indicating that the PI3K pathway is downstream of axonal transport defects. Additionally, dominant negative PI3K disrupts the normal motility of HTT suggesting that the PI3K pathway is directly linked to axonal transport. Intriguingly, proteins in the PI3K pathway show functional interactions with motor proteins and increased levels of glycogen synthase kinase 3β (GSK3β), a downstream effector of PI3K, is observed in larvae expressing expanded amounts of polyQ repeats and in motor protein mutations. Taken together these observations suggest that axonal transport defects likely activates the PI3K pathway to decrease oxidative stress induced neuronal cell death and degeneration.

ReferencesArvind K. Shukla, Prakash Pragya, Hitesh S. Chaouhan, D.K. Patel, M.Z. Abdin, Debapratim Kar Chowdhuri, “A mutation in Drosophila methuselah resists paraquat induced Parkinson-like phenotypes.” Neurobiology of Aging, Volume 35, Issue 10, October 2014, Pages 2419.e1-2419.e16

Dolma K, Iacobucci GJ, Zheng KH, Shandilya J, Toska E, White JA 2nd, Spina E, Gunawardena S. (2013) Presenilin influences Glycogen Synthase Kinase-3beta (GSK-3β) for kinesin-1 and dynein function during axonal transport. Hum Mol Genet. 2013 Oct 8. Gunawardena, S. and Goldstein, L.S.B. (2001). "Disruption of axonal transport and neuronal viability by amyloid precursor protein mutations in Drosophila." Neuron 32:389-401. Gunawardena, S., Her, L., Laymon, R.A., Brusch, R.G., Niesman, I.R., Sintasath, L., Bonini, N.M., and Goldstein, L.S.B. (2003) "Disruption of axonal transport by loss of huntingtin or expression of poly Q protein in Drosophila." Neuron 40:25-40. Martindale, J.L., Holbrook, N.J. (2002) “Cellular response to oxidative stress: Signaling for suicide and survival” J. Cel.. Physiol. 192: 1-15.

AcknowledgementsSpecial thanks to everyone at the Gunawardena Lab, as well as the UB Center for Undergraduate Research and Creative Activities for funding this project.

Figure 5.

p-GSK3β (S9)

Total GSK3β

Tubulin

ApplG

al4

Roblk

-/-

PI3K.

CAAX

PI3K.21

B

Htt128

Q

Htt138

Q

MJDQ77

MJDQ78

APPs

we

p-Akt (S473)

Total Akt

A.

B. C.

Figure 5. Levels of p-GSK3β (S9) but not p-Akt(S473) is increased in both motor mutants and PolyQ disease genotypes.A. Western blot analysis of a dynein motor mutant (Rob1k -/-), PolyQ and APP disease genotypes (HTT128Q, HTT138Q, MJDQ77, MJDQ78, APPswe), and excess of PI3K (PI3K.CAAX, PI3K21B) probed with antibodies against GSK3Beta (S9) which probes the activation of the PI3K pathway, Total Akt, and Tubulin are also probed as a control. B-C. Quantitative analysis reveals that levels of p-GSK3Beta (S9) are increased in both the dynein motor mutant as well as the disease genotypes, while levels of p-Akt (S472) remained unchanged. N = 1 gel. Figure 6.

CSPA

WT KLC+/- Roblk +/-

PI3K92E PI3K92E;KLC PI3K92E,Roblk

Akt.Exel Akt.Exel;KLC Akt.Exel, Roblk

tor.WT tor.WT;KLC tor.WT,Roblk

B

C

D

E

F

14-3-3Zeta[07103] +/-

14-3-3Zeta[12BL] +/-

14-3-3Zeta[07103]; KLC

14-3-3Zeta[07103],

Roblk

14-3-3Zeta[12BL]; KLC

14-3-3Zeta[12BL], Roblk

G.G

*

***

* **

**

*

nsFigure 6. Proteins in the PI3K/Akt signaling pathway genetically interact with kinesin and dynein. A: Wild type larval segmental nerves show smooth staining (CSP). kinesin light chain (KLC +/-) and dynein light chain (Roblk +/-) also show smooth staining Bar = 50 μm. B. Larvae expressing PI3K92E.CAAX with 50% reduction in kinesin or dynein show axonal blocks (Arrows). C. Larvae expressing Akt show smooth staining, while larvae expressing Akt with either a 50% reduction OF kinesin or dynein show axonal blocks (Arrows). D. Larvae expressing Tor show smooth staining, while larvae expressing Tor with either a 50% reduction in kinesin or dynein show axonal blocks (Arrows). E-F. loss of function OF 14-3-3Zeta (12BL), or partial loss of function (07103) show smooth staining in segmental nerves. When combined with 50% reduction in either kinesin or dynein both show axonal blocks (Arrows). G-H. Quantitative analysis reveals that axonal accumulations between the following genotypes are significant as compared to the Wild Type control: UAS-Akt.Exel;KLC +/- (p=0.015), UAS-Akt.Exel, Roblk+/- (p=0.0004), UASPI3K92E;KLC +/- (p=0.049), UASPI3K92E, Roblk +/- (p=0.002), UAStorWT; KLC +/- (p=0.007), UAStorWT, Roblk +/- (p=0.02), 14-3-3Zeta[12BL] +/-, KLC +/- (p=0.0007), 14-3-3Zeta[07103] +/-, KLC +/- (p=0.02). N = 5 larvae.

H.Gns

ns

**

p=0.038

N=6 N=3

Htt138QmRFP TUNEL Merged

PI3K92E.CAAX;Htt138QmRFP

A B

Figure 3.

C

D E F

G.Figure 3. Expression of active PI3K suppresses neuronal cell death induced by expression of expansion of polyQ repeats A-C. Expression of HTT138QmRFP causes neuronal cell death as measured by the TUNEL assay. D-F. Larvae expressing active PI3K (PI3K92E.CAAX) with HTT138QmRFP decreases the amount of neuronal cell death G. Quantitative analysis reveals that the amount of cell death seen in PI3K.CAAX;HTT138QmRFP larvae are significantly less compared to larvae expressing HTT138QmRFP alone (p = 0.038.)

Figure 4.

HTT15Q

HTT15Q

CSP

CSP

Overlay

CSP

HTT15QmRFP

HTT15Q-mRFP: PI3K.DNHTT15Q-mRFPFigure 4. Dominant negative PI3K disrupts the normal motility of HTT within axonsA. Expression of HTT15QmRFP normally shows smooth CSP staining within larval axons similar to Wild type larvae, Note that HTT also is smooth within axons B. Expression of the dominant negative PI3K, or PI3K.DN with HTT15QmRFP causes CSP and HTT blockages.

PI3K/Akt signaling is overactive in motor mutants as well as numerous neurodegenerative disease genotypes.

Proteins in the PI3K pathway show functional interactions with motor proteins.

Expression of excess polyQ repeats causes axonal transport defects and cell death.

Expressing constitutively active PI3K protein is able to rescue HTT138Q induced neuronal cell death, but not axonal transport defects.

Paraquat ingestion causes axonal transport defects and cell death.

Expressing constitutively active PI3K protein with Paraquat ingestion has no effect on axonal defects but decreases neuronal cell death.

Expression of dominant negative P13K causes axonal transport defects.

PI3k acts downstream of axonal transport.

P13K pathway is likely activated due to axonal transport defects and an early oxidative stress response.

PI3K.CAAX;Htt138QmRFP

B

Htt138QmRFP

AHtt138Q CSP Merged

Figure 2.

Figure 2: . Expression of active PI3K does not affect axonal transport defects induced by expression of expansion of polyQ repeats. A. Expression of HTT138QmRFP causes accumulations of mutant huntingtin and cysteine string protein (CSP) (arrows). Note that accumulations of CSP co-localize with huntingtin (yellow dots, merged image.) B. Larvae expressing PI3K92E.CAAX with HTT138QmRFP also contain accumulations of both mutant huntingtin and CSP. Figure C-D. Quantified analysis reveals that the number of are not significantly different between larvae expressing HTT138QmRFP, and larvae expressing both HTT138QmRFP and PI3K92E.CAAX indicating that active PI3K does not have an effect on axonal transport defects and that the PI3K pathway is downstream of axonal transport. N = 5 larvae.

C.

ns

D.ns

Figure 1. Ingestion of Paraquat causes axonal transport defects and neuronal cell death. Expression of active PI3K suppresses Paraquat-mediated cell death but not axonal transport defects . A, B. Wild Type (APPLGAL4) Drosophila larvae raised on 0mM and 20mM Paraquat. Note axonal blocks in 20mM Paraquat. C, D, E. Drosophila larvae expressing PI3K92E.CAAX raised on 0mM and 20 mM Paraquat. Note that Paraquat-mediated axonal blocks are not rescued by excess P13K. (p = 0.4879.)F, G. Wild Type (APPLGAL4) Drosophila larvae raised on 0mM and 20mM Paraquat show statistically significant amount of neuronal cell death as assayed by the TUNEL assay. (p =0.0165.) H, I, J. Expression of PI3K significantly suppresses Paraquat induced cell death. (p = 0.0013).

Figure 1.A.

C.

B.

D.

F G

H I

ns

ns

ApplGal4 0mM CSP ApplGal4 20mM CSP

PI3K92E.CAAX 0mM CSP PI3K92E.CAAX 20mM CSP

E.

ApplGal4 0mM ApplGal4 20mM

PI3K92E.CAAX 20mM PI3K92E.CAAX 0mM

J.

p = 0.0165

*

p = 0.0013

**

N=9 N=10 N=6 N=10

N=12 N=5 N=12 N=10

A. B.

Paraquat &PolyQ Expansion

Conclusions