Introduction

Mitochondrial dysfunction is a common feature of numerous

neurodegenerative diseases. Furthermore, several disease-associated

mutations have been identified in genes regulating the selective

degradation of damaged mitochondria by autophagy (aka mitophagy),

suggesting the involvement of mitophagy in driving disease

pathogenesis. Selective mitophagy is critically important for neuronal

survival as it maintains an optimal cellular energy production whilst

avoiding the toxic accumulation of damaged/ dysfunctional

mitochondria, which can lead to cell death. The discovery of mutations

in the PINK1 and PRKN (Parkin) genes in Parkinson’s disease has

facilitated mechanistic understanding of the mitophagy process. For

inducing selective mitophagy, PINK1 and PRKN interact with various

autophagic adaptor proteins including SQSTM1 (p62), which has been

shown to be mutated in patients with frontotemporal dementia (FTD).

The precise role of SQSTM1 in mitochondrial function and clearance

remains to be clarified. In particular, it is unclear whether SQSTM1 is

essential in neuronal mitophagy. To address this knowledge gap, we

are assessing the importance of mitophagy in human iPSC derived

neurons with or without genetic alterations in SQSTM1.

Result 2: Generation of iPSCs lacking SQSTM1

Result 3: The differentiation capacity of iPSCs into cortical

neurons appears unaffected by the loss of SQSTM1.

Result 5: Depletion of SQSTM1 results in reduced

expression of genes involved in the respiratory chain

and impairs mitochondrial respiration in human iPSC-

derived cortical neurons.

Result 7: Mitochondrial PINK1 recruitment and loss of

mitochondria in iPSC-derived cortical neurons occur in

the absence of SQSTM1 following mitochondrial

depolarization.

Conclusions and Future Direction

Increase in PINK1 localization to the

mitochondria after 18h of oligomycin

and antimycin (O+A) treatment and

mitochondrial fractionation →

Investigating the role of SQSTM1 (p62) in mitochondrial function and

clearance in cortical neurons

Figure 1. SQSTM1 (p62) has been identified as an adaptor for

mitophagy (Martinez-Vicente 2017, Shahheydari et al., 2017).

Mutations have been shown to be causative for FTD/ALS.

Result 1: SQSTM1 is expressed in both axons and

dendrites of human iPSC-derived cortical neurons.

SQSTM1 / MAP2 / DAPI 50µm

◦ SQSTM1 is a regulator of mitochondrial respiratory chain function.

◦ Despite its important role in autophagy, SQSTM1 is not essential for the clearance of

damaged mitochondria/ mitophagy.

◦ Future work will examine other arms of the autophagy pathway and cargo

removal mediated by SQSTM1 in our human neuronal model.

Clearance of depolarised mitochondria as indicated by the depletion of mitochondrial proteins in

whole neuron lysates following 36-62h O+A treatment ↓

Aim 1: Generate in vitro human neuron model lacking SQSTM1

Methods: Derivation of human cortical neurons from induced pluripotent stem cells

(iPSCs) from SQSTM1 knockout (KO) and wild type (WT) lines.

Suppliers of WT (RBi001-A) line: Roslin Cells Limited and the European Bank for

induced pluripotent Stem Cells.

Aim 2: Determine the effect of SQSTM1 depletion on mitochondrial function in cortical neurons

Methods: Seahorse XFe96 mitochondria respiration assay, MitoTracker, RNA-Seq.

Methods: induction of mitophagy using 2µM oligomycin and 1µM of antimycin,

mitochondrial enrichment, immunoblotting, real-time PCR to assess mtDNA level.

Aim 3: Assess the effect of SQSTM1 depletion on mitophagy in cortical neurons

SQSTM1 / MAPT(Tau) / DAPI

50µm

SQSTM1/ Hoechst 33342

Non

-edi

ted

WT

SQ

ST

M1

KO

SQSTM1/ Hoechst 33342

50µm

50µm

BCL11B(CTIP-2) / TUBB3(TUJ1) / DAPI

WT

KO

50µm

50µm

SLC17A7(VGLUT1) / TUBB3(TUJ1) / DAPI

WT

KO

50µm

50µm

MitoTracker Deep Red FM / TUBB3(TUJ1) / DAPI

WT

KO

50µm

50µm50µm

Anna Poon1, Gregory A. O’Sullivan1, Hélène Plun-Favreau2, Selina Wray2, Lee Dawson*, & James McCarthy1

1Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, UK2Institute of Neurology, UCL, London, WC1N 3BG, UK

Project Overview

Poster presented at the 14th International Conference on Alzheimer's and Parkinson's Diseases. Poster available to download from Astex’s website at www.astx.com

* The current affiliation of co-author L.D. is Cerevance, UK.

© Astex Pharmaceuticals

Result 6: Depletion of SQSTM1 reduces autophagy flux

with less LC3-II accumulation in iPSC-derived cortical

neurons following autophagy inhibition.

10µm

10µm

Result 4: Depletion of SQSTM1 does not appear to

significantly alter mitochondrial biogenesis and

distribution in iPSC-derived cortical neurons.

-10.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

OC

R (

pm

ol/

min

/Nu

cle

ic

Ac

id)

Basal

0.0

10.0

20.0

30.0

40.0

50.0

60.0

OC

R

(pm

ol/

min

/Nu

cle

ic …

ATP Production

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

OC

R

(pm

ol/

min

/Nu

cle

ic …

Maximal Respiration

0.0

100.0

200.0

300.0

OC

R

(pm

ol/

min

/Nu

cle

ic …

Spare Respiratory

Capacity*P=0.0285 *P=0.0091

p=0.0019

p=0.0026

DM

SO

36h O+A

48h O+A

60h O+A

DM

SO

36h O+A

48h O+A

60h O+A

0.0

0.5

1.0

Fo

ld c

han

ge o

f T

OM

M20

leve

ls c

om

pare

d t

o D

MS

O

WT SQSTM1 KO

p=0.0154

p=0.0022

p=0.0085

p=0.0021

p=0.0004

p=0.0003

DM

SO

36h O+A

48h O+A

60h O+A

DM

SO

36h O+A

48h O+A

60h O+A

0.0

0.5

1.0

Fo

ld c

han

ge o

f T

IM23

levels

co

mp

are

d t

o D

MS

O

WT SQSTM1 KO

p=0.0002

p<0.0001

p<0.0001

p<0.0001

DM

SO

36h O

+A

48h O

+A

62h O

+A

0

20000

40000

60000

80000

MtD

NA

C

op

y N

um

ber

WT

P<0.0001

P<0.0001

P<0.0001

DM

SO

36h O

+A

48h O

+A

62h O

+A

0

500

1000

1500

2000

MtD

NA

C

op

y N

um

ber

SQSTM1 KO

P=0.0035

P=0.0065

P=0.0016 ← Mitochondrial clearance as

indicated by the depletion in

mitochondrial DNA relative to

nuclear DNA in neurons following

36-62h O+A treatment.

WT

SQSTM1 KO

0

5

10

15

20

Full Medium

LC

3-I

I / β

-Acti

n

p=0.0308

WT

SQSTM

1 KO

0

1

2

3

4

Starvation Medium

LC

3-I

I / β

-Acti

n

TBR1 / TUBB3(TUJ1) / DAPI

WT

KO

50µm

50µm

← Alteration in

expression levels of

several genes

associated with cortical

development suggests

SQSTM1 may

modulate neuronal

subtype specification

in the cerebral cortex

(* p< 0.05).

Complex IIComplex I

ND

UFA

6N

DU

FS

4TIM

MD

C1

SD

HA

F4

SD

HA

F3

ETFA

CY

CS

CO

X18

CO

X7A

2LC

OX

6A1

CO

X4I

1A

TP

5F1E

ATP

5MC

2A

TP

5PB

ATP

5F1A

ATP

5MC

3A

TP

5PD

SLC

25A

14

0.0

0.5

1.0

Rela

tiv

e t

ran

scri

pt

leve

ls

WT

SQSTM1 KO

Complex IV Complex V UCP

* * * * * * * * * * * * * * * * * *

* p< 0.05

(KO compared to WT)

Mitochondrial

BiogenesisMtDNA

Maintenance

Mitochondrial

Translation

PPARG

C1A

PPARG

C1B

PPARG

NRF1

MPV17

PO

LG

PNPT1

SSBP1

MRPL4

MRPL9

MRPL15

MRPL19

MRPL23

MRPL37

MRPL41

MRPL46

MRPL47

MRPL48

MRPL50

EEF1A

1

TSFM

LARS2

TRM

U

0.0

0.5

1.0

1.5

Rela

tiv

e t

ran

scri

pt

leve

ls

WT

SQSTM1 KO

* * p< 0.05

(KO compared to WT)

MAP2

MAPT

TUBB3

DLG

4SYP

RBFO

X3

RELN

CUX1

PO

U3F

2

BCL11

B

FOXP2

SATB

2

TBR1

NEURO

G2

FEZF2

RO

RB

PAX6

EM

X2

FOXG

1

0

1

2

3

Rela

tiv

e t

ran

scri

pt

leve

ls

WT

SQSTM1 KO

Pan neuronal Cortical

*

* *

* * * *

*

*

Figure source: Martinez-Vicente 2017