Stella Gagliardi

Genomic and post-Genomic Center, IRCCS Mondino Foundation, Pavia

IL METABOLISMO DELL’RNA NELLE MALATTIE NEUROLOGICHE, 6 luglio 2018, Pavia

Presentation Overview

Introduction: ALS disease and lncRNAs

Experimental Plan

Results:1. Deep sequencing lncRNAs expression profiles in sporadic ALS

patients2. Deep sequencing lncRNAs expression profiles in mutated ALS

patients3. Work in progress

Conclusions

Amyotrophic lateral sclerosis (ALS) is a rare late-onsetneurodegenerative disease

Selective degeneration of motor neurons in the spinal cord,brainstem and cerebral cortex

Approximately 90-95% of cases are classified as sporadic ALS (SALS)

The remaining 5-10% of cases is familial (FALS)

Genetic Features in both FALS and SALS

Amyotrophic Lateral Sclerosis (ALS)

Renton et al., 2014

Brown et al., 2017

Amyotrophic Lateral Sclerosis (ALS)

Shahheydari et al., 2017

Bar et al., 2016

ALS and lncRNAs

ALS and lncRNAs

ALS and AS: NEAT1_1 and C9orf72 lncRNAs

NEAT1_1 Pathological alterations in the nucleus of an ALS

motor neuron

Nishimoto et al., 2013

C9orf72 ASSchematic diagram of C9ORF72 gene and antisense

transcripts

Zu et al. ,2013

NEAT1_2 lncRNA forms paraspeckle structure with RNA-bindingproteins consisting of TDP-43 and FUS/TLS, and other paraspeckleproteins including. TDP-43 and FUS/TLS were enriched inparaspeckles and bound to NEAT1_2 lncRNA directly..

C9ORF72 antisense transcripts are elevated in the brains ofC9(+) patients, and antisense GGCCCC (G2C4) repeat-expansionRNAs accumulate in nuclear foci in brain.Sense and antisense foci accumulate in blood and are potentialbiomarkers of the disease.

Experimental plan

FUS

TARDBP

SOD1

SALS

CTRs

PBMCsisolation

RNA extractionand NGS

Data analysis

Experimentaldesign

NextGeneration Sequencing

Whole transcriptome

analysis

Long non coding RNAs

Coding RNAs

Validation by RT-PCR

In PBMCs and spinal cord

Aim of the work

lncRNAs

coding/non-coding

co-expression analysis

mRNAs

Deep RNAs Profiling in PBMCs and Spinal Cord from ALS patients

All comparisons are given between the disease state and the control samples. We kept only the lncRNAs showing |log2(disease sample/healthy donor)|≥1 and a False Discovery Rate ≤ 0.1. In heatmap, all differentially expressed mRNAs (A) and lncRNAs (B) were considered.

B - lncRNAsLIA - mRNAsLI

Expression profiles of differently expressed lncRNAsand RNAs in SALS and healthy controls

Gagliardi et al., 2018

CTRs

SALS

mRNAs lncRNAs

UP-regulated 57 183

DOWN-regulated 30 110

total 87 29363%

27%

7%

3%

SALS

antisense

lincRNA

processedtranscript

sense intronic

RNA-seq expression profiles in SALS patients

Gagliardi et al., 2018

CTRSALS

SALS: Pathway analysis

GO pathway analysis for DEGs in SALS patients compared to healthy controls.TOP10 enriched GO terms for Biological process.

ZEB1-AS

ZEB1-AS

The length of the bar represents the significance of that specific gene-set or term. In addition, the brighter the color, the more significant that term is.

Transcription Factors

MINCR and MYCBPOncogenes

SALS: Top 10 deregulated lncRNAs

Transcript ID Gene Name geneType

ENST00000377540.1 TTC25 processed_transcript

ENST00000425493.1 RP11-475I24.8 lincRNA

ENST00000609619.1 RP11-38M8.1 lincRNA

ENST00000423714.1 ZEB1-AS1 processed_transcript

ENST00000563897.1 CTB-58E17.1 lincRNA

ENST00000607333.1 Xbac-BPG252P9.10 (IER3-AS1) antisense

ENST00000508106.1 RP11-526A4.1 lincRNA

ENST00000310358.7 SPON1 processed_transcript

ENST00000536865.1 ZBTB11-AS1 antisense

ENST00000609178.1 CTA-384D8.35 lincRNA

Gagliardi et al., 2018

ZEB1 and ZEB1 AS

Li et al., 2016

ZEB1 coding

ZEB1 (Zinc Finger E-Box Binding

Homeobox 1) acts as a

transcriptional repressor.

Su et al., 2017

ZEB1 AS(in cancer)

ZEB1 AS and ALS (genes expression)

NT

H 2O 2

0.0

0.5

1.0

1.5

2.0

RN

A F

old

Ch

an

ge

ZEB1-AS

****

NT

H 2O 2

0.0

0.5

1.0

1.5

2.0

RN

A F

old

Ch

an

ge

ZEB1

NT

H 2O 2

0

1

2

3

4

mir200c

RN

A F

old

Ch

an

ge

*

SH-SY5Y cells treated with 1mM H2O2 for 1h

Project in collaboration with Di Giulio’s group (UniMi)

ZEB1

ALS

CTR

0.0

0.5

1.0

1.5ALS N=20

CTR N=20

mR

NA

(rq

)

PBMCs from ALS patients and controls

Cancer and neurodegeneration: pathogenetic convergences

Plun-Favreau et al., 2010Hiroyoshi Ariga, 2015

MINCR and MYCBPOncogenes

C-MYC and MYC-induced long noncoding RNA (MINCR)

Iaccarino et al., 2017

lncRNAs involved in the control of MYC transcriptional program

Stine et al., 2015

C-MYC coding MINCR

MINCR and ALS (genes expression)

*p<0,05*p<0,05

*p<0,05

PBMCs from ALS patients and controls

MINCR and ALS (proteins level)

PBMCs from ALS patients and controls

C-MYC

GAPDH

CTR ALS

MYCBP

GAPDH

CTR ALS

MAX

GAPDH

CTR ALS

CTR ALS

c-MYC

MINCR

CANCER ALS

Cell trasformation

tumorigenesis

cancer

RNA-seq expression profiles in mutated ALS patients

FUS TARDBP SOD1

mRNAs lncRNAs mRNAs lncRNAs mRNAs lncRNAs

35 16 10 6 14 0

87 5 20 9 4 2

122 21 30 15 18 2

UP-regulated

DOWN-regulated

total

lncRNAs classification for biotypes and statusFUS TARDBP SOD1

gene biotype

antisense 11 7 2lincRNA 6 6 0

processed transcript 2 1 0

sense intronic 2 1 0

FUS

TARDBP

SOD1

CTRs

PAX Binding Protein AS is up-regulated in FUS patients.

It is fundamental for skeletal muscle development and it is already described as involved

in ALS (Villar et al., 2014)

SNAP25 is involved in axonal repair and synaptic vesicle

processing and it is deregulated in ALS patients

(Ikemoto et al., 2002)

Mutation in SOD1 which may lead to reduced creatine

kinase activity by inactivation of important target enzymes, including

Mitochondrial creatinekinase (Lee et al., 2015)

Mutated patients: the most Deregulated AS

Work in progress: increasing the sample size

CTR N= 14SALS N=15SOD1 N=4FUS N=3TARDBP N=2C9orf72 N=1VCP N=1

Work in progress: fast and slow progressor

SALS N=15

N=8 Slow ProgressorN=5 Fast progressor

SALS

mRNAs FAST SLOW

UP-regulated 475 140

DOWN-regulated 257 183

total 732 323

68 DEG in common between fast and slow progressor ALS

B - lncRNAsLIA - mRNAsLI

Conclusions

The most implicated pathway is the regulation of transcription, include unexpected factor such as oncogenes

Sense and antisense regulation may have an important role in ALS pathogenesis

Antisense regulation in ALS affect both directly the sense gene than indirectly the involvedsense pathway

ALS mutated patients showed a RNA regulation different from SALS unmutated patients

Patients with Fast disease progressor showed a major RNA deregulation compared to patients with Slow disease progressor.

Genomic and Post-Genomic Center, IRCCS Mondino Foundation, Pavia

Cristina Cereda

Susanna ZuccaOrietta Pansarasa

Daisy SprovieroMarialuisa ValenteMassimo Plumari

Alessia AsaroGaetano GriecoMatteo BordoniMarta GianniniCecilia PandiniJessica Garau

Valentina Fantini

Division of General NeurologyProf. Mauro Ceroni

Luca Diamanti

Collaborations:

Department of Molecular Biotechnology and Health Sciences, Bioinformatic and Genomic Unit, University of TurinProf. Raffaele Calogero, Maddalena Arigoni

Acknowledgements

TRANS-ALS group:

Department of Health Sciences, Laboratory of Pharmacology, University of Milan: Stephana Carelli

Neurology IV – Neuroimmunology and Neuromuscular Diseases Unit, Foundation Neurological Institute "Carlo Besta“, Milan: Pia Bernasconi, Stefania Marcuzzo

Department of Neurology-Stroke Unit and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan, Italy: Antonia Ratti

Dino Ferrari Center, University of Milan: Stefania Corti

Department of Pharmacological Science and Biomolecular (DISFeB),University of Milan: Prof. Angelo Poletti, Valeria Crippa