Pavia, 27-28th June 2019

RNA profiling in neurodegenerative diseases:

extracellular vesicles characterization reveals a

distinct microRNA signature

IL METABOLISMO DELL’RNA NELLE MALATTIE NEUROLOGICHEDaisy Sproviero

Neurodegenerative Diseases (NDs)

• Neurodegenerative diseases (NDs) are a heterogeneous group ofdiseases of the Central Nervous System in which neuronsdegenerate.

Brain cortexAlzheimer's disease

Pick's diseaseFronto-temporal dementia

Progressive supranuclear palsyCortico-basal degeneration

Vascular dementia

Basal ganglia and brain stemParkinson's diseaseDementia with Lewy bodiesHuntington's disease

Cerebellar spino degenerationSpino-cerebellar ataxia

Friedrich's ataxiaAtaxia telengiectasia

Motoneuronal degenerationAmyotrophic lateral sclerosisBulb-spinal atrophySpinal-muscular atrophy

Specificity in the cerebral area

Specificity in the neuronal cells

Specificity in hallmarkproteins

Clinical overlap

Genetic Overlap

Pathways overlap

In NDs the pathological specificity is related to the selective processof neuronal death that affects specific parts of the Central NervousSystem

NDs: Cerebral specificity

Bertram et Tanzi, J Clin Invest., 2005

In the NDs to the specificity of the area of the central nervous system affected is also correlated the cellular specificity that is the type of neuron that degenerates

NDs: Neuronal Specificity

ALS motor neuronnot directly

testedtheir activation

precedesnot directly

tested

cortical and hippocampal

neurons

not directlytested

microglialdysfunction

contributes to pathogenesis

not directlytested

striatal neuronsmutant expression

renders neuronsvulnerable in culture

their activationoccurs early and progresses with

disease

not directlytested

Pukinje cellsmutant expressionin Bergmann glia

suffices for disease

not directlytested

not directlytested

corticalneurons

PrPC expressionsufficies for

disease

microglialactivation

decreases prioninfection

probably notimportant for pathogenesis

dopaminergicneurons

expressesenzyme that

induces toicity

Their activationprecedes

neurodegeneration

elevated expressionin oligodendrocytessuffices for disease

Prion disease

Spinocerebellar ataxia

Huntington’s disease

Parkinson’s disease

Alzheimer’s disease

primary target neuron astrocytes microglial cells

Schwann cells or oligodendrocytes

Depending on the type of disease,neuronal degeneration caninvolve different clinical signs:

Cognitive Deficits Dementia

Motor alterations Behavioural disorders Psychological disorders

NDs: Clinical Overlap

Ganash MA, J Proteomics Bioinform, 2017

Ahmed RM et al., J Neurol Neurosug Psychiatry, 2016

Common genetic factors and pathways

in neurodegenerative diseases

Arneson et al. J Genet 2018; 97(3): 795–806.

Ganash MA. J Proteomics Bioinform 2017; 10: 135-143.

Anthony and Shiels, Transplant Res, 2013 2:10

, Annexin V

and Alix

Extracellular Vesicles as possible biomarkers

Spinelli et al., Non-Coding RNA 2019, 5(1), 1.

Extracellular Vesicles as Conduits of

Non-Coding and coding RNA

CTRALS

Aim of the study and methods

Exos MVs Exos MVs Exos MVs Exos MVsmiRNAs

UP-regulated 84 134 48 111 20 9 93 89

DOWN-regulated 26 68 39 13 14 3 18 20

total 110 202 87 124 34 12 111 109

wholetranscriptomemRNAs

UP-regulated 480 32 194 64 0 0 0 2

DOWN-regulated 43 56 33 23 0 0 0 10

total 523 88 227 87 0 0 0 12

lncRNAs

UP-regulated 16 14 15 17 0 0 0 0

DOWN-regulated 0 4 0 1 0 0 0 1

total 16 18 15 18 0 0 0 1

PDALS FTD AD

miRNAs and RNA in EVs from plasma of PD, AD,

ALS and FTD patients

miRNA

Whole transcriptome

CTRL

PKAD

FTD

ALS

FTDPK

ALS

miRNA

RNA

miRNAs and RNA in extracellular vesicle from PD, AD, ALS and FTD patients

ALSALS

EXOs MVs

Gagliardi et al., unpublished

CTRL

N=9

PK

N=9

FTD

N=9

ALS

N=6

AD

N=6

6

hsa-miR-133a-3p

hsa-miR-543

hsa-miR-4451

hsa-miR-6889-5p

hsa-miR-4781-3p

hsa-miR-323b-3p

miRNA EXOs

hsa-miR-1262

hsa-miR-3152-3p

hsa-miR-7856-5p

hsa-miR-365a-5p

hsa-miR-4433b-5p

hsa-miR-6068

hsa-miR-767-3p

miRNA MVs

SDPR

PPBP

MAP3K7CL

B2M

RGS18

TUBB1

MAP3K7CL

AP003068,23

Common miRNAs and RNA in Evs from plasma of PD, AD,

ALS and FTD patients

Parkin-Ubiquitin Proteasomal System

Chemokine signalling pathway

Ubiquitin mediated proteolysis

MAPK signaling pathway

Toll-like receptor signaling pathway

TGF-beta signaling pathway

MAPK signaling pathway

Neurotrophin signaling pathway

Glycosphingolipid biosynthesis

Ras signaling pathway

RNA EXOs RNA MVs

miRNAs and RNA in extracellular vesicle from PD patients

PD MVs PD EXOs

miRNA

PD MVs PD EXOs

AMPK signaling pathways

AMPK signaling pathways

Calcium signaling pathway

Sphingolipid signaling

Signaling pathways regulating pluripotency

of stem cellsMAPK signaling

pathway

miRNAs and RNA in extracellular vesicle from AD patients

AD MVs AD EXOs

miRNA

AD MVs AD EXOs

LysosomecGMP-PKG signaling

patway

Focal adhesionVEGF signaling

pathway

Hippo signaling pathway Lysosome

miRNAs and RNA in extracellular vesicle fromFTD patients

FTD MVs FTD EXOs

miRNA RNAFTD MVs FTD EXOs

miRNA RNA

FTD MVs FTD EXOs FTD MVs FTD EXOs

Chemokine signalingpathway Axon guidance

Positive regulation of neuron death

mRNA splicing

VEGF signaling pathway Rap1 signaling pathwaymRNA splice site selection mRNA processing

FoxO signaling pathway ErbB signaling pathway

Positive regulation of phosphorylation of RNA POL II

mRNA containingribonucleoprotein complex export from nucleus

Hippo signaling pathway Dopaminergic synapse

Regulation of phosphorylation of RNA POL II C-terminal domain RNA metabolic process

miRNAs and RNA in extracellular vesicle from ALS patients

ALS MVs ALS EXOs

miRNA RNA

ALS MVs ALS EXOs

miRNA RNA

ALS MVs ALS EXOs ALS MVs ALS EXOs

cGMP-PKG signaling pathway Axon guidance Spliceosome

Ubiquitin mediated proteolysis

PI3K-Akt signalingpathway

Hippo signalingpathway

Pathohenicinfection Spliceosome

Hippo signalingpathway

ErbB signalingpathway

Herpes Simplex infection

mRNA surveillance pathway

Amyotrophiclateral sclerosis

(ALS)MAPK signaling

pathway Mismatch RepairDopaminergic

synapse

Study of ALS in blood: RNA and Long non coding RNAs in PBMCs

lncRNAs showing |log2(disease sample/healthy

donor)| ≥1

False Discovery Rate ≤ 0.1

lncRNAsImRNAsI

Moreover some miRNAs, strongly upregulatedin EXOs derived from ALS patients, like miR-200, miR-141 and miR-429 negatively controlZEB1, transcription factor that represses T-lymphocyte-specific IL2 gene expression, andits antisense was found downregulated in thewhole transcriptome published in Gagliardi etal. 2018.

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

ENST00000536865.1 ZBTB11-AS1 antisense

EVs transcriptome is linked to PBMC transcriptome

Wellner, Brabletz and Keck, Cancers 2010, 2(3), 1617-1628

In collaboration with Dr. Carelli and Prof. Di Giulio (UniMi)

Higher nuclear SOD1 is protective against aggregation

Soluble

Insoluble

Higher nuclear SOD1 is protective against oxidative stress damage

SOD1, TARDBP and FUS mutations showed protein relocalization and aggregation in ALS LCLs

SOD-1 TARDBP FUS

Extracellular vesicles in Amyotrophic Lateral Sclerosis

Greater presence of SOD1, TDP-43 and FUS in plasma derived MVs of ALS patients compared to CTRLs

Leukocyte derived MVs are overrepresented in ALS patients and are carriers of SOD1

CD45-Leukocyte CD31- Endothelial CD235a-erythrocyte CD61-platelets

FP-Rate(1-Specificity)00.20.40.60.81

1

TP-Rate(1-Sen

sitivity)

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

1) Our data showed that miRNAs cargo was different among the four neurodegenerativediseases.

2) miRNAs are the most interesting subpopulation of transcripts transported by plasmaderived EXOs in neurodegenerative diseases.

3) On the other hand, miRNAs split the group of PD patients in two, one overlapped withAD and the other with FTD patients. Also FTD patients showed two subgroups, oneoverlapping to PD and the other to ALS.

4) The mean size both for MVs and for EXOs resulted increased in ALS patients comparedto controls. MVs derived from ALS patients were enriched in proteins compared toCTRLs. MVs are the most interesting subpopulation of vesicles for the transport ofproteins in neurodegenerative diseases.

5) Leukocyte derived MVs (LMVs) were mostly present in ALS patients compared to ADpatients and healthy donors.

Summary

Acknowledgements

Bioinformatics and Genomics Unit, University of Turin, Italy

Raffaele CalogeroMaddalena Arigoni

IRCCS Mondino Foundation, GpG Center

Cristina Cereda Stella GagliardiOrietta PansarasaSabrina La SalviaSusanna ZuccaMarta GianniniMatteo Bordoni

IRCCS Mondino Foundation, Neurology DepMauro CeroniLuca DiamantiAlfredo CostaClaudio PacchettiElena Sinforiani Roberta Zangaglia

Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), University of MilanAngelo PolettiValeria Crippa

Humanitas Clinical and Research Center –IRCCS, Rozzano

Michela MatteoliFederico ColomboEliana Lauranzano

Neurodegeneration Group, Queen Mary University of LondonAndrea Malaspina

Dipartimento di Scienze Biomediche e Cliniche "L. Sacco”Fabio CorsiRaffaele Allevi Carlo Morasso

Dino Ferrari Centre, Neuroscience Section, University of Milan, IRCCS Foundation Ca' Granda Ospedale MilanStefania CortiMonica Nizzardo

Università degli Studi di Milano, Dipartimento di Scienze della SaluteAnna Maria Di GiulioStephana Carelli