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  • 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 neuron not directlytested

    their activationprecedes

    not directlytested

    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 109wholetranscriptome

    mRNAs

    UP-regulated 480 32 194 64 0 0 0 2DOWN-regulated 43 56 33 23 0 0 0 10

    total 523 88 227 87 0 0 0 12lncRNAs

    UP-regulated 16 14 15 17 0 0 0 0DOWN-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