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