multiple layers of cdk5r1 regulation in alzheimer’s disease

Venturin Marco

RNA 2019

PAVIA, 27-28 giugno 2019

Multiple layers of CDK5R1 regulation in Alzheimer’s disease

Venturin Marco

IL METABOLISMO DELL’RNA NELLE MALATTIE NEUROLOGICHE


Venturin Marco

RNA 2019


CDK5R1

RNA-binding proteins

MicroRNAs

Long non-coding RNAs

Outline

Venturin Marco

RNA 2019


Cyclin-dependent kinase 5, regulatory subunit 1 (p35)

- highly expressed in CNS

- encodes p35, a specific activator of the CDK5 kinase

CDK5R1

CDK5R1

p35

p35

CDK5

p35/CDK5 FUNCTIONS

Venturin Marco

RNA 2019


Implication in the pathology of Alzheimer’s disease

and other neurodegenerative diseases

THE ‘DARK SIDE’ OF p35/CDK5

Venturin Marco

RNA 2019


924 pb 2725 pb

5’-UTR 3’-UTRCDS

CDK5R1 GENE

Structure and conservation

3’-UTR

Long

Highly conserved

AU-rich elements

Regulation at

post-transcriptional

level?

Venturin Marco

RNA 2019


CDK5R1


MicroRNAs


Outline

Venturin Marco

RNA 2019


Identification of a CDK5R1 3’-UTR U-rich region bound by protein factors

SK-N-BE

Zuccotti et al., BBA 2014

Venturin Marco

RNA 2019


nELAV proteins interact with the R1 region of CDK5R1 3′-UTR and have a

positive regulatory activity on CDK5R1 expression

SK-N-BESK-N-BE

HEK-293

HEK-293


Venturin Marco

RNA 2019


hnRNPA2/B1 protein interacts with the R1 and R2 regions of CDK5R1 3′-UTR

and displays a negative regulatory activity on its expression

SK-N-BE

hnRNPA2/B1


Venturin Marco

RNA 2019


CONCLUSIONS - I

A. nELAVs and hnRNPA2/B1 proteins bind a specific U-rich region in

CDK5R1 3’-UTR

B. nELAVs have a stabilizing effect on CDK5R1 transcript and

positively regulate its expression

C. hnRNPA2/B1 has a destabilizing effect on CDK5R1 mRNA and

negatively regulates its expression

Venturin Marco

RNA 2019


CDK5R1


MicroRNAs


Outline

Venturin Marco

RNA 2019


Prediction of miRNA target sites within CDK5R1 3′-UTR

The miR-15/107 familyPiCTar predictions

Venturin Marco

RNA 2019


The miR-15/107 family

Expression miR-15/107 targets

Targets and molecular processes in AD

APP (miR-16, miR-195)

BACE1 (miR-107, miR-195)

Amyloidogenesis (miR-195)

APP/Tau phosphorylation (miR-15a, miR-16)

Venturin Marco

RNA 2019


miR-15/107 family modulates CDK5R1 expression - I

Moncini et al., Mol. Neurobiol. 2017

Venturin Marco

RNA 2019


miR-15/107 family modulates CDK5R1 expression - II

SK-N-BE


Venturin Marco

RNA 2019


miR-15/107 miRNAs do not influence CDK5 expression but its kinase activity

Thr668-


Venturin Marco

RNA 2019


miR-15/107 family is downregulated in AD temporal cortex and hippocampus

CDK5R1 mRNA levels are increased in AD hippocampus


Venturin Marco

RNA 2019


Inverse correlation between miR-15/107 and

CDK5R1 expression in hippocampus


Venturin Marco

RNA 2019


CONCLUSIONS - II

A. The miR-15/107 group of miRNAs is involved in CDK5R1 regulation

B. miR-15/107 miRNAs are able to reduce APP phosphorylation at

threonin 668, suggesting their role in the regulation of CDK5 activity

(without affecting CDK5 levels)

C. The miR-15/107 family is downregulated and CDK5R1 levels are

upregulated in AD brains

Venturin Marco

RNA 2019


CDK5R1P

-

miR-15/107

nELAVhnRNPA2/B1

+-

CDK5

Microtubules TAU

APP

Neurofilaments

p35

AD (Aβ?)

AD AD

The picture so far…

Venturin Marco

RNA 2019


CDK5R1


MicroRNAs


Outline

Venturin Marco

RNA 2019


LSD1REST

CoREST

nLSD1

-

CDK5R1P

-

miR-15/107

nELAVhnRNPA2/B1

+-NEAT1

MALAT1

HOTAIR (interagisce con il complesso LSD1/CoREST/REST)

MALAT1 (modula lo splicing alternativo)

Candidate lncRNAs

Toffolo et al.,

J. Neurochem. 2014

Tsai et al., Science 2010

Tripathi et al., Mol. Cell 2010

Venturin Marco

RNA 2019


NEAT1, MALAT1 and HOTAIR regulate CDK5R1 and miR-15/107 expression

HeLa

Spreafico et al., Int. J. Mol. Sc. 2018

Venturin Marco

RNA 2019


MALAT1 modulates the nLSD1/LSD1 ratio

pBS-Splicing MG800

% 8a = h nLSD1/(h LSD1 + h nLSD1)

ASO MALAT1

rqf-PCR

Unpublished data

Venturin Marco

RNA 2019


CDK5R1P

BDNF

NGF

TNF-α

ERKEGR1

+

MALAT1 positively regulates EGR1 levels

MALAT1

+


Chen et al., 2016

Venturin Marco

RNA 2019


No difference of MALAT1 expression between AD patients and controls


Venturin Marco

RNA 2019


CN AD0

1

2

3

4

5

6

NE

AT

1 n

orm

alize

d f

old

ch

an

ge

Hippocampus

*

CN AD0.0

0.5

1.0

1.5

2.0

2.5

NE

AT

1 n

orm

alized

fo

ld c

ha

ng

e

Cerebellum

*

NEAT1 is Upregulated in AD Temporal Cortex and Hippocampus


Venturin Marco

RNA 2019


Unpublished data

nLSD1/LSD1 ratio in AD vs control brains

Venturin Marco

RNA 2019


Unpublished data

Negative correlation between nLSD1/LSD1 ratio and CDK5R1

expression in AD hippocampus

Hippocampus

Venturin Marco

RNA 2019


CONCLUSIONS - III

A. NEAT1, HOTAIR, and MALAT1 long non-coding RNAs differently

regulate CDK5R1 expression

B. NEAT1, HOTAIR, and MALAT1 upregulate miR-15/107 expression

C. MALAT1 enhances CDK5R1 expression by upregulating EGR1

transcription factor

D. NEAT1 is upregulated in AD temporal cortex and hippocampus:

protective or pathogenic mechanism?

Venturin Marco

RNA 2019


What is missing?

RBPs

lncRNAs

miRNAs

Interplay?

Interaction?

1

3 CDK5R1 mRNA subcellular localization

starBase starBase

2 NEAT1_1 vs NEAT1_2, paraspeckles in AD?

ZIP codes? Role of ELAV proteins?

THANKS TO…

Prof. Paola Riva

Silvia Moncini

Paola Zuccotti

Marco Spreafico

Andrea Barbieri

Marta Lunghi

Alice Valmadre

Prof. Elena Battaglioli

Francesco Rusconi

Barbara Grillo

Venturin Marco

RNA 2019


Prof. Cecilia Gelfi

Sara De Palma

Daniele Capitanio

Prof. Antonia Ratti

Claudia Colombrita

Prof. Michela A. Denti

Margherita Grasso

Francesca Fontana

Valerio Del Vescovo

multiple layers of cdk5r1 regulation in alzheimer’s disease

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