Dalla morte cellulare alle malattie

neurodegenerative…e dal passato uno sguardo al

futuro.

Pierluigi Nicotera MD PhD

1. The calcium hypothesis of cell death.2. Longevity, Ageing and Neurodegeneration.3. Epigenetics and data science.

Prof. Malcovati.Prof. Ventura.Prof. Fratino- Dr. Bellomo.Prof. NappiProf. Finardi.Prof. Luigi ManzoProf. Blandini

proliferation

death

differentiation

death

Regional death programmes Loss of specialised functions

senescence

Cell Death in Multicellular Organisms

Because it is unlikely that active death programmes were programmed in true unicellular species, the first death mechanisms must have originated from the simple failure of survival systems (i.e., ion pumps, channels, energy metabolism, anti-oxidant defence)

Primordial unicellular organisms developed defence mechanism against the environment:

1. Ion pumps and channels to create a sustainable micro-environment different from the Na+ and Ca2+-rich ocean waters.

2. Antioxidant systems as a defence from an oxygen-rich environment.

Vert

ical

evo

luti

on

Horizontal gene transfer

As evolution progressed, initial multicellular organisms developed multiple, more specialised protective functions. Eventually death routines developed to shape the development of multicellular organisms and tissue differentiation.

The Calcium Hypothesis of Cell Death

Calcium overload and neuronal death

Glutamate-releasingnerve ending

AMPA-R

Voltage-gated channel

Other channels

Ca2+

NMDA-R

Ca2+

Excitotoxicity: excess glutamate accumulation

at synaptic or extrasynaptic terminals Bano et al Cell, 2005

Dypbukt et al., JBC, 1994 Bonfoco et al., PNAS, 1995Ankarcrona et al., Neuron, 1995Nicotera, P., Bano D. , Cell 2003

Time (h)0 3 6 9 12

100

75 control = 0.78

Glutamate

Intensity of the insult

Dypbukt et al., JBC, 1994 Bonfoco et al., PNAS, 1995

Cell

Dea

th

Ankarcrona et al., Neuron, 1995

Oxidative stress and different death routines

Different programmes or simply distinct death subroutines?

Leist, M., Single, B., Castoldi, A.F., Kuhnle, S., and Nicotera, P. (1997). J. Exp. Med. 185, 1481–1486.

Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Cell. 1997, 90:405-13

Eguchi Y, Shimizu S, Tsujimoto Y. Cancer Res. 1997, 57(10):1835-40.

+ ATP

apoptosisnecrosis

- ATP

ATP

Intracellular ATP level: a switch in the apoptotic and necrotic cell death routines.

Up to 1 metre50 µm

Text

Synaptic death

Cell body death Axo-dendritic death

Localized death programmes

Synaptic damage isinflicted by BoNT/C

BoNT/ABoNT/C

The actin and microtubule networks aredamaged, tau is abnormally phosphorylated

Mitochondria in the projections remainintact and retain membrane potential

Mitochondria in the cell bodylyse and release cytochrome c

Caspase-3

Caspase-3 execute apoptosisin the cell body

Neurite degeneration is not causedby the block of neurotransmitterrelease and it is not modified bytrophic stimulation

Spatial Control of Cell Death inCentral Neurons

Microglia,activated bydegeneratingneurites mayreleasefactors killingthe cell body

Microglia activation to clear debris?

Berliocchi et al. J.Cell. Biol. (2005) 168, 207

Spatial & temporal distribution of neuronal clearing by microglia in neurodegeneneration

Calcium triggers different death programmes, but it is essential for plasticity and survival

Ca2+

Plasticity

CREBPBcl-2

Ca2+Inhibition of Plasticity

p-AktmTOR

PLCγPIP2

DAG

Ca2+

Ca2+

Extracellular space

Ca2

(1.3 mM)

Endoplasmic reticulum

Receptor

SERCA pump

Ins(1,4,5)P3R

Ca2+

Voltagesensitivechannels

Ca2+

Store-operated channel

Ca2+Ca2+

PMCA

Ca2+

(100 nM)

Mitochondria

PT pore

Ca2+

RyR receptor

Ca2+

Ca2+

Na+

NCX

Ca2+

Glutamate channels

Ins(1,4,5)P3R

Fig. 1

Na+/H+

Ca2+

Uniporter

Calreticulin/Calsequestrin

Intracellular Ca2+ signalling in living cells

Regulation of cell death: the calcium-apoptosis link Sten Orrenius, Boris Zhivotovsky and Pierluigi Nicotera Nat. Rev. Mol. Cell Biol. 4, 552-565, 2003

The enemy at the gates: Ca2+ entry through TRPM7 channels and anoxic neuronal death Pierluigi Nicotera and Daniele Bano Cell: 115, 768-769 (2003)

*

*

Short and long-term nicotine signaling

a4β2a7

Ca2+

CREBPcalcineurin

RyR2

RyR2

Ca2+

nicotine

glutamate

Pre- and post-synaptic nicotine signaling

P

CREBP

•The short term CREB phosphorylation is in part pre-synaptic.

RyR2

CREBP

•The long-term CREB phosphorylation is post-synaptic and sustained by a feed-forward loop involving RyR2.

CICR •Upregulation of the RyR2 amplifies Ca2+-induced-Ca2+ release (CICR).

Nicotine long-term effects on plasticity

require RyR2 upregulation

RyR2 knockout mice

wt/wt KO/KO

➢ FullKOofRyR2resultsinembryoniclethality(~E12.5)

RyR2flox/flox

RyR2 Genomic

locusloxP loxP

RyR2 Genomic

locus

Exon 1 2 3 4 5 6 7 9

loxP

Cre recombinase

Exon 1 2 3 4 5 6 7 8 9

I. Synapsin-Cre II. CamKIIα-Cre

neuron-specific forebrainexcitatoryneurons

-RyR2KOmiceshowdendriticandspinealterationinpyramidalneurons!

-Thelossofspineisconsistentamongthedifferentmodelsanditisnotneurodevelopmentallyrelated!

-ItoccursinabsenceofchangesinLTP

Outcomes

Centers for Disease Control)

Defeating early onset diseases

Rapidly improving lifestyle faster than genetic adaptation

Epigenetic regulation (transcriptome) can adapt faster and be inherited

Increasing Longevity & Adaptation

Longevity can be reprogrammed

mTORsignalingeIF2signalingRegula1onofeIF4andp70S6KsignalingProteinubiqui1na1onpathwayHypoxiasignalinginthecardiovascularsystemNADHrepairPTENsignalingCTLA4signalingincytotoxicTlymphocytes

Enrichedpathwaysamongdifferen4allymethylatedpromotersinagedspermandoldfatheroffspringincluded:

Old>21months

Young4months

F1offspringofoldfathers

Young4months

Young4months

F1offspringofyoungfathers

Exacerbatedaging-associatedpathologyinoldfatheroffspringmice:

renal

tubula

r atro

phy

thyroi

d foll

icle m

orpho

logy

myoca

rdial

fibros

is

muscle

fiber

atrop

hy

microg

ranulo

ma

liver

steato

sis

glomeru

loscle

rosis

bronc

hus-a

ssoc

iated

lymph

atic t

issue

arteri

al ela

stic f

iber c

onten

t

age-r

elated

glan

d like

struc

tures

vasc

ular h

yalin

osis

testis

atro

phy

brain

nitrot

yrosin

e con

tent

-3

-2

-1

0

1

2

3

Coh

en's

d (e

ffect

of a

ging

) Young father offspringOld father offspring

Heritable epigenetic alterations in longevity regulators.

Dan Ehninger & Colleagues

Exacerbated (age-associated) pathology in old father offspring mice

Longevity & Heterogeneous Genomes

Science,2015 350(6256), 94–98. In neurons mutations can occur independent of replication in transcriptionally active chromatin.

Mutations can occur in individual neurons and are not easily detected by normal genomic screens

Epigenetic changes

Damage accumulation

Genetic and Epigenetic factors

Mod. from Benayoun, B.A.& Brunet A. (2015) Nature Reviews16, 593-610

Increased life span increases the risk of genomic vulnerability to stress factors or random errors

What epigenetic factors are most relevant?

Heneka M and Nicotera P, Thoughts on Obesity and Brain Glucose, Cell 165 (2016) Jais, A., … Brüning J, et al. (2016). Cell 165

Metabolism & Inflammation

The role of the innate immune system and cold inflammation Heneka, Latz,

Can we fight chronic inflammation? Linking Pharmaco-epidemiology and Neurodegenerative Diseases

Key result: Anti-diabetic drugs influence risk of developing dementia (people diagnosed with diabetes, data from health insurance company)

145,000 data sets for individuals above 60 yr. Three drugs: Metformin, Rosiglitazone, Pioglitazone

27

A revolution in the life and medical sciences: Single cell genomics

SingleCellGenomics:ausecasethatcurrentlytransformsandrevolu2onizesthebiomedicalsciences

•  inherentlysparsedata

•  profilethousandstomillionsofcellsleadstobigdata

•  verystandardizableusecasetodevelopalgorithmsforsparsedatathatbecomebig

•  Ourmajorusecasetogetstarted

J.&H. Schultze

28

Major computational issues in SCG sparse data

§  Highlevelofmissingdata§  Datawithlowsignal-to-noisera0o§  Biologicalandtechnicalvariabilityinherenttothedata§  Lossofspa0alinforma0onduringdatacapturing§  Currentalgorithmsandso9wareinfrastructuresdonotscalewell§  Hardwarememoryandmemoryperformanceislimi;ngcomputeefficiency

Large data – a human genome

Challenges in biomedical analytics

Bigmedicaldata-genomics

-transcriptomics-epigenomics -metabolomics-microbiomics

SignificantinterconnectionDozensofadditional

externalscientificdatabases

(large,continuousupdates)

DevelopmentofuserinterfacesUsabilityandinteractivityForscientistsandphysicians:Directlyexperienceknowledge

Reliabilityofstatisticalmodelsandanalysis

Memory-Driven Computing

31Architecture for the future of computing

GPU DSP

x86A

SIC

Phot

onic

NeuroQuantum

POWER

SPARC

RIS

C

VAR

M

FPGA

Memory

32

MemoryMemory

Mem

oryM

emory

Mem

ory

MemoryMemory

Memory

Mem

ory

Mem

ory

Mem

ory

Memory

SoC SoC

SoCS

oC

SoC

SoCSoC

SoC

SoC

SoC

SoC

SoC

Future architecture

Memory-Driven Computing

Today’s architecture

From processor-centric computing

33

Mesh-computing infrastructure enables large data sharing while protecting privacy

The German Centre for Neurodegenerative Diseases (DZNE)