Thomas ArendtPaul Flechsig Institute of Brain Research

University of Leipzig, Germany

Alzheimer´s disease -

an evolutionary perspective

“Nothing in biology makes sense except in the light of evolution”(Th. Dobzhansky)

3rd Alzheimer’s Focused #C4CT Concussion Awareness Summit at United Nations

secretases

COOHNH2

loss of synapsesAß-peptide

Prevailing concepts amyloid cascade hypothesis

PSEN1/2 & APP FAD mutations

Aß-aggregates

hyperphosphorylation and aggregation of tau-protein

APP

Aß-plaques

neurofibrillary tangles

Neuronal dysfunction and death

Cerebralization:accelerated brain growth in hominid evolutionincrease in:

Why do we get Alzheimer´s disease ?

1500 cm3

1000 cm3

500 cm3

brain volume

million years-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0

Homo sapiens

Homo erectus

Australopithecus afarensischimpanzee

• AD is unique to human• major genetic risk factor: ApoE polymorphism is unique to human

• cortical synapses • brain plasticity • cognitive capacity

Why do we get Alzheimer´s disease ?

Katerina Semendeferi & Hanna Damasio, J. Human Evolution (2000) 38:317-332.

Allometric volume plots

brain regions, highly vulnerable to AD-pathology have been elaborated in most recent hominid evolution

acceleration

AD vulnerability

deceleration

parie

to-o

ccip

ital

front

al

tem

pora

l

cere

bellu

m

Development of neurofibrillary degenerationinversely recapitulates brain development

brain structure: last in – first out progression of myelination

[acc. to Flechsig]

progression of neurofibrillarydegeneration [acc. to Braak]

limbic cortex

non-primary association cortex

primary sensory association cortex

primary sensory & motor cortex

degeneration / regression?

development

Reversal of developmental behavioural hierarchy in AD „ last in - first out “

Reversal of developmental behavioural hierarchy in AD „ last in - first out “

Sequence at which function is acquired during development

(J. Piaget)

Sequence at whichfunction is lost in AD

•Perform complex activities of daily life

•Put on clothing properly• Perform mechanics of toileting correctly• Maintain urinary continence

•Maintain fecal continence

•Say a few intelligible words

•Walk independently

•Sit up independently

•Smile

•Hold up head independently

How to make a bigger brain ?

Kornack & Pasko Rakic PNAS (1998) 95: 1242-1246.Krubitzer & Kahn Prog. Neurobiol. (2003) 70: 33-52.Hill & Walsh; Nature (2005) 437: 64-67.

expansion and laminar elaboration of primate neocortex:

accelerated cell-cycle kinetics withdelayed maturation

• extended duration of cell cycle• more total rounds of cell division

Achilles heel• increased risk of mitotic errors• special requirements of differentiation control

Re-expression of cell cycle markers in AD

CDK 4

cyclin D

p27Kip1

p16INK4a

p18INK4c

G1

S

M

G0

cell death CDK4cyclin D

p16INK4a

p27Kip1

cyclin A p27Kip1

CDK1

p18INK4c

CDK5

cyclin E

cyclin A

CDK 1

cyclin E

Gärtner et al. Neuroreport (1995)Arendt et al. Hoppe Seyler (1993), Neurorep. (1995), Neurosci. (1996)

10µm

2n 4n

Mosch et al. J.Neurosci. 2007; Arendt et al. Am.J.Pathol. 2010

Chromosom 7Chromosom 17

10µm

The human brain is a mosaic with hyperploid neuronsThe human brain is a mosaic with hyperploid neurons

controlAD

30

4

8

12

hybr. spots

*

*

4

(%)

Slide based cytometry

Lenz et al. Progr.Biomed.Opt.Imaging 2004, 5322:146-56.Mosch et al. Cytometry A. 2006, 69:135-8Mosch et al. J. Neurosci. 2007, 27:6859-67Arendt et al. Int. J.Mol.Sci. 2009, 10:1609-27

AD: 50% increase in single cell DNA content

AD 29% 1.1%Control 12% 0.4%

0

10

20

30

40

4n

10

20

30

40

10

20

30

40

2n - 4n

*

*

num

ber o

f neu

rons

[%]

PCR amplification of alu repeats

0

10

20

30

0

10

20

30

rela

tive

frequ

ency

(%)

AD

control

4n

2n

single-neuron DNA content (pg)

2n

controlAD

control preclinical AD

mild AD severe AD

CDR 0 0 0.5 3

Braak 0 I-II B

III-IV B-C

V-VI C

CERAD normal possible probable definite

NIA negative low-intermediate

Intermed. high

Arendt et al. Am.J.Pathol. 2010, 177: 15-20

Hyperploidy is an early (preclinical) event

controlpreclinical ADmild ADsevere ADnu

mbe

r of h

yper

ploi

d ne

uron

s (m

m-2

)

disease progression

Arendt et al. Am.J.Pathol. 2010, 177: 15-20

Hyperploidy occurs prior to cell deathHyperploidy occurs prior to cell death

additional challenge: consuming reparative/compensatory capacity

•concussion•oxidative stress•chronic intoxication•metabolic imbalances•latent infections

num

ber o

f hyp

erpl

oid

neur

ons

(mm

-2)

controlpreclinical ADmild ADsevere AD

Selective cell death of hyperploid neurons

entorhinal cortex (n=28)

disease progressionArendt et al. Am.J.Pathol. 2010, 177: 15-20

~90%

~10%

Cell cycle proteins subserve alternative functions in differentiated neurons: regulation of synaptic plasticity

Schmetsdorf et al. Cerebral Cortex (2007) Schmetsdorf et al. Eur.J.Neurosci. (2009)

CDK 4 regulates structural plasticity

siRNA CDK4

control

100 20050 1500relative neurite length (%)

*

anti-Cdk4

cyclin D CDK 4constitutive expression

CDK 4 Cyclin D

axonal localisation

control siRNA CDK 4

The dual functions of cell cycle regulators in neurons

development adult

“canonical cell cycle regulators”(e.g. CDKs, cyclins, CDK-inhibitors …)

alternative effector pathways

control of

the cell cycle

control of

synaptic

plasticity

maturation (differentiation)

developmental switch

neurodegeneration (de-differentiation)

degenerative switch

'Dr. Jekyll & Mr. Hyde concept'Is the risk of a phylogenetic regression based on the persistence ofdevelopmentally primitive aspects in a highly developed biological system.

non-neuronal dividing cells

(e.g. fibroblasts)

loss of neighbouring cells

loss of contaction inhibition

proliferation

tissue repair

postmitotic neurons

(non-dividing)Cellular evolution:synapse formation on the expense of the ability to proliferate

Regression:loss of synaptic input is mis-regarded as loss of contact inhibition

triggers ancient programm of repair that involves cell cycle activation

network re-organisation

re-activation of proliferation

neuronal death

functional deafferentation

Hibernation: a model for repeated cycles of synaptic regressionHibernation: a model for repeated cycles of synaptic regression600

400

200

0

40

35

30

25

20

15

10

586420

time (h)

met

ablo

icra

te (m

lO2

h-1 )

hear

t rat

e (B

MP)

body

tem

pera

ture

(°C

)

sep oct nov dec jan feb mrt

40

30

20

10

body

tem

pera

ture

(°C

)

0

euthermic torpor arousal

spin

e de

nsity

(spi

nes/

µm)

metabolic rate depression

body temperature

energy expenditure

reversible „brain shrinkage“

metabolic rate depression

body temperature

energy expenditure

reversible „brain shrinkage“

AD (PHF)-like phosphorylation of tau in hibernation

Brown bear (Ursus arctos)

European ground squirrel

Arendt et al. J.Neurosci. (2003)Härtig et al. Eur.J.Neurosci. (2007)

Stieler et al. (2008), Stieler et al. PlosOne (2011)

euthermic torpor arousal

Arctic ground squirrel(Spermophilus parryii)

pS202+pT205 (AT8)

NeocortexHippocampusCerebellumMidbrainBrainstem

Linking metabolic depression (diabetes ?) to AD-type pathology

Hamster (Cricetinae)

euthermic torpor longtorpor short arousal short arousal long

synaptophysin (stratum lucidum): mossy fibre input

MAP 2 (green) / PHF-tau: AT8 (red) synapticdetachement

gradual re-appearance of synaptic afferentation

torpor

Accumulation of PHF-tau at postsynaptic sites coincideswith synaptic detachement of excitatory afferentation

0

2x105

lesionvolume(mm)

3

lesi

onvo

lum

e(µ

m3 )

p16I

NK

4ain

duce

dp1

6IN

K4a

repr

esse

d

1x105

0

2x105

lesionvolume(mm)

3

lesi

onvo

lum

e(µ

m3 )

p16I

NK

4ain

duce

dp1

6IN

K4a

repr

esse

d

1x105

Blocking cell cycle activation by p16INK4A is neuroprotectiveBlocking cell cycle activation by p16INK4A is neuroprotective

conditional (tet), neuron-specific (CamKII) expression of p16INK4A

protects against NMDA- induced cell deathconditional (tet), neuron-specific (CamKII) expression of p16INK4A

protects against NMDA- induced cell death

p16INK4A Fluoro-Jade B

INK4A

INK4A

300µm

no lesion

no lesion

NMDA

NMDA

B300µm

no lesion

no lesion

AA 300µm

p16

in

duce

dIN

K4A

p16

re

pres

sed

INK

4A

p16INK4A Fluoro-Jade B

INK4A

INK4A

300µm

no lesion

no lesion

NMDA

NMDA

B300µm

no lesion

no lesion

AA 300µm

p16

in

duce

dIN

K4A

p16

in

duce

dIN

K4A

p16

re

pres

sed

INK

4Ap1

6

repr

esse

dIN

K4A

expression of the transgene excitotoxic cell death

cell death

S

G0

G1

M

p16INK4a

CDK4

Arendt 2000; 2003

lesion volume (µm3)

p16I

NK

4Ain

duce

d

p1

6IN

K4A

repr

esse

d

ischemic cell death (middle cerebral artery occlusion; MCAO)

5

0

4

3

2

1

lesi

on v

olum

e (m

m3 )

MC

AO

+ p

16 re

pres

sed

MC

AO

+ p

16 in

duce

d

*

late

ralis

atio

n (%

)

MC

AO

+ p

16 re

pres

sed

MC

AO

+ p

16 in

duce

d

no M

CA

O

*

0

20

40

60

80

100

Expression of p16INK4a as universal mechanism of neuroprotection

MC

AO

+ p

16IN

K4A

repr

esse

dM

CA

O +

p16

INK

4A in

duce

d

lesion volume

(mm3)lateralisation

(%)

1mm

MCAO + p16INK4A repressed

MCAO + p16 INK4A induced 1mm1mm

MCAO + p16INK4A repressed

MCAO + p16 INK4A induced 1mm1mm

MCAO + p16INK4A repressed

MCAO + p16 INK4A induced 1mm1mm

MCAO + p16INK4A repressed

MCAO + p16 INK4A induced 1mm

MC

AO

+ p

16IN

K4A

repr

esse

dM

CA

O +

p16

INK

4A in

duce

d

no M

CA

O

Arendt 2000; 2003

p16INK4A

injection side

anti‐p16

Fluoro-Jade

Fluoro-Jade

anti‐p16

NMDA-inducedneurone death(Fluoro-Jade)

1mm

1mm 1mm

A

Lesi

on v

olum

e [%

]

0

10

20

30

*

p<0.01

Our vision: neuroprotection by gen-transfer of p16INK4AOur vision: neuroprotection by gen-transfer of p16INK4A

Stieler et al. Neuroreport (2001) 12:3969-3972

Cell cycle dysregulation on peripheral lymphocytes as diagnostic blood biomarker of ADCell cycle dysregulation on peripheral lymphocytes as diagnostic blood biomarker of AD

CD69 expression after mitogenic stimulation (PHA, 12µg/ml)FACscan flow cytometry

ADcontrol

stim

ulat

ion

inde

x

CD4+ CD19+st

imul

atio

n in

dex

CD4+ CD19+

mini mental scale

r=0.73

r=0.63

LymPro Test®AD patient

AD - patient

Normal subject

AD: MCI: control: n=43; n=14; n=18n=27; n=45

synaptic detachement & abnormal synaptic turnover

phylogenetic regression

cell cycle activation & partial DNA replication

second challenge –consuming reparative capacity

neuronal death

LymPro Test®

The evolutionary 'Dr. Jekyll & Mr. Hyde concept‘ of ADand emerging diagnostic & therapeutic targets

p16INK4a

gentherapy

amyloid cascade

University of LeipzigPaul Flechsig Institute of

Brain Research

Birgit MoschMarkus MorawskiUwe UeberhamMartina BrücknerMax HolzerJens StielerWolfgang HärtigUlrich GärtnerStefanie SchmetsdorfTorsten Bullmann

Support by the families of our patientsis gratefully acknowledged

University of FlorenceSandro Sorbi

Benedetta Nacmias

University of JenaOtto W. Witte

University of BristolJames Uney

Stephen KellyL.F.Wong

Polish Academy of Science

Barbara Nawrot

University of AlaskaBrian Barnes

Franziska KohlOivind Toien

Freistaat Sachsen

Freistaat Sachsen

Amarantus BioscienceHolding, Inc.

Gerald E. Commissiong