Male Wistar Rats (n=55)

Age at recruitment; 8 weeks

• 18 received a control diet• 19 received a Hcy enriched diet (1.7% homocystine)

• 18 received a methionine rich diet (2.4% methionine)

Duration of the study; 5 months

Blood samples were collected

Brain was isolated and one part (about 50 mg from the frontal area and the brain stem)was homogenized in acetic acid for SAM and SAH assay

Phosphorylated Tau protein was tested in brain tissues, by using immunoflourescence microscope

Effect of Dietary HHCY on Brain Metabolites; in-vivo Experiments

Brain stem

Frontal cortex

Pla

sma t

Hcy

, m

ol/L 225

200

175

150

125

100

75

50

25

0

Metn=18

Hcyn=18

Controln=19

7.0 = median

74.4

112.9*

*

Diet

* p<0.001 compared to the control diet; † compared to the Hcy group

Effect of Dietary HHCY on Brain Metabolites; in-vivo Experiments

1.7% homocystine

2.4% methionine

Male Wistar Rats (n=55)Age at recruitment; 8 wk duration 5 months

Obeid et al., submitted

The diets caused disturbances in methylation markers in the brain (frontal cortex)

Obeid et al., submitted

20

16

12

8

4

0

SAM/SAH ratio/g tissue

10.2

7.7

4.0

HCYN=18

MethN=18

Control N=19

*

*

1.7% homocystine

2.4% methionine

Male Wistar Rats (n=55)Age at recruitment; 8 wk duration 5 months

* P<0.01

Plasma folate, nmol/L

SA

M/S

AH

rati

o in b

rain

tis

sues

300280260240220200180160140

25

20

15

10

5

0

Frontal cortex

R=0.36, p=0.007

Methylation Potential in Rat Brain correlates with plasma vitamins

900800700600500400300

25

20

15

10

5

0

Plasma vitamin B12, pmol/L

Frontal cortex

R=0.37, p=0.006

Experimental HHCY caused P-tau accumulation in rats brain

16B Control diet 1B Methionine rich diet (2.4%)26B HCY rich diet (1.7%)

Fontal Cortex, left hemisphere, fluorescence detection using PHF-Tau phosphorylated at serine 396 + Alexa flour 488

Axio Vision REL 4.4 Carl Zeiss®

Homocystine diet

N = 6

Methionine dietN = 6

Control dietN = 9

Opti

cal densi

ty o

f P-t

au p

rote

in,

pix

el

1250

1000

750

500

250

0

312

912

891

* *

• Experimental hyperhomocysteinemia decreases the methylation potential and increases P-tau accumulation in rat brain

• The methylation potential in rat brain correlates directly with serum folate and B12 but inversely with Hcy

• A reduced methylation potential correlates with higher concentrations of markers of neurodegeneration, P-tau, APP, -amyloid and α-synuclein

• α-synuclein a new marker of neuronal degeneration in PD correlated inversely with SAM but positively with amyloid-β

• The cognitive performance in PD correlated directly with the methylation potential but inversely with amyloid-β

• HHCY in rats causes significant P-tau accumulation in brain

• The brain methylation potential correlates with serum folate and B12

• P-tau accumulation in rats brain correlates with HCY + inversely with folate

• PP2A activity in rats brain correlates with folate + inversely with P-tau

• In conclusion, B-vitamin deficiency has via hypomethylation a significant impact on the process of neurodegeneration

Summary / Final Comments

↓ Methylation

Protein P

P

P

P

↑ Phosphorylation

↓ PP2A- activity

Fats, oil

sweets

↓ FolateB12, B6, Choline

Tangling disease

Functional proteinsTau P-tau APP pAPP

NF-HpNF-H

Impaired repair/controlmechanisms

• DNA methylation• β-Amyloid• α-Synuclein• PC;SP/PE• Ubiquitin proteasome system• PIMT; Pin-1

CH3

C

G

X

Acknowledgements

PD Dr. Markus Herrmann

Dipl. Chem. J.P. Knapp

Dr. Natascha Umanskaja

Department of Clinical Chemistry

M. Kasoha

J. Schlundt

Department of Neurology

Financial Support

HOMFOR

PhD students

Prof. K. Fassbender

PD Dr. U. Dillmann P. Kostopoulus

Jun.-Prof. Dr. Rima Obeid

A Schadt