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