Dr. Gabor Juhasz of ELTE University in Budapest discusses use of Pathway Studio in the study of neurodegenerative diseases such as Alzheimer’s Disease.

1. We are interested in knowing how the brain works in health
and ill-health at molecular level

2. Neuroscience: Alzheimer's disease
Lennart Mucke
Nature 461, 895-897(15 October 2009)

3. (A) Aggregate sizes in 200 μM Aβ(1–42) solutions after 0-h, 24-h, and 72-h incubation in ACSF at room
temperature. Each bar represents measurement from at least 200 individual aggregates. (B) ThT
fluorescence of Aβ solutions after the indicated incubation periods in ACSF. The elevated ThT fluorescence
intensity suggests an increase in the amyloid-like structure content with incubation time (* p < 0.05). (C– E)
AFM images of Aβ samples after 0-h, 24-h, and 72-h incubation in ACSF, respectively. The color code on the
left indicates the height-trace range.
Characterization of the size, morphology and
amyloid-like properties of Aβ aggregates.

4. (B) Time course of the effect of Aβ solutions (1 μl, 200 μM) on the pSpike (n = 5 for each group) (* p < 0.05).
The effects of the 24-h and 72-h solutions become statistically significant compared to the control (ACSF)
group from 30 min after the end of the injection period. (C) Lack of effect of the Aβ solutions on the pEPSP
of freely moving rats.
Effects of Aβ solutions on the pSpike
amplitude in freely moving rats.
0.0 0.8 1.6 2.3 3.1 3.9 4.7 5.5 6.2 7.0
0.0
0.5
1.0
1.5
fEPSP slope
mV
msec
population spike amplitudestim

5. Intracellular amyloid- in Alzheimer's disease
Frank M. LaFerla, Kim N. Green & Salvatore Oddo
Nature Reviews Neuroscience 8, 499-509 (July 2007)
doi:10.1038/nrn2168
The clinical maze of mitochondrial neurology
Salvatore DiMauro, Eric A. Schon, Valerio Carelli &
Michio Hirano
Nature Reviews Neurology 9, 429-444 (August 2013)
doi:10.1038/nrneurol.2013.126
MAM: mitochondria associated membrane

6. Int J Alzheimers Dis. 2011 Mar 15;2011:925050.
doi: 10.4061/2011/925050.
Amyloid-Beta interaction with mitochondria.
Pagani L1, Eckert A.

7. Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18670-5.
doi: 10.1073/pnas.1006586107. Epub 2010 Oct 11.
Early deficits in synaptic mitochondria in an
Alzheimer's disease mouse model.
Du H1, Guo L, Yan S, Sosunov AA, McKhann GM, Yan SS.

8. Representative electron microscopic images of non-synaptic (A1–3) and synaptic (B1–3) mitochondrial samples.
Mitochondria occupy the majority of the preparations. The area percentages of mitochondria (the area occupied by
mitochondria/the area occupied by all structure (mitochondria + mostly unidentified debris)) are shown for non-
synaptic (C) and synaptic (D) samples. Scale bars = 1 μm for A1 and B1, 0.5 μm for A2 and B2, and 0.25 μm for A3
and B3.
Validation of the purity of the mitochondria preparations with
electron microscopy.

9. The FSC (forward scatter)/SSC (side scatter) dot plot (A) shows the gating strategy for mitochondria particles, which
was the same for all samples. Representative dot plot of unstained (B1) and double stained with NAO and DiIC6 (B2)
samples are also shown. The quadrant analysis demonstrates the purity/homogeneity of the samples. The markers
used to select events positive for NAO (C and D) or DiIC1 (E and F) show highly selective and homogenous staining of
both non-synaptic and synaptic mitochondria.
Representative FACS analysis of synaptic (A, B1, B2, D and F) and
non-synaptic (C and E) mitochondria samples (NAO: mitochondrial
staining, DiIC1: mitochondrial membrane potential staining).

10. Functional clustering of protein differences between synaptic an
non-synaptic mitochondria

11. Immunopositive bands are shown at 50 kDa for Sucla2, and 40 kDa for Idh3a, 33 kDa for C1qbp, and 25 kDa for Sod2
in synaptic and non-synaptic mitochondria (A). Densitometric analysis was performed for all 4 proteins (n = 6–6). For
Idh3a, both bands were combined for the analysis. The results are shown in B–E. The expressions of Sucla2 (B) and
C1qbp (D) were significantly increased while Idh3a (C) and Sod2 (E) expressions were significantly decreased in the
synaptic compared to the non-synaptic mitochondria samples (***: p < 0.001, **: p < 0.01). Error bars indicate s.e.m.
Western blot validation of changes in Sucla2 and Idh3a expression
in synaptic mitochondria.

12. White arrow indicates amyloid plaque, that appears in 6-, and spread in 9 months old APP/PS1 mice brain. Higher
magnification image of an amyloid plaque stained with reduced osmium (d). Low power electron micrograph of
the same amyloid plaque as shown in d (h). Scale bars: a, b, c, e, f, g – 500 µm, d, h – 20 µm (CA1: cornu ammonis
1, DG: dentate gyrus, cc: corpus callosum).
Representative light microscopy images of 3, 6 and 9 months old
B6 control (a, b, c) and APP/PS1 transgenic mice (e, f, g)
hippocampal and cortical brain regions.

13. Representative 2D-DIGE image with labeled locations of non-synaptic mitochondrial
significant protein changes on the gel (e). Fold changes of the non-synaptic
mitochondrial proteins of 3 (f), 6 (g) and 9 months old animals (h).

14. Representative 2D-DIGE image with labeled locations of synaptic mitochondrial
significant protein changes on the gel (a). Fold changes of the synaptic
mitochondrial proteins of 3 (b), 6 (c) and 9 months old animals (d).

15. Mitochondrial localization of altered non-synaptic mitochondrial proteins in 3, 6
and 9 months old APP/PS1 brain. Green, yellow and orange background indicates
proteins from 3, 6 and 9 months old APP/PS1 animals, respectively.

16. Mitochondrial localization of altered synaptic mitochondrial proteins in 3, 6 and 9
months old APP/PS1 brain. Green, yellow and orange background indicates
proteins from 3, 6 and 9 months old APP/PS1 animals, respectively.

17. Western blot validation of changes in Ethe1 and Htra2 expression in synaptic
mitochondria of 3, 6 and 9 months old APP/PS1 mice.
Densitometric analysis was performed for Ethe1 and HtrA2 (n=6). The levels of Ethe1 and Htra2 were significantly
decreased in synaptic mitochondria of 3 and 9, while significantly increased in 6 months old APP/PS1
mice Representative immunopositive bands are shown under the diagram. (Student’s t-tests, ***: p < 0.001; **: p <
0.01; error bars indicate s.e.m.).

18. Common regulator analysis of altered mitochondrial proteins
Blue edges indicate the relationships between common regulators/targets and altered mitochondrial proteins. Yellow
indicates mitochondrial proteins that were significantly changed and have common targets or regulators. Green
indicates the common regulator (a) or target (b) proteins.

19. Common target analysis of altered mitochondrial proteins
Blue edges indicate the relationships between common regulators/targets and altered mitochondrial proteins. Yellow
indicates mitochondrial proteins that were significantly changed and have common targets or regulators. Green
indicates the common regulator (a) or target (b) proteins.

20. The Tnf-α (common regulator and target protein) induced extrinsic and the Aβ-
mediated Htra2, Ethe1, Pebp1 and Vdac1 related mitochondrial apoptotic
pathways connections
The regulation pathways suggest
the importance of Aβ effect on
NFκB signaling and caspase
cascade pathways.

21. • Close juxtaposition between the mitochondria and ER -> MAM
• Intracellular lipid raft-like microdomain
• Known functions: Lipid- , Glucose- ,Cholesetherol-metabolism , regulation of
Ca2+ homeostasis , formation of autophagosomes
• Possibly implicated in the regulation of glucose metabolism and viral infactions
formation of autophagosomes

22. Involvement of MAM in lipid synthesis and transport
MAM localized proteins involved in lipid
synthesis:
• DGAT(Diglyceride acyltransferase)
• PSS (phophatidyl serine synthase)
• ACAT1(acetyl-CoA acetyltransferase 1)
• FACL4 (Long-chain-fatty-acid--CoA ligase 4)
MAM localized proteins involved in
lipis transport:
• ERMES: Mdm34,Mdm10,Mdm12,Mmm1 (ER-
mitochondria encounter structure)
• S100B
• apoE, apoB, apoC (apolipoprotein E,B,C)
• Sigma1R

23. MFN2 : Mitofusin 2
GRP75(Hspa9): glucose-regulated protein
VDAC1 : voltage-dependent anion selective
channel protein 1
IP3R :inositol-1,4,5-trisphosphate receptor
FACL:Long-chain-fatty-acid--CoA ligase
PML 1 :RING finger protein 71
->inactivate Akt 1
PACS1 :Phosphofurin acidic cluster sorting
protein 1
Involvement of MAM in the regulation of Ca2+ homeostasis
Rosario Rizzuto, Diego De Stefani, Anna Raffaello & Cristina Mammucari , 2012, Nature
In the presence of mitochondrial Ca2+ buffering activity, the initial increase of cytosolic Ca2+ levels again
induce Ca2+ release from the ER .However, as mitochondrial Ca2+ uptake reduces the [Ca2+]c at the
mouth of the channel, no negative feedback by cytosolic Ca2+ is exerted and, thus, Ins(1,4,5)P3R activity
is sustained and Ca2+ release from the ER is prolonged.

24. Involvent of MAM dyfunction in the pathogenesis of
Alzheimer’s disease

25. Eric A. Schon and Estela Area-Gomez 2012
Increased function and length of regions of contact between
ER and mitochondria

28. Representative gel image

29. Pathway Studio Analyse– Possibly implicated in insulin
resistance?

30. Conclusion
• There are early changes in mitochondria and
MAM fare before behavioral symptoms
develop in AD
• Early changes are molecular: possibility of
early biomarkers.
• All early changes are small changes in several
proteins: protein network fine tuning is
probably the earliest change in the
mitochondria in AD.

31. Very rich who has good questions in science,
but very poor who has only answers

32. Questions opened
• How far a mouse model reflects the early
mechanaism of the human AD?
• Is there a realistic way to get human brain
samples before AD symptoms develop?
• When AD begins as a mitochondrial protein
network miss-tuning, how can we detect it at
the level of metabolism?
• Could brain metabolic imaging be AD specific?

33. Katalin Völgyi
Balázs Györffy
Edina Udvari
Kata Badics
Vilmos Tóth
Adrienna Katalin Kékesi, PhDProf. Gábor Juhász
Lilla Ravasz
Péter Gulyássy
Antonia Arszovszki, PhD
Zsolt Borhegyi, PhD
Dóra Madarasi
Flóra Fedor
Gréti KozocsayMihail Todorov