Download Sequencing by Synthesis

Article #3: A High-Coverage Genome Sequence from
an Archaic Denisovan Individual Science 338:222(2012)
Made Possible By:
Improved Ancient DNA
Recovery Method
(from ssDNA)
Improved DNA
Sequencing Technology
Denisova Cave in Siberia
-source of bone fossils from
Neandertal and Denisovan
Archaic Human Groups
Next Generation Sequencing Technology
(Sequencing by Synthesis)
Genetic Features Unique to Modern Humans
-became fixed after divergence from Denisovans and Neandertals
111,812 Single Nucleotide Changes (SNCs)
9,499 insertions and deletions
260 SNCs result in amino acid change, 72 affect splicing
patterns, 35 affect transcription
Among 23 most conserved changes in modern human
populations, eight affect brain function or nervous system
function (cell adhesion, energy metabolism, microtubule
assembly, neurotransmission)
Some implicated in autism and other neurological disorders
Article #4: Mitochondrial Alterations near Amyloid Plaques in an
Alzheimer’s Disease Mouse Model Journal of Neuroscience 33:17042 (2013)
What was known before the study?
In vitro studies suggested a link between Amyloid β plaques and
mitochondrial dysfunction.
What new question is the study trying to answer?
All evidence suggesting this link comes from in vitro or
post mortem studies. An animal model is needed for in vivo
studies.
What reagents or techniques were needed for the study?
APPswe:PSEN1dE9 transgenic mice-already made
Multiphoton Microscopy
Amyloid β Plaques in Alzheimer’s Disease
Aβ42
tau
outside
cells
inside
cells
Mutations associated w/ familial
early onset Alzheimer’s:
APP and γ-secretase subunits (PSEN)
APPswe:PSEN1dE9
human disease-associated alleles
Multiphoton Microscopy
Another super-resolution microscope with low phototoxicity
Uses two laser beams of low energy, long λ infrared light to
simultaneously excite a fluorochrome to emit higher energy,
short λ light in fluorescent range
-optical sectioning effect without pinhole aperture (at point of 2 laser
beam dissection): improving resolution
-low energy long λ light minimizes phototoxicity and allows
penetration to greater depths in specimen (500 µm in this study)
(lattice light sheet 100 µm)
Fig. 1 Mitochondrial loss and structural abnormalities in living APP/PS1 transgenic mouse
brain.
A) mt-GFP observed in layer II-IV
neuronal mitochondria (Lentiviral
Infection)
-no toxicity from mtGFP
B) mtGFP staining absent w/in
16 μm zone of Methoxy X04labeled Aβ plaques
C) AAV mediated expression of
neuronal cytoplasmic GFP &
mtGFP: similar absence of mtGFP
near plaques
D) dystrophic morphology of
GFP-labeled neurites lacking
intact mitochondria near plaques
E) Decrease in COX IV immunostaining near plaques
Fig. 2 Mitochondrial fragmentation in living APP/PS1 transgenic mouse brain.
Tg
wt
Xie H et al. J. Neurosci. 2013;33:17042-17051
-mt-GFP in cable-like structures (2-30 µm long) of axons in wt mice
-mtGFP in shorter fragmented cables (2-4 μm long) near plaques only in Tg mice
Fig. 3 MMP was not altered in areas far from amyloid plaques in the APP/PS1 transgenic mice.
Tests of MMP-sensitive (MTR
and JC-1 aggregate)
vs MMP-insensitive (MTG
and JC-1 monomer)
mitochondrial stains
-FCCP treatment used to perturb
MMP: both dyes can be used
to monitor MMP by
Multiphoton Microscopy
No MMP defects observed in
brain areas far from plaques
Xie H et al. J. Neurosci. 2013;33:17042-17051
Fig. 4 Impairment of MMP near amyloid plaques in living APP/PS1 transgenic mouse.
A)
-reduction in both J-a and J-m
near plaques (fewer
mitochondria)
-also reduction in J-a/J-m ratio
near plaques (w/in 20 µm zone)
B) Similar observation w/
TMRE (MMP-sensitive)
C) Similar observation w/
MTR (MMP-sensitive) vs
MTG (MMP-insensitive)
D) Similar mitochondrial
defects NOT observed near
amyloid plaques in smooth
muscle cells of leptomeningeal
vessels in Cerebral Amyloid Angiopathy
Fig. 5 Reduced MMP in dystrophic neurites in living APP/PS1 transgenic mouse.
TMRE staining was observed in GFP-labeled neurites (even in dystrophic
ones near plaques) but intensity reduced in those near plaques
Fig. 6 Oxidative stress accompanied by mitochondrial dysfunction in living APP/PS1 mouse.
-used redox-sensitive GFP
variants to assess oxidative
stress near plaques
roGFP: excites at 900nm
when reduced and
at 800 when oxidized
Oxidative stress roGFPex800
observed in dystrophic
neurites and neurons
near plaques and
contain mitochondria with
reduced MMP (TMRE)
Xie H et al. J. Neurosci. 2013;33:17042-17051
What did we learn from the study?
The majority of mitochondria in brains of mice with amyloid plaques
are NORMAL. Defects in mitochondrial density, composition, MMP,
redox and ROS status only observed in zone surrounding plaques.
What
remaining/new
questions
need to be
addressed?
(Differs
from
in vitro work
where amyloid
plaque
burden artificially high)
Establish a timeline for Amyloid β accumulation, mitochondrial
abnormalities, oxidative stress, and altered intracellular Ca2+ levels
(cause/effect
relationship)
Are there any
caveats to the conclusions?
Potential for therapies aimed at mitochondrial function (See article on
Sirtuins in treatment of Parkinsons Disease)
Cause vs. Effect?