Article #3: A High-Coverage Genome Sequence from an Archaic Denisovan Individual Science 338:222(2012)

Denisova Cave in Siberia-source of bone fossils from Neandertal and Denisovan Archaic Human Groups

Next Generation Sequencing Technology (Sequencing by Synthesis)

Made Possible By:

Improved Ancient DNARecovery Method(from ssDNA)

Improved DNASequencing Technology

Genetic Features Unique to Modern Humans-became fixed after divergence from Denisovans and Neandertals

111,812 Single Nucleotide Changes (SNCs)9,499 insertions and deletions260 SNCs result in amino acid change, 72 affect splicing patterns, 35 affect transcription

Some implicated in autism and other neurological disorders

Among 23 most conserved changes in modern human populations, eight affect brain function or nervous system function (cell adhesion, energy metabolism, microtubule assembly, neurotransmission)

What was known before the study?

What new question is the study trying to answer?

What reagents or techniques were needed for the study?

In vitro studies suggested a link between Amyloid β plaques and mitochondrial dysfunction.

All evidence suggesting this link comes from in vitro orpost mortem studies. An animal model is needed for in vivostudies.

APPswe:PSEN1dE9 transgenic mice-already made

Multiphoton Microscopy

Article #4: Mitochondrial Alterations near Amyloid Plaques in anAlzheimer’s Disease Mouse Model Journal of Neuroscience 33:17042 (2013)

Aβ42

outsidecells

tau

insidecells

Mutations associated w/ familial early onset Alzheimer’s:APP and γ-secretase subunits (PSEN)

Amyloid β Plaques in Alzheimer’s Disease

APPswe:PSEN1dE9human disease-associated alleles

Multiphoton Microscopy

Another super-resolution microscope with low phototoxicity

Uses two laser beams of low energy, long λ infrared light tosimultaneously 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 (LentiviralInfection)-no toxicity from mtGFP

B) mtGFP staining absent w/in16 μm zone of Methoxy X04-labeled Aβ plaques

C) AAV mediated expression of neuronal cytoplasmic GFP & mtGFP: similar absence of mtGFPnear plaques

D) dystrophic morphology ofGFP-labeled neurites lackingintact mitochondria near plaques

E) Decrease in COX IV immuno-staining near plaques

Fig. 2 Mitochondrial fragmentation in living APP/PS1 transgenic mouse brain.

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

Tgwt

Fig. 3 MMP was not altered in areas far from amyloid plaques in the APP/PS1 transgenic mice.

Xie H et al. J. Neurosci. 2013;33:17042-17051

Tests of MMP-sensitive (MTRand JC-1 aggregate) vs MMP-insensitive (MTGand JC-1 monomer) mitochondrial stains

-FCCP treatment used to perturb MMP: both dyes can be usedto monitor MMP byMultiphoton Microscopy

No MMP defects observed inbrain areas far from plaques

Fig. 4 Impairment of MMP near amyloid plaques in living APP/PS1 transgenic mouse.

A)-reduction in both J-a and J-mnear plaques (fewer mitochondria)-also reduction in J-a/J-m rationear plaques (w/in 20 µm zone)

B) Similar observation w/TMRE (MMP-sensitive)

C) Similar observation w/MTR (MMP-sensitive) vsMTG (MMP-insensitive)

D) Similar mitochondrialdefects NOT observed nearamyloid plaques in smoothmuscle cells of leptomeningealvessels 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 dystrophicones near plaques) but intensity reduced in those near plaques

Fig. 6 Oxidative stress accompanied by mitochondrial dysfunction in living APP/PS1 mouse.

Xie H et al. J. Neurosci. 2013;33:17042-17051

-used redox-sensitive GFPvariants to assess oxidativestress near plaquesroGFP: excites at 900nm when reduced and at 800 when oxidized

Oxidative stress roGFPex800

observed in dystrophic neurites and neuronsnear plaques and contain mitochondria with reduced MMP (TMRE)

What did we learn from the study?

What remaining/new questions need to be addressed?

Are there any caveats to the conclusions?

The majority of mitochondria in brains of mice with amyloid plaquesare NORMAL. Defects in mitochondrial density, composition, MMP, redox and ROS status only observed in zone surrounding plaques.

(Differs from in vitro work where amyloid plaque burden artificially high)

Establish a timeline for Amyloid β accumulation, mitochondrialabnormalities, oxidative stress, and altered intracellular Ca2+ levels(cause/effect relationship)

Cause vs. Effect?

Potential for therapies aimed at mitochondrial function (See article onSirtuins in treatment of Parkinsons Disease)