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 DNA Recovery Method (from ssDNA) Improved DNA Sequencing 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 deletions 260 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 or post mortem studies. An animal model is needed for in vivo studies. APPswe:PSEN1dE9 transgenic mice-already made Multiphoton Microscopy Article #4: Mitochondrial Alterations near Amyloid Plaques in an Alzheimer’s Disease Mouse Model Journal of Neuroscience 33:17042 (2013)

Aβ42 outside cells tau inside cells Mutations associated w/ familial early onset Alzheimer’s: APP and γ-secretase subunits (PSEN) Amyloid β Plaques in Alzheimer’s Disease 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 X04- labeled 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 immuno- staining near plaques

Fig. 2 Mitochondrial fragmentation in living APP/PS1 transgenic mouse brain. Xie H et al. J. Neurosci. 2013;33: 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 Tg wt

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: 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

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. Xie H et al. J. Neurosci. 2013;33: used redox-sensitive GFP variants to assess oxidative stress near plaques roGFP: excites at 900nm when reduced and at 800 when oxidized Oxidative stress roGFP ex800 observed in dystrophic neurites and neurons near 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 plaques are 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, mitochondrial abnormalities, oxidative stress, and altered intracellular Ca 2+ levels (cause/effect relationship) Cause vs. Effect? Potential for therapies aimed at mitochondrial function (See article on Sirtuins in treatment of Parkinsons Disease)