1. Alzheimer's Disease and the Mitochondria
PHM Fall 2018
Instructor: Ms. Maya Latif
Coordinator: Dr. J. Henderson
-introduce ourselves & topic
Felicia Leung, Manmohit Gill, Jonathan Tsou, Han Nguyen

2. Alzheimer’s Disease (AD)
Neurodegenerative disorder
2 types: early & late onset
Progressive disease
Symptoms: learning deficits, memory loss, confusion, trouble with once familiar tasks

3. Amyloid Precursor Protein (APP)
Large membrane protein
Role in neural growth and repair
Sends signals through G protein system
Binds to structural molecules

4. Amyloid Beta Plaques
Amyloid beta formed from proteolysis of APP by secretases
Normally, protein fragments broken down and eliminated
In those with AD, fragments accumulate to form hard, insoluble plaques

5. Tau Proteins-Neurofibrillary Tangles
Stabilization of microtubules
In those with AD, abnormal accumulations of tau protein (neurofibrillary tangles)
Symptoms of AD shown to correlate with accumulation of plaques and tangles
Amyloid beta and tau proteins used as biomarkers

6. Mitochondria: The Powerhouse of the Cell
Surrounded by two membranes and has its own genome
Divides independently from the cell in which it resides in
Generates ATP at a rate of 4.7bn ATP/second per cortical neuron
ATP used in the CNS for: synaptic transmission, action potential, formation of synapses, neuronal branching and growth
Neurons highly rely on the function of mitochondria

7. Mitochondrial DNA (mtDNA)
mtDNA contains 37 genes - 13 of the genes code for making enzymes involved in oxidative phosphorylation while the others code for the production of rRNA and tRNA
No histones on mtDNA
Exhibits maternal inheritance
Heteroplasmy can occur

8. Reactive Oxygen Species (ROS) are Produced by the Mitochondria
ROS are a component of the killing response of the immune system to microbial invasion
Most ROS are formed as byproducts during mitochondrial ETC of aerobic respiration
ROS are necessary intermediates of metal catalyzed oxidation reactions
Recent studies indicate ROS act as messengers in normal cell signal transduction and cell cycling

9. Mitochondrial Generation of Reactive Oxygen Species (ROS)
ETC generates ATP when NADH donates electrons
ROS such as a superoxide radical forms when the donated electrons escape and combine with oxygen
Superoxide radical is a proximal mitochondrial ROS that is destructive to cellular components

10. Protection from Superoxide
Our bodies contain superoxide dismutase (SOD) to reduce superoxide to H2O2
Catalase then converts H2O2 to water and oxygen

11. Mitochondria dysfunction in patients with Alzheimer’s Disease (Outline)
Point 1: Associating AD pathology with mitochondria
The mechanism by which amyloid beta is introduced into the mitochondria
Point 2: The effects of ROS in neurodegenerative disease (NDD)
How DNA damage leads to apoptosis
The Electron Transport Chain (ETC) enzymes implicated in AD or ROS overproduction
ROS cause cytokines to be released from neurons to activate microglia and astrocytes
Transcript:
What goes wrong in mitochondria/immune system for a patient with Alzheimer’s -Jon
Now I will endeavour to describe what may go wrong in mitochondria in a patient with Alzheimer’s Disease (AD).
((AD patients are known to have reduced numbers of mitochondria. (1, ref 33). In rat models of AD, where wildtype Amyloid Precursor protein (APP) was overexpressed, mitochondria dysfunction was also seen (1, ref 44). And in mouse models of AD, accumulation of amyloid beta led to neuronal dysfunction (1, ref32).
Its believed that mitochondria dysfunction exacerbates amyloid-beta and tau pathology by increasing plaque production and aggregation (2, ref 27). So in short, mitochondria may be implicated in the pathology of AD. ))
There are two major topics which I would like to introduce: the first being the mechanism by which amyloid beta accumulates in mitochondria, and the second being how ROS (which can be produced in mitochondria) are implicated in neurodegenerative disease (NDD). In the latter I will briefly talk about three effects of ROS: how apoptosis is induced, where the electron transport chain may be effected, and what cytokines are activated by ROS.
The first point illustrates that AD pathology may be associated with mitochondria, and the second point describes how one process in mitochondria, ROS production, being awry results in pathology through apoptosis, etc dysfunction, and neuroinflammation.
(So, two points, the latter being divided into 3.)
Show all figures in miniature without circles

12. Q: How does Amyloid Beta enter the mitochondria? (Point 1)
Picture above:
Mancuso M, Calsolaro V, Orsucci D, Carlesi C, Choub A, Piazza S, Siciliano G. Mitochondria, cognitive impairment, and Alzheimer's disease. Int J Alzheimers Dis Jul 6;2009. pii: doi: /2009/ PubMed PMID: ; PubMed Central PMCID: PMC
Picture below:
Hansson Petersen CA, Alikhani N, Behbahani H, et al. The amyloid β-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(35):13145–13150.

13. Q: How does Amyloid Beta enter the mitochondria? (Point 1)
APP is transported by the translocase of outer membrane (TOM) Complex and the translocase of inner membrane (TIM) complex.
TOM: 3 receptors (Tom20, 22, 70), and 1 pore (Tom40).
In rats, neutralizing antibodies against receptor proteins led to reduced transport of amyloid beta (abeta).
Mancuso M, Calsolaro V, Orsucci D, Carlesi C, Choub A, Piazza S, Siciliano G. Mitochondria, cognitive impairment, and Alzheimer's disease. Int J Alzheimers Dis Jul 6;2009. pii: doi: /2009/ PubMed PMID: ; PubMed Central PMCID: PMC
Transcript:
To introduce the first point, I present you with a leading question: “How would amyloid beta (an aberrant protein which accumulate in AD) even enter the mitochondria?”
((The picture is of an immunostain for DNA (DAPI) in light blue, mitochondria (mitotracker) in red, and externally-introduced, fluorescently-labelled amyloid beta protein (Abeta1-40) in green. Yellow indicates overlap (surrounding the mitochondria). ))
These images were taken from human neuroblastoma cells (SH-SY5Y cell line, presumably from an AD patient) and show that abeta colocalizes with mitochondria (but doesn’t show their uptake very well) (point to bottom immunostain pic).
(Recall that in AD pathology, there is a progressive loss neurons accompanied by formation of plaques with amyloid beta (cleaved from amyloid precursor protein (APP)), as well as neurofibrillary tangles (NFT) of hyperposphorylated Tau protein. This is the amyloid beta cascade hypothesis; a model of familial (inherited) AD (ref 1, 23).)
Amyloid precursor protein (APP, the amyloid-beta precursor) is transported by the tranlocase of outer membrane (TOM) protein, to cross the outer membrane of the mitochondria from the cytosol. That’s this protein here and complex of proteins here (point to APP and TOM). From there, APP crosses the inner leaflet via the translocase of the inner membrane (TIM) (point to TIM). (1, ref40)
The TOM protein consists of 3 receptor and 1 pore protein. The receptor proteins are Tom20, Tom22, and Tom70. The import pore is Tom40. (In rat liver mitochondria,) Neutralizing antibodies against the receptor proteins reduce the import of amyloid beta (abeta). (1, ref40)
(Other fun facts:
The import of abeta is not affected by disruption in inner membrane potential (e.g. not affected by the drug vancomycin.
Amyloid beta inhibits COX protein (enzyme for generation of prostaglandins
The experiments done in the paper). (1, ref40)
Show figure from ref1
Show figure from 1, ref40

14. Priming Questions (Point 2)
DNA-damage and Apoptosis
How does oxidative stress lead to DNA damage and apoptosis?
ETC dysfunction
Are there any defects in ETC enzymes known to be associated with AD?
Neuroinflammation
What cytokines are released by neurons in NDD that cause neuroinflammation?
For the second point: we have three priming questions:
How does oxidative stress lead to
DNA damage and apoptosis? through P53, BCL-XL, caspase 3,7
Are there any defects ETC enzymes known to be associated with AD? What ETC enzymes are affected by an overproduction of ROS? No. Complex 1,3,4. (4 also implicated in AD)
What cytokines are released by neurons and act on resident immune cells (microglia and astrocytes) that cause neuroinflammation? Answer: IL-1beta, IL-6, TNF-alpha
Transcript:
For the first,
ROS leads to lipid peroxidation, protein oxidation, and DNA damage. (point to each in figure).
DNA damage can lead to DNA-repair, cell cycle arrest and apoptosis. Double-strand breaks lead to apoptosis via P53, BCL2 proteins, and caspases. (point to each in figure). (ref 2)
For the second,
There are no known causative mutations linked to AD, but genetic abnormalities in complex IV (cytochrome oxidase) were found in AD patients. (1) In a study where cells were depleted of endogenous mitochondria and replaced with mtDNA from AD patients, the cells overproduced amyloid beta and were more susceptible to apoptosis (1, 47). In general, pathogenic mtRNAcan be found in a variety of cognitive disorders (1, 57).
mtDNA damage leads to complex 1 and complex 3 defects, which cause formation of superoxide anion. ( 2 ref 16). Nitric oxide (NO; a ROS) modifies the activity of complex 1 and complex 4 by interfering with the oxygen-binding Iron-sulphate centre. (2, 20; 1, 43)
For the last,
ROS stimulate the release of cytokines IL-1beta, IL-6, and TNF-alpha. NO also activates immune cells in the brain. These cytokines activate microglia and astrocytes causing release of more ROS and chronic inflammation. (2, ref 29). This neuroinflammation increases the formation of neurofibraillary tangles of (tau protein). (2, ref 30) (You may recall from lecture 1 that chronic inflammation involves cycles of repair and inflammation.)

15. How does oxidative stress lead to DNA damage and apoptosis?
Mancuso M, Calsolaro V, Orsucci D, Carlesi C, Choub A, Piazza S, Siciliano G. Mitochondria, cognitive impairment, and Alzheimer's disease. Int J Alzheimers Dis Jul 6;2009. pii: doi: /2009/ PubMed PMID: ; PubMed Central PMCID: PMC

16. How does oxidative stress lead to DNA damage and apoptosis?
dsDNA break
→ P53 stimulated
→ alter regulation of BCL2 family (e.g. antiapoptotic BCL- XL downregulated)
→ upregulation of caspases 3, 6, 7.
→ apoptosis
Fdsfesf
alter regulation of BCL2 family of apoptotic proteins: downregulation of antiapoptic protein Bcl- xl; upregulation of apoptosis-inducing BCL-XL → upregulation caspase 3, 6 and 7, causing cell death
Islam MT. Oxidative stress and mitochondrial dysfunction-linked neurodegenerative disorders. Neurol Res Jan;39(1): Epub 2016 Nov 3. Review. PubMed PMID:

17. Are there any defects in ETC enzymes known to be associated with AD?
No causative mutations linked to AD currently known 1
But,
Complex 4 mutations found in AD patients 1
Replacing mitochondria with mtDNA from AD patients → amyloid beta overproduction, susceptibility to apoptosis 2
Pathogenic mtDNA in a variety of cognitive disorders 3
mtDNA damage → complex 1, complex 3 defects → O2- formed 4
NO modifies activity of complex 1 and 4 (interferes with Fe-S centre) 5
References for this slide:
Picture and reference 1:
Mancuso M, Calsolaro V, Orsucci D, Carlesi C, Choub A, Piazza S, Siciliano G. Mitochondria, cognitive impairment, and Alzheimer's disease. Int J Alzheimers Dis Jul 6;2009. pii: doi: /2009/ PubMed PMID: ; PubMed Central PMCID: PMC
2. Khan SM, Cassarino DS, Abramova NN, et al. Alzheimer’s disease cybrids replicate β-amyloid abnormalities through cell death pathways. Annals of Neurology. 2000;48(2):148–155.
3. Wang J, Xiong S, Xie C, Markesbery WR, Lovell MA. Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer’s disease. Journal of Neurochemistry. 2005;93(4):953–962.
4. Castro MR, Suarez E, Kraiselburd E, et al. Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys. Exp Gerontol. 2012;47:29–37.
5. Andreazza AC, Shao L, Wang JF, et al. Mitochondrial complex I activity and oxidative damage to mitochondrial proteins in the prefrontal cortex of patients with bipolar disorder. Arch Gen Psychiatr. 2010;67:360–368

18. Are there any defects in ETC enzymes known to be associated with AD?
No causative mutations linked to AD currently known 1
But,
Complex 4 mutations found in AD patients 1
Replacing mitochondria with mtDNA from AD patients → amyloid beta overproduction, susceptibility to apoptosis 2
Pathogenic mtDNA in a variety of cognitive disorders 3
mtDNA damage → complex 1, complex 3 defects → O2- formed 4
NO modifies activity of complex 1 and 4 (interferes with Fe-S centre) 5
References for this slide:
Picture and reference 1:
Mancuso M, Calsolaro V, Orsucci D, Carlesi C, Choub A, Piazza S, Siciliano G. Mitochondria, cognitive impairment, and Alzheimer's disease. Int J Alzheimers Dis Jul 6;2009. pii: doi: /2009/ PubMed PMID: ; PubMed Central PMCID: PMC
2. Khan SM, Cassarino DS, Abramova NN, et al. Alzheimer’s disease cybrids replicate β-amyloid abnormalities through cell death pathways. Annals of Neurology. 2000;48(2):148–155.
3. Wang J, Xiong S, Xie C, Markesbery WR, Lovell MA. Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer’s disease. Journal of Neurochemistry. 2005;93(4):953–962.
4. Castro MR, Suarez E, Kraiselburd E, et al. Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys. Exp Gerontol. 2012;47:29–37.
5. Andreazza AC, Shao L, Wang JF, et al. Mitochondrial complex I activity and oxidative damage to mitochondrial proteins in the prefrontal cortex of patients with bipolar disorder. Arch Gen Psychiatr. 2010;67:360–368

19. Which cytokines are released by neurons and act on microglia to cause neuroinflammation?
ROS → release IL-1beta, IL-6, TNF-alpha (nitric oxide also acts on immune cells) 1
→ More ROS, chronic inflammation → neuron loss 2
Neuroinflammation increases NFT formation 3
References for this slide:
Islam MT. Oxidative stress and mitochondrial dysfunction-linked neurodegenerative disorders. Neurol Res Jan;39(1): Epub 2016 Nov 3. Review. PubMed PMID:
McGeer PL, Itagaki S, Boyes BE, et al. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology. 1988;38:1285–1285.
Benilova I, Karran E, De Strooper B. The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes. Nat Neurosci. 2012;15:349–357.

20. Which cytokines are released by neurons and act on microglia to cause neuroinflammation?
ROS → release IL-1beta, IL-6, TNF-alpha (nitric oxide also acts on immune cells) 1
→ More ROS, chronic inflammation → neuron loss 2
Neuroinflammation increases NFT formation 3
References for this slide:
Islam MT. Oxidative stress and mitochondrial dysfunction-linked neurodegenerative disorders. Neurol Res Jan;39(1): Epub 2016 Nov 3. Review. PubMed PMID:
McGeer PL, Itagaki S, Boyes BE, et al. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology. 1988;38:1285–1285.
Benilova I, Karran E, De Strooper B. The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes. Nat Neurosci. 2012;15:349–357.

21. Mitochondria dysfunction in patients with Alzheimer’s Disease (Summary)
Point 1:
Amyloid beta is introduced into the mitochondria by APP protein-transport mediated by TOM and TIM (1, ref40)
Point 2:
Apoptosis may be mediated by P53, BCL-2 proteins, caspases 3, 6 and 7.
There are currently no known mutations in ETC proteins linked to AD, but ROS lead to mutations or modify the activity of Complexes 1, 3 and 4
ROS causes the release of cytokines IL-1beta, IL-6, TNF-alpha to be released from neurons to activate microglia and astrocytes
There are currently no known mutations in ETC proteins linked to AD, but other NDDs may have Complex 1 and 3 mutations, and ROS modifies the activity of Complex 1 and 4

22. Pharmacology/Treatments
Cholinesterase inhibitors
donepezil, rivastigmine, and galantamine
N-methyl-D-aspartate antagonist
memantine
SSRIs, Serotonin Reuptake Inhibitors
fluoxetine, sertraline, paroxetine, citalopram, fluvoxamine
TCA, Tricyclic Antidepressants
desipramine, nortriptyline
SNRIs, Selective Norepinephrine Reuptake Inhibitors
mirtazapine, venlafaxine, duloxetine
Benzodiazepine
lorazepam, oxazepam, temazepam, olanzapine

23. The Amyloid Hypothesis of Alzheimer’s Disease
To block the progression of the disease they have to interfere with the pathogenic steps responsible for the clinical symptoms:
Deposition of extracellular amyloid β plaques and intracellular neurofibrillary tangle formation
Inflammation
Oxidative damage
Iron deregulation
Cholesterol metabolism

24. Summary AD is the progressive degeneration and/or death of nerve cells
Symptoms include learning deficits, memory loss, confusion
Amyloid beta formed from proteolysis of APP by secretases, accumulate to form plaques
Abnormal accumulations of tau protein called neurofibrillary tangles
Symptoms of AD shown to correlate with accumulation of plaques and tangles
ROS is formed when the electrons donated to the ETC by NADH during aerobic respiration escapes and combines with oxygen
SOD reduces superoxide to form H2O2 which is then converted to water and oxygen by catalase
ROS causes the release of  cytokines IL-1beta, IL-6, TNF-alpha to be released from neurons to activate microglia and astrocytes
Amyloid beta is introduced into the mitochondria by APP protein-transport mediated by TOM and TIM
Apoptosis may be mediated by P53, BCL-2 proteins, caspases 3, 6 and 7.
There are currently no known mutations in ETC proteins linked to AD, but ROS lead to mutations  or modify the activity of Complexes 1, 3 and 4  
Current treatments include cholinesterase inhibitors, NMDA antagonists, and antipsychotics/antidepressants, with further research to block the progression of the disease at a biochemical level

25. References
Kim E. et al. Cholinesterase Inhibitor Donepezil Increases Mitochondrial Biogenesis through AMP-Activated Protein Kinase in the Hippocampus. Neuropsychobiology. 2016; 73:
Selkoe D. J., & Hardy J. The amyloid hypothesis of Alzheimer's disease at 25 years. EMBO Molecular Medicine. 2016; 8:
Xin, S.; Tan, L.; Cao, X.; Yu, J.; Tan, L. Clearance of Amyloid Beta and Tau in Alzheimer’s Disease :
From Mechanisms to Therapy. 2018, 733–748.
Yiannopoulou K. G., & Papageorgiou S. G. Current and future treatments for Alzheimer’s disease. Ther Adv Neurol Disord. 2013; 6(1):
Mancuso M, Calsolaro V, Orsucci D, Carlesi C, Choub A, Piazza S, Siciliano G. Mitochondria, cognitive impairment, and Alzheimer's disease. Int J Alzheimers Dis Jul 6;

26. References cont.
ros-in-cells/
Islam MT. Oxidative stress and mitochondrial dysfunction-linked neurodegenerative disorders. Neurol Res Jan;39(1):73-82.
Khan SM, Cassarino DS, Abramova NN, et al. Alzheimer’s disease cybrids replicate β-amyloid abnormalities through cell death pathways. Annals of Neurology. 2000;48(2):148–155.
Wang J, Xiong S, Xie C, Markesbery WR, Lovell MA. Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer’s disease. Journal of Neurochemistry. 2005;93(4):953–962.

27. References cont.
Castro MR, Suarez E, Kraiselburd E, et al. Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys. Exp Gerontol. 2012;47:29–37.
Andreazza AC, Shao L, Wang JF, et al. Mitochondrial complex I activity and oxidative damage to mitochondrial proteins in the prefrontal cortex of patients with bipolar disorder. Arch Gen Psychiatr. 2010;67:360–368.
Hansson Petersen CA, Alikhani N, Behbahani H, et al. The amyloid β-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae. PNAS. 2008;105(35):13145–13150.