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Evaluating Alzheimer’s Disease
Therapeutics on mitoNEET Expression
Rachel M. Hemmerlin, Kayle J. Marsh, and Dr. Ashley M. Loe
Department of Chemistry, Slippery Rock University of Pennsylvania,
Slippery Rock, PA, United States
Hallmarks of Alzheimer’s Disease (AD)
• Neurodegenerative disease
• Common symptoms include
dementia, cognitive impairment,
and progressive memory loss
• Hallmarks of AD include the
formation of senile plaques and
neurofibrillary tangles
Mitochondrial dysfunction and its role in cells
• Mitochondria play a role in many cell
cycles including energy metabolism,
antioxidant production, and cell
survival
• Mitochondrial dysfunction leads to the
formation of reactive oxygen species
(ROS)
• Impaired mitochondrial pathways
include ATP generation, ROS
formation and defense, calcium
buffering, morphology and dynamics,
mPTP opening, and cytochrome c
release.
Current AD therapeutics
• AD therapeutics are
cholinesterase inhibitors
which inhibit
acetylcholinesterase (AChE)
and therefore increase the
level of available
acetylcholine
• They are limited to alleviating
the symptoms of AD by trying
to counterbalance the
neurotransmitter disturbance
What is mitoNEET and its role in cells
• Outer mitochondrial membrane protein
with iron sulfur clusters
• Plays a role in iron homeostasis,
regulating energy metabolism, formation
of inter-mitochondrial junctions, and
production of ROS
• Novel target for treating mitochondrial
dysfunction and associated diseases
Experiment Scheme
Imaging using Fluorescence Microscopy
Figure 1. N2a cells expressing mitoNEET-GFP (a) exposed to 5 µM (b), 50 µM (c), or 500 µM
(d), donepezil for 24 hours.
Expression of mitoNEET in response to
donepezil, rivastigmine, and galantamine
mitoNEET-GFP ± rivastigmine
200
150
100
50
0
Control
5 µM
50 µM
250
200
150
100
50
0
Control
500 µM
mitoNEET-GFP ± donepezil
0.5 uM
5 uM
50 uM
500 uM
800000
600000
400000
200000
0
500 µM
*
100
50
0
0.1 uM
1 uM
10 uM
100 uM
1200000
1000000
800000
600000
400000
200000
1000000
800000
600000
*
*
400000
200000
0
0
50 µM
*
150
mitoNEET-GFP ± galantamine
Raw Integrated Density
Raw Integrated Density
1000000
5 µM
200
Control
1200000
Control
250
mitoNEET-GFP ± rivastigmine
1200000
Raw Integrated Density
mitoNEET-GFP ± galantamine
Mean Fluorescence Intensity (a.u.)
250
Mean Fluoresence Intensity (a.u.)
Mean Fluorescence Intensity (a.u.)
mitoNEET-GFP ± donepepzil
Control
0.5 uM
5 uM
50 uM
500 uM
Control
0.1 uM
1 uM
10 uM
100 uM
Conclusion and Future Direction
• Donepezil and rivastigmine did not statistically impact the expression whereas
certain concentrations of galantamine slightly statistically down regulated
mitoNEET expression.
• These finding suggest that current AD drugs do not change mitoNEET expression
and therefore do not target or impact mitochondrial dysfunction in cells.
• Future studies will explore the expression of mitoNEET in response to Aβ and
oxidative stress.
References
•
2017 Alzheimer's Disease Facts and Figures. www.alz.org/facts/.
•
Lovell, M. A.; Markesbery, W. R., Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease. Nucelic Acids Res, 2007, 35 (7497-7504)
•
Fox, N. C.; al., e., Serial magnetic resonance imaging of cerebral atrophy in preclinical Alzheimer's disease. Lancet, 1999, 353, 2125.
•
LaFerla, F. M., Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease. Nat. Rev. Neurosci, 2002, 3, 862-872.
•
Bossers, K.; al., e., Concerted changes in transcripts in the prefrontal cortex precede neuropathology in Alzheimer's disease. Brain: A Journal of Neurology, 2010, 133, 3699-3723.
•
N.C. Berchtold, e. a., Synaptic genes are extensively downregulated across multiple brain regions in normal human aging and Alzheimer's disease. Neurobiol. Aging, 2013, 35, 1653-1661
•
Mane, Deepali J.; Kanase, Vanita; Ansari, Imtiyaz. An overview of treatment for Alzheimer’s disease. European Journal of Biomedical and Pharmaceutical Science, 2018, 5 (6), 1-6.
•
•
•
Singh, Sandeep Kumar; Srivastav, Saurabh; Yadav, Amarish Kumar; Srikrishna, Saripella, Perry, George. Overview of Alzheimer’s Disease and Some Therapeutic Approaches Targeting Aβ by Using Several Synthetic and Herbal Compounds. Oxidative Medicine and Cellular
Longevity, 2016.
Cheignon, C.; Tomas, M.; Bonnefont-Rousselot, D.; Faller, P.; Hureau, C.; Collin, F. Oxidative stress and the amyloid beta peptide in Alzheimer’s disease. Redox Biology, 2018. 14. 450-464.
Mudher, A.; Lovestone, S., Alzheimer's disease - do tauists and baptists finally shake hands? Trends Neurosci, 2002, 25 (1), 22-26.
•
Terry, A. V.; Buccafusco, J. J., The cholinergic hypothesis of age and Alzheimer's disease-related cognitive deficits: recent challenges and their implications for novel drug development. Journal of Pharmacolgy and Experimental Therapeutics, 2003, 306 (3), 821-827.
•
Kril, J. J.; al., e., Neuron loss from the hippocampus of Alzheimer's disease exceeds extracellular neurofibrillary tangle formation. Acta Neuropathol, 2002, 103, 370-376.
•
Jones, Simon Vann Kounatidis. Nuclear factor kappa B and Alzheimer disease, unifying genetic and environmental risk factors from cell to humans. Frontiers in Immunology, 2017, 8, 1805/1-1805/9.
•
Xie, Y; Zheng, J; Li, S; Li, H; Zhou, Y; Zheng, W; Zhang, M; Liu, L; Chen, Z, GLP-1 improves the neuronal supportive ability of astrocytes in Alzheimer’s disease by regulating mitochondrial dysfunction via the cAMP/PKA pathway. Biochemical Pharmacology, 2021, 188.
•
Paddock, Mark L.; Wiley, Sandra E.; Axelrod, Herbert L.; Cohen, Aina E.; Roy, Melinda; Abresch, Edward C.; Capraro, Dominique; Murphy, Anne N.; Nechushtai, Rachel; Dixon, Jack E.; Jennings, Patricia A. MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane
protein stabilized by pioglitazone. PNAS, 2007, 104(36), 14342-14347.
•
Vodickova, A; Kore, S, A; Wojtovich, A, Site-specific mitochondrial dysfunction in neurodegeneration. Mitochondrion, 2022, 64, 1-18.
•
Moreira, P. I.; Carvalho, C.; Zhu, X.; Smith, M. A.; Perry, G., Mitochondrial dysfunction is a trigger of Alzheimer's disease pathophysiology. Biochimica et Biophysica Acta, 2010, 1802, 2-10.
•
Ehret, M. J; Chamberlin, K. W; Current practices in the treatment of alzheimer disease: where is the evidence after the phase III trials? Clinical Therapeutics, 2015, 37, 1604-1616.
•
Geldenhuys, W.J; Piktel, D.; Moore, J. C.; Rellick, S. L.; Meadows, E.; Pinti, M. V.; Hollander, J. M.; Ammer, A. G.; Martin, K. H.; Gibson, L. F., Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia. Free
Radical Biology and Medicine, 2021, 175, 226-235.
Tasnim, H.; Landry, A. P.; Fontenot, C. R.; Ding, H., Exploring the FMN binding site in the mitochondrial outer membrane protein mitoNEET. Free Radical Biology and Medicine, 2020, 156, 11-19.
•
•
Yonutas, H. M.; Hubbard, W. B.; Pandya, J. D.; Vekaria, H. J.; Geldenhuys, W. J.; Sullivan, P. G., Bioenergetic restoration and neuroprotection after therapeutic targeting of mitoNEET: New mechanism of pioglitazone following traumatic brain injury. Experimental Neurology, 2020,
327.
•
Li, X.; Wang, Y.; Tan, G.; Lyu, J.; Ding, H., Electron transfer kinetics of the mitochondrial outer membrane protein mitoNEET. Free Radical Biology and Medicine, 2018, 121, 98-104.
•
Habener, Anika; Chowdhury, Arpita; Echtermeyer, Frank; Lichtinghagen, Ralf; Theilmeier, Gregor; Herzog, Christine. MitoNEET protects HL-1 cardiomyocytes from oxidative stress mediated apoptosis in an in vitro model of hypoxia and reoxygenation. PLoS One, 2016, 11 (5),
e0156054/1-e0156054/18.
•
mitoNEET (AT1A8): sc-517413, Santa Cruz Biotechology, Inc, https://datasheets.scbt.com/sc-517413.pdf.
•
•
Knowles, Joanne; Donepezil in alzheimer’s disease: an evidence-based review of its impact on clinical and economic outcomes. PMC, 2006, 1(3), 195-219.
Birks, J. S.; Evans, J. G., Rivastigmine for alzheimer’s disease. Cochrane Library, 2015.
•
Razay, G.; Wilcock, G. K., Galantamine in alzheimer’s disease. Expert Review of Neurotherapeutics, 2014, 8, 9-17.
•
Yiannopoulou, K. G.; Papageorgiou, S. G., Current and future treatments for alzheimer’s disease. Therapeutic Adavances in Neurological Disorders, 2013, 6 (1), 19-33.
•
Kusminski, C. M.; Holland, W. L.; Sun, K.; Park, J.; Spurgin, S. B.; Lin, Y.; Askew, G. R.; Simcox, J. A.; McClain, D. A.; Li, C.; Scherer, P. E., MitoNEET, a key regulator of mitochondrial function and lipid homeostasis. Nat. Med, 2012, 18(10), 1539-1549.
Therapeutics on mitoNEET Expression
Rachel M. Hemmerlin, Kayle J. Marsh, and Dr. Ashley M. Loe
Department of Chemistry, Slippery Rock University of Pennsylvania,
Slippery Rock, PA, United States
Hallmarks of Alzheimer’s Disease (AD)
• Neurodegenerative disease
• Common symptoms include
dementia, cognitive impairment,
and progressive memory loss
• Hallmarks of AD include the
formation of senile plaques and
neurofibrillary tangles
Mitochondrial dysfunction and its role in cells
• Mitochondria play a role in many cell
cycles including energy metabolism,
antioxidant production, and cell
survival
• Mitochondrial dysfunction leads to the
formation of reactive oxygen species
(ROS)
• Impaired mitochondrial pathways
include ATP generation, ROS
formation and defense, calcium
buffering, morphology and dynamics,
mPTP opening, and cytochrome c
release.
Current AD therapeutics
• AD therapeutics are
cholinesterase inhibitors
which inhibit
acetylcholinesterase (AChE)
and therefore increase the
level of available
acetylcholine
• They are limited to alleviating
the symptoms of AD by trying
to counterbalance the
neurotransmitter disturbance
What is mitoNEET and its role in cells
• Outer mitochondrial membrane protein
with iron sulfur clusters
• Plays a role in iron homeostasis,
regulating energy metabolism, formation
of inter-mitochondrial junctions, and
production of ROS
• Novel target for treating mitochondrial
dysfunction and associated diseases
Experiment Scheme
Imaging using Fluorescence Microscopy
Figure 1. N2a cells expressing mitoNEET-GFP (a) exposed to 5 µM (b), 50 µM (c), or 500 µM
(d), donepezil for 24 hours.
Expression of mitoNEET in response to
donepezil, rivastigmine, and galantamine
mitoNEET-GFP ± rivastigmine
200
150
100
50
0
Control
5 µM
50 µM
250
200
150
100
50
0
Control
500 µM
mitoNEET-GFP ± donepezil
0.5 uM
5 uM
50 uM
500 uM
800000
600000
400000
200000
0
500 µM
*
100
50
0
0.1 uM
1 uM
10 uM
100 uM
1200000
1000000
800000
600000
400000
200000
1000000
800000
600000
*
*
400000
200000
0
0
50 µM
*
150
mitoNEET-GFP ± galantamine
Raw Integrated Density
Raw Integrated Density
1000000
5 µM
200
Control
1200000
Control
250
mitoNEET-GFP ± rivastigmine
1200000
Raw Integrated Density
mitoNEET-GFP ± galantamine
Mean Fluorescence Intensity (a.u.)
250
Mean Fluoresence Intensity (a.u.)
Mean Fluorescence Intensity (a.u.)
mitoNEET-GFP ± donepepzil
Control
0.5 uM
5 uM
50 uM
500 uM
Control
0.1 uM
1 uM
10 uM
100 uM
Conclusion and Future Direction
• Donepezil and rivastigmine did not statistically impact the expression whereas
certain concentrations of galantamine slightly statistically down regulated
mitoNEET expression.
• These finding suggest that current AD drugs do not change mitoNEET expression
and therefore do not target or impact mitochondrial dysfunction in cells.
• Future studies will explore the expression of mitoNEET in response to Aβ and
oxidative stress.
References
•
2017 Alzheimer's Disease Facts and Figures. www.alz.org/facts/.
•
Lovell, M. A.; Markesbery, W. R., Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease. Nucelic Acids Res, 2007, 35 (7497-7504)
•
Fox, N. C.; al., e., Serial magnetic resonance imaging of cerebral atrophy in preclinical Alzheimer's disease. Lancet, 1999, 353, 2125.
•
LaFerla, F. M., Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease. Nat. Rev. Neurosci, 2002, 3, 862-872.
•
Bossers, K.; al., e., Concerted changes in transcripts in the prefrontal cortex precede neuropathology in Alzheimer's disease. Brain: A Journal of Neurology, 2010, 133, 3699-3723.
•
N.C. Berchtold, e. a., Synaptic genes are extensively downregulated across multiple brain regions in normal human aging and Alzheimer's disease. Neurobiol. Aging, 2013, 35, 1653-1661
•
Mane, Deepali J.; Kanase, Vanita; Ansari, Imtiyaz. An overview of treatment for Alzheimer’s disease. European Journal of Biomedical and Pharmaceutical Science, 2018, 5 (6), 1-6.
•
•
•
Singh, Sandeep Kumar; Srivastav, Saurabh; Yadav, Amarish Kumar; Srikrishna, Saripella, Perry, George. Overview of Alzheimer’s Disease and Some Therapeutic Approaches Targeting Aβ by Using Several Synthetic and Herbal Compounds. Oxidative Medicine and Cellular
Longevity, 2016.
Cheignon, C.; Tomas, M.; Bonnefont-Rousselot, D.; Faller, P.; Hureau, C.; Collin, F. Oxidative stress and the amyloid beta peptide in Alzheimer’s disease. Redox Biology, 2018. 14. 450-464.
Mudher, A.; Lovestone, S., Alzheimer's disease - do tauists and baptists finally shake hands? Trends Neurosci, 2002, 25 (1), 22-26.
•
Terry, A. V.; Buccafusco, J. J., The cholinergic hypothesis of age and Alzheimer's disease-related cognitive deficits: recent challenges and their implications for novel drug development. Journal of Pharmacolgy and Experimental Therapeutics, 2003, 306 (3), 821-827.
•
Kril, J. J.; al., e., Neuron loss from the hippocampus of Alzheimer's disease exceeds extracellular neurofibrillary tangle formation. Acta Neuropathol, 2002, 103, 370-376.
•
Jones, Simon Vann Kounatidis. Nuclear factor kappa B and Alzheimer disease, unifying genetic and environmental risk factors from cell to humans. Frontiers in Immunology, 2017, 8, 1805/1-1805/9.
•
Xie, Y; Zheng, J; Li, S; Li, H; Zhou, Y; Zheng, W; Zhang, M; Liu, L; Chen, Z, GLP-1 improves the neuronal supportive ability of astrocytes in Alzheimer’s disease by regulating mitochondrial dysfunction via the cAMP/PKA pathway. Biochemical Pharmacology, 2021, 188.
•
Paddock, Mark L.; Wiley, Sandra E.; Axelrod, Herbert L.; Cohen, Aina E.; Roy, Melinda; Abresch, Edward C.; Capraro, Dominique; Murphy, Anne N.; Nechushtai, Rachel; Dixon, Jack E.; Jennings, Patricia A. MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane
protein stabilized by pioglitazone. PNAS, 2007, 104(36), 14342-14347.
•
Vodickova, A; Kore, S, A; Wojtovich, A, Site-specific mitochondrial dysfunction in neurodegeneration. Mitochondrion, 2022, 64, 1-18.
•
Moreira, P. I.; Carvalho, C.; Zhu, X.; Smith, M. A.; Perry, G., Mitochondrial dysfunction is a trigger of Alzheimer's disease pathophysiology. Biochimica et Biophysica Acta, 2010, 1802, 2-10.
•
Ehret, M. J; Chamberlin, K. W; Current practices in the treatment of alzheimer disease: where is the evidence after the phase III trials? Clinical Therapeutics, 2015, 37, 1604-1616.
•
Geldenhuys, W.J; Piktel, D.; Moore, J. C.; Rellick, S. L.; Meadows, E.; Pinti, M. V.; Hollander, J. M.; Ammer, A. G.; Martin, K. H.; Gibson, L. F., Loss of the redox mitochondrial protein mitoNEET leads to mitochondrial dysfunction in B-cell acute lymphoblastic leukemia. Free
Radical Biology and Medicine, 2021, 175, 226-235.
Tasnim, H.; Landry, A. P.; Fontenot, C. R.; Ding, H., Exploring the FMN binding site in the mitochondrial outer membrane protein mitoNEET. Free Radical Biology and Medicine, 2020, 156, 11-19.
•
•
Yonutas, H. M.; Hubbard, W. B.; Pandya, J. D.; Vekaria, H. J.; Geldenhuys, W. J.; Sullivan, P. G., Bioenergetic restoration and neuroprotection after therapeutic targeting of mitoNEET: New mechanism of pioglitazone following traumatic brain injury. Experimental Neurology, 2020,
327.
•
Li, X.; Wang, Y.; Tan, G.; Lyu, J.; Ding, H., Electron transfer kinetics of the mitochondrial outer membrane protein mitoNEET. Free Radical Biology and Medicine, 2018, 121, 98-104.
•
Habener, Anika; Chowdhury, Arpita; Echtermeyer, Frank; Lichtinghagen, Ralf; Theilmeier, Gregor; Herzog, Christine. MitoNEET protects HL-1 cardiomyocytes from oxidative stress mediated apoptosis in an in vitro model of hypoxia and reoxygenation. PLoS One, 2016, 11 (5),
e0156054/1-e0156054/18.
•
mitoNEET (AT1A8): sc-517413, Santa Cruz Biotechology, Inc, https://datasheets.scbt.com/sc-517413.pdf.
•
•
Knowles, Joanne; Donepezil in alzheimer’s disease: an evidence-based review of its impact on clinical and economic outcomes. PMC, 2006, 1(3), 195-219.
Birks, J. S.; Evans, J. G., Rivastigmine for alzheimer’s disease. Cochrane Library, 2015.
•
Razay, G.; Wilcock, G. K., Galantamine in alzheimer’s disease. Expert Review of Neurotherapeutics, 2014, 8, 9-17.
•
Yiannopoulou, K. G.; Papageorgiou, S. G., Current and future treatments for alzheimer’s disease. Therapeutic Adavances in Neurological Disorders, 2013, 6 (1), 19-33.
•
Kusminski, C. M.; Holland, W. L.; Sun, K.; Park, J.; Spurgin, S. B.; Lin, Y.; Askew, G. R.; Simcox, J. A.; McClain, D. A.; Li, C.; Scherer, P. E., MitoNEET, a key regulator of mitochondrial function and lipid homeostasis. Nat. Med, 2012, 18(10), 1539-1549.