See the published version of this preprint at Fluids and Barriers of the CNS.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
Research
Abraham Al-Ahmad
Texas Tech University Health Sciences Center
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7463-3204
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Alzheimer’s disease (AD) is the most common form of neurodegenerative disease. It is an irreversible condition marked by irreversible cognitive loss, commonly attributed to the loss of hippocampal neurons due to the formation of senile plaques and neurofibrillary tangles. Although the sporadic form is the most prevalent, the presence of familial form (involving several genes such as APP, PSEN1 and PSEN2) of the disease is commonly used as a model for understanding the pathophysiology of the disease. The aim of this study is to investigate the effect of mutation on PSEN1 and PSEN2 genes on the BBB function using induced pluripotent stem cells (iPSCs).
Methods: iPSC lines from patients suffering from familial form of Alzheimer’s disease and harboring mutations in PSEN1 or PSEN2 were used in this study and compared to a control iPSC line. Cells were differentiated into brain microvascular endothelial cells (BMECs) following established differentiation protocols. Barrier function was assessed by measuring TEER and fluorescein permeability, drug transporters activity was assessed by uptake assay, glucose uptake and metabolism assessed by cell flux analyzer, mitochondrial potential by JC-1 and lysosomal acidification by acridine orange.
Results: iPSC derived BMECs from the FAD patient presenting a mutation in PSEN1 gene showed impaired barrier function compared to the FAD patient harboring a mutation in PSEN2 and to control group. Such impaired barrier function correlated with poor tight junction complexes and reduced drug efflux pump activity. In addition, both PSEN1 and PSEN2-BMECs displayed reduced bioenergetics, lysosomal acidification, autophagy, while showing an increase in radical oxygen species (ROS) production. Finally, PSEN1- and PSEN2-BMECs showed an elevated secretion of Ab1-40 peptides compared to control-BMECs.
Conclusion: Our study reports that iPSC-derived BMECs obtained from FAD patients showed impaired barrier properties and BMECs metabolism. In particular, PSEN1 mutation was associated with a more detrimental phenotype than the PSEN2 mutation, as noted by a reduced barrier function, reduced drug efflux pump activity and diminished glucose metabolism. Therefore, assessing the contribution of genetic mutations associated with Alzheimer’s disease will allow us to better understand the contribution of the BBB in dementia, but also other neurodegenerative diseases.
Keywords
iPSCs, blood-brain barrier, PSEN, familial form of Alzheimer’s disease (FAD)
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- FBCNSFADBBBR2FiguresSupplementary.pdf
Supplementary Figure 1: Phenotype of iPSC-derived neurons differentiated from the iPSC lines. Cells were differentiated into neurons following existing protocol (24, 42). Neurons were stained against nestin (red), III-tubulin. DAPI was used as nuclear counterstaining. Supplementary Figure 2: Effect of PSEN1 and PSEN2 mutations on astrocytes glucose uptake and cell metabolic activity. (A) Glucose uptake assay in iPSC-derived astrocytes. Note the similar decrease in glucose uptake as observed in BMECs. Note the efficacy of GTI as a pharmacological inhibitor for GLUTs, as all three cell lines showed a significant decrease in glucose uptake. (B) Cell metabolic activity in astrocytes and neurons using an MTS-assay. Note the higher cell metabolic activity reported in PSEN1-neurons compared to other groups, such higher metabolic rate being absent in astrocytes. (C) DCF levels in control BMECs following incubation with pyocyanin or N-acetyl-cystein (NAC).
Loading...
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Fluids and Barriers of the CNS.
No community comments so far
Editor assigned
On 03 Dec, 2020
Editorial decision: Accept
On 03 Dec, 2020
Submission checks complete
On 03 Dec, 2020
Editor invited
On 03 Dec, 2020
No comments provided
Review #1 received
Received 20 Nov, 2020
Reviewer #3 agreed
On 19 Nov, 2020
Reviewer #2 agreed
On 19 Nov, 2020
Review #3 received
Received 19 Nov, 2020
Review #2 received
Received 19 Nov, 2020
Editor assigned
On 19 Nov, 2020
Reviewers invited
Invitations sent on 19 Nov, 2020
Reviewer #1 agreed
On 19 Nov, 2020
Submission checks complete
On 19 Nov, 2020
Editor invited
On 19 Nov, 2020
Version 3
Posted 04 Nov, 2020
No comments provided
Editorial decision: Major revision
On 11 Nov, 2020
Editor assigned
On 28 Oct, 2020
Reviewers invited
Invitations sent on 28 Oct, 2020
Reviewer #1 agreed
On 28 Oct, 2020
Reviewer #2 agreed
On 28 Oct, 2020
Reviewer #3 agreed
On 28 Oct, 2020
Review #1 received
Received 28 Oct, 2020
Review #3 received
Received 27 Oct, 2020
Review #2 received
Received 27 Oct, 2020
Submission checks complete
On 27 Oct, 2020
Editor invited
On 27 Oct, 2020
No comments provided
Editorial decision: Major revision
On 10 Jan, 2020
Editor assigned
On 09 Jan, 2020
Submission checks complete
On 08 Jan, 2020
Editor invited
On 08 Jan, 2020
No comments provided
Editorial decision: Major revision
On 15 Dec, 2019
Review #3 received
Received 05 Dec, 2019
Review #1 received
Received 05 Dec, 2019
Review #2 received
Received 05 Dec, 2019
Reviewers invited
Invitations sent on 02 Dec, 2019
Reviewer #1 agreed
On 02 Dec, 2019
Reviewer #2 agreed
On 02 Dec, 2019
Reviewer #3 agreed
On 02 Dec, 2019
Reviewer #4 agreed
On 02 Dec, 2019
Editor assigned
On 01 Dec, 2019
Submission checks complete
On 30 Nov, 2019
Editor invited
On 30 Nov, 2019
First submitted
On 29 Nov, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
Learn more about our company.
A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
See the published version of this preprint at Fluids and Barriers of the CNS.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Abraham Al-Ahmad
Texas Tech University Health Sciences Center
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7463-3204
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Fluids and Barriers of the CNS.
No community comments so far
Editor assigned
On 03 Dec, 2020
Editorial decision: Accept
On 03 Dec, 2020
Submission checks complete
On 03 Dec, 2020
Editor invited
On 03 Dec, 2020
No comments provided
Review #1 received
Received 20 Nov, 2020
Reviewer #3 agreed
On 19 Nov, 2020
Reviewer #2 agreed
On 19 Nov, 2020
Review #3 received
Received 19 Nov, 2020
Review #2 received
Received 19 Nov, 2020
Editor assigned
On 19 Nov, 2020
Reviewers invited
Invitations sent on 19 Nov, 2020
Reviewer #1 agreed
On 19 Nov, 2020
Submission checks complete
On 19 Nov, 2020
Editor invited
On 19 Nov, 2020
Version 3
Posted 04 Nov, 2020
No comments provided
Editorial decision: Major revision
On 11 Nov, 2020
Editor assigned
On 28 Oct, 2020
Reviewers invited
Invitations sent on 28 Oct, 2020
Reviewer #1 agreed
On 28 Oct, 2020
Reviewer #2 agreed
On 28 Oct, 2020
Reviewer #3 agreed
On 28 Oct, 2020
Review #1 received
Received 28 Oct, 2020
Review #3 received
Received 27 Oct, 2020
Review #2 received
Received 27 Oct, 2020
Submission checks complete
On 27 Oct, 2020
Editor invited
On 27 Oct, 2020
No comments provided
Editorial decision: Major revision
On 10 Jan, 2020
Editor assigned
On 09 Jan, 2020
Submission checks complete
On 08 Jan, 2020
Editor invited
On 08 Jan, 2020
No comments provided
Editorial decision: Major revision
On 15 Dec, 2019
Review #3 received
Received 05 Dec, 2019
Review #1 received
Received 05 Dec, 2019
Review #2 received
Received 05 Dec, 2019
Reviewers invited
Invitations sent on 02 Dec, 2019
Reviewer #1 agreed
On 02 Dec, 2019
Reviewer #2 agreed
On 02 Dec, 2019
Reviewer #3 agreed
On 02 Dec, 2019
Reviewer #4 agreed
On 02 Dec, 2019
Editor assigned
On 01 Dec, 2019
Submission checks complete
On 30 Nov, 2019
Editor invited
On 30 Nov, 2019
First submitted
On 29 Nov, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Fluids and Barriers of the CNS.
Background: Alzheimer’s disease (AD) is the most common form of neurodegenerative disease. It is an irreversible condition marked by irreversible cognitive loss, commonly attributed to the loss of hippocampal neurons due to the formation of senile plaques and neurofibrillary tangles. Although the sporadic form is the most prevalent, the presence of familial form (involving several genes such as APP, PSEN1 and PSEN2) of the disease is commonly used as a model for understanding the pathophysiology of the disease. The aim of this study is to investigate the effect of mutation on PSEN1 and PSEN2 genes on the BBB function using induced pluripotent stem cells (iPSCs).
Methods: iPSC lines from patients suffering from familial form of Alzheimer’s disease and harboring mutations in PSEN1 or PSEN2 were used in this study and compared to a control iPSC line. Cells were differentiated into brain microvascular endothelial cells (BMECs) following established differentiation protocols. Barrier function was assessed by measuring TEER and fluorescein permeability, drug transporters activity was assessed by uptake assay, glucose uptake and metabolism assessed by cell flux analyzer, mitochondrial potential by JC-1 and lysosomal acidification by acridine orange.
Results: iPSC derived BMECs from the FAD patient presenting a mutation in PSEN1 gene showed impaired barrier function compared to the FAD patient harboring a mutation in PSEN2 and to control group. Such impaired barrier function correlated with poor tight junction complexes and reduced drug efflux pump activity. In addition, both PSEN1 and PSEN2-BMECs displayed reduced bioenergetics, lysosomal acidification, autophagy, while showing an increase in radical oxygen species (ROS) production. Finally, PSEN1- and PSEN2-BMECs showed an elevated secretion of Ab1-40 peptides compared to control-BMECs.
Conclusion: Our study reports that iPSC-derived BMECs obtained from FAD patients showed impaired barrier properties and BMECs metabolism. In particular, PSEN1 mutation was associated with a more detrimental phenotype than the PSEN2 mutation, as noted by a reduced barrier function, reduced drug efflux pump activity and diminished glucose metabolism. Therefore, assessing the contribution of genetic mutations associated with Alzheimer’s disease will allow us to better understand the contribution of the BBB in dementia, but also other neurodegenerative diseases.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- FBCNSFADBBBR2FiguresSupplementary.pdf
Supplementary Figure 1: Phenotype of iPSC-derived neurons differentiated from the iPSC lines. Cells were differentiated into neurons following existing protocol (24, 42). Neurons were stained against nestin (red), III-tubulin. DAPI was used as nuclear counterstaining. Supplementary Figure 2: Effect of PSEN1 and PSEN2 mutations on astrocytes glucose uptake and cell metabolic activity. (A) Glucose uptake assay in iPSC-derived astrocytes. Note the similar decrease in glucose uptake as observed in BMECs. Note the efficacy of GTI as a pharmacological inhibitor for GLUTs, as all three cell lines showed a significant decrease in glucose uptake. (B) Cell metabolic activity in astrocytes and neurons using an MTS-assay. Note the higher cell metabolic activity reported in PSEN1-neurons compared to other groups, such higher metabolic rate being absent in astrocytes. (C) DCF levels in control BMECs following incubation with pyocyanin or N-acetyl-cystein (NAC).
Loading...