Antiaggregation
potential of 6-hydroxy-L-nicotine from Paenarthrobacter
nicotinovorans pAO1 against amyloid peptide: in vitro
and in vivo studies (NICAGREG), PN-III-P4-PCE-2021-1692, No.
PCE 49/2022
- Research
project funded by the Ministry of Research, Innovation
and Digitization, CNS-UEFISCDI, project number
PN-III-P4-PCE-2021-1692, within PNCDI III;
- Funding
amount: 1.200.000 lei (250.000 euros);
- Funding period: 31 months (02/06/2022 – 31/12/2024).
Project summary:
Alzheimer's disease (AD) is characterized by progressive degradation of memory processes, being associated with three major changes that occur in the brain: i) the formation of intra- and extracellular beta-amyloid deposits; ii) the appearance of neurofibrillary tangles and iii) the death of cholinergic neurons and a significant decrease in acetylcholine level. The identification of neuroprotective therapies for AD was dominated by the hypothesis of amyloid cascade and tau proteins. Unfortunately, both approaches have so far failed to provide an effective therapeutic strategy. The involvement of α7 and α4β2 subtypes of nicotinic receptors (nAChRs) in the pathogenesis of AD has led to the proposal of a new therapeutic approach. Immunohistochemical studies in the brains of patients with sporadic AD have shown that Aβ1-42 and α7nAChR are present in neuritic plaques and this interaction may be inhibited by α7nAChRs ligands. Nicotine, through its ability to bind to nAChRs is the ideal molecule for the development of new derivatives with therapeutic applications. The project aims to test the antiaggregating potential against Aβ1-42 of 6-hydroxy-L-nicotine (6HLN), derived from the metabolism of nicotine in the microorganism Paenarthrobacter nicotinovorans pAO1. To achieve this goal, the project activities will focus on evaluating the action of 6HLN in vitro on cell lines and in vivo on 5xFAD transgenic mice, in order to identify its therapeutic potential.
Project members:
| Alexandru Ioan Cuza
University of Iași |
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|
Project coordinator | U-1700-039M-8193 | ||
|
Biochemistry and Molecular Biology | U-1700-038N-4243 | ||
|
Biochemistry and Molecular Biology | U-1800-055L-7447 |
||
|
Ph.D. student, Molecular Biology |
U-2000-065K-8850 | ||
|
Ph.D. student, Molecular Biology | U-2100-068P-2027 | ||
| Center
for Fundamental Research and Experimental
Development in TranslationMedicine - Transcend, Regional Institute of Oncology, Iași |
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|
Cellular and Molecular Biology |
U-1700-030C-6243 |
||
|
Cellular and Molecular Biology |
U-1700-035A-8848 | |
|
| Paula Alexandra Stache |
Cellular and Molecular Biology | U-1700-037P-9545 | ||
| Project's objectives and activities: | Publications |
Project's objectives and activities:
General objective - The objectives and the activities of the project focus on the effects and mechanism by which the nAChRs activation by 6HLN affects Aβ production in both cell lines and laboratory mice brain, with particular attention on cytotoxicity, cognitive deficits, neuroinflammation and brain oxidative stress. To achieve this, a biotechnology based on a P. nicotinovorans strain will be used to produce 6HLN. Once the 6HLN is obtained, the mechanism by which the nAChRs activation by 6HLN affects Aβ production and cytotoxicity in the cell lines will be assessed. Next, the effects of 6HLN on Aβ deposition, memory, neuroinflammation and oxidative stress in laboratory mice brain will be evaluated.
Activities in 2022:
Step 1. IN VITRO EVALUATION OF THE ANTIAGGREGATING POTENTIAL OF 6HLN ON THE Aβ1-42Act 1.3. Production of 6HLN using a Paenarthrobacter nicotinovorans based biotechnology
Act 1.4. Antiaggregating and disintegration potential assay of 6HLN against mature Aβ1-42 fibril formation
Act 1.5. Evaluation of Aβ1-42 level in cell lines under the action of 6HLN
Act 1.6. Perform the required data analysis and literature survey
Act 1.7. Disseminate the results
Step 2. IN VIVO ASSESSING OF THE 6HLN ANTIAGGREGATING POTENTIAL ON THE Aβ1-42. IN VIVO ASSESSING OF THE 6HLN AGAINST Aβ1-42-INDUCED CYTOTOXICITY. IN VIVO ASSESSING OF THE 6HLN AGAINST Aβ1-42-INDUCED MEMORY DEFICITS.
Act 2.1. Acquisitions: reagents and consumables, 5xFAD transgenic mice, 4 months-old, from Jackson Laboratories, USA
Act 2.2. Quantification of Aβ1-42 level in mice hippocampal homogenates after exposure to 6HLN.
Act 2.3. Evaluation of antiapoptotic activity of 6HLN in mice hippocampal homogenates by the DNA fragmentation method.
Act 2.4. Assessment of short-term memory by means of the Y-maze task.
Act 2.5. Assessment of working and reference memory by means of radial arm-maze task.
Act 2.6. Assessment of anxiety-like behavior by means of elevated plus maze task.
Act 2.7. Assessment of depressive behavior by means of forced swimming test.
Act 2.8. Perform the required data analysis and literature survey.
Act 2.9. Disseminate the results.
Activities from 2024 performed in advance in 2023:
Step 3. IN VIVO ASSESSING OF THE 6HLN AGAINST Aβ1-42-INDUCED NEUROINFLAMMATION. IN VIVO ASSESSING OF THE 6HLN AGAINST Aβ1-42-INDUCED OXIDATIVE STRESS.
Act 3.1. Acquisitions: 5xFAD transgenic mice, 4 months-old, from Jackson Laboratories, USA
Step 3. IN VIVO ASSESSING OF THE 6HLN AGAINST Aβ1-42-INDUCED NEUROINFLAMMATION. IN VIVO ASSESSING OF THE 6HLN AGAINST Aβ1-42-INDUCED OXIDATIVE STRESS.
Act. 3.2. Performing immunostaining for GFAP and IBA1 for astrocytes and microglia
Act. 3.3. Evaluation of the levels of inflammatory markers: TLR4, NF-κβ, TNF-α, and IL6 in hippocampal homogenates of mice after exposure to 6HLN.
Act. 3.4. Evaluation of specific activities of acetylcholinesterase (AChE) and antioxidant enzymes (SOD, GPX, CAT) and total reduced GSH content in hippocampal homogenates of mice after exposure to 6HLN.
Act. 3.5. Evaluation of MDA levels (lipid peroxidation) and carbonylated proteins (protein oxidation) in hippocampal homogenates of mice after exposure to 6HLN.
Act. 3.6. Analysis of experimental data and evaluation of specialized literature (part 3)
Act. 3.7. Dissemination of obtained results (part 3)
Contact
Project manager:
Lucian Hrițcu, PhD
E-mail: hritcu@uaic.ro
Phone: 40(0)232201102 - 1666
Fax: 40(0)232201472
Further details: here
