Rolul proteinei postsinaptice gephyrin in patogeneza bolii Alzheimer – Acronim proiect: Gephyrin in Alzheimer, Cod proiect: PN-III-P4-ID-PCE-2016-0052, drector proiect: Șef lucr. Dr. Kiss Eva
| Title project | THE ROLE OF GEPHYRIN POSTSINAPETIC PROTEIN IN THE PATHOGENESIS OF ALZHEIMER DISEASE |
| The Name Program from PN III | Program 4 – Fundamental and Border Research |
| Type of project | Research Projects Exploratory |
| Action | 1.1.4 Attracting staff with advanced skills from abroad |
| Code Project | PN-III-P4-ID-PCE-2016-0052, Acronim: gephyrin in Alzheimer |
| Duration contract: | 30 months |
| Beneficiary | University of Medicine and Pharmacy Tirgu Mureş |
| Scope (purpose) | The studies planned under this project aim to provide new information on the role of inhibitory synapses and the network of inhibitory cells in the hippocampus in Alzheimer’s disease in order to identify possible new targets for early diagnosis and treatment. |
| Steps: | Step 1 – Study of changes in different populations of interneurons in the hippocampus in APPS1 mice at different ages, corresponding to the early and late stages of the disease. Characterization of postsyanptic clusters of the gephyrin protein – the scaffold protein necessary for agglomeration of glycine and GABA receptors on postsynaptic density, total and phosphorylated, selective in these populations of hippocampal interneurons in APPPS1 mice, depending on the stages of disease progression. Step 2 – Study of the relationship between CDK5 enzyme activity and phosphorylation and agglomeration of gephyrin in the hippocampus in APPPS1 mice (CDK5 phosphorylates gephyrin in vitro). Stage 3 – Correlation of phenotypic changes between the mice model and the hippocampus of patients with Alzheimer’s disease using post mortem material. Testing whether gephyrin can be detected in cerebrospinal fluid in Alzheimer’s disease as a possible biological early diagnosis indicator. |
| Results foreseen | 1. Detection of selective and specific changes in different populations of hippocampal interneurons in APPPS1 mice depending on the stages of evolution of the disease. 2. Existence of a correlation between CDK5 enzyme activity and gephyrin expression and postsyanptic agglomeration. 3. The existence of qualitative and quantitative changes in gephyrin clusters in the human hippocampus with Alzheimer’s disease (post-mortem material) equivalent to those found in the mouse model. 4. The presence of gephyrin, phosphorylated gephyrin or gephyrin fragments in the cerebrospinal fluid in Alzheimer’s disease depending on the stage of the disease. |
| Start date | 12/07/2017 |
| Implementation period | 12.07.2017 – 31.12.2019 |
| Total budget value | 999,589.00 lei |
| Scientific Report: | PN-III-P4-ID-PCE-2016-0052 |
2017-2018/1
Main focus: The analysis of gephyrin cluster formation in different populations of interneurons in the hippocampus in APPPS1 mice at early and late stages of the disease in comparison to age-matched wild type (WT) mice.
For the characterization of the cell type-specific distribution of gephyrin clusters during AD pathogenesis double and/or triple immunstainings for postsynaptic gephyrin and cell-specific markers of GABAergic interneuron subpopulations (parvalbumin, somatostatin, calbindin) and/or presynaptic VGAT has been optimized and performed with the main focus on 3-month-old APPPS1 and WT mice. The experiments for 12- month-old animals are in progress.
Confocal images has been aquired and digital image analysis has been partially done (parvalbumin, VGAT, mAb7, GAD67) or is ongoing.
The expression pattern of proteins indicated has been investigated by Western blot analysis following protein isolation of freshly prepared and frozen hippocampus of APPPS1- and WT mice.
A part of the innovative results has been presented (POSTER PRESENTATION) at the. „11th FENS Forum of Neuroscience in Berlin, 7-11 July 2018”. The Abstract is attached (F18-2139).
The corresponding manuscript has been edited and submitted for publication (August 2018).
2018/2
Main focus: Analysis of the expression of CDK5 and functionally related proteins (p25/p35) in correlation to gephyrin and specific proteins of inhibitory synapses as well as the expression, phosphorylation and clustering of gephyrin in the hippocampus in APPPS1- and WT mice.
Immunostainings of brain sections from 3-month-old mice has been done for CDK5 and p25/p35 as well as different forms of phosphorylated tau. The experiments for 12- month-old animals are in progress.
Confocal images has been aquired and digital image analysis has been partially done or is ongoing.
Western blot analysis for the above mentioned proteins has been performed from freshly prepared and frozen hippocampus of 3-month-old APPPS1- and WT mice. The experiments for 12- month-old animals are in progress.
Continuation and completion of the experiments from „2018/1“
Revision and resubmission of the manuscript (November 2018) (see „2018/1“)
2019/1
January 2019: publication of the Manuscript: Early alterations in hippocampal perisomatic GABAergic synapses and network oscillations in a mouse model of Alzheimer’s disease amyloidosis. Hollnagel JO, Elzoheiry S, Gorgas K, Kins S, Beretta CA, Kirsch J, Kuhse J, Kann O, Kiss E. PLoS One. 2019 Jan 15;14(1):e0209228.
Continuation and completion of the experiments from „2018/1-2“with Main focus (1) on the evaluation of p25/p35 and CDK5 expression and their correlation to gephyrin and specific proteins of inhibitory synapses in the hippocampus in APPPS1- and WT mice by in vivo and in vitro experiments:
Double and triple immunostainings of brain sections from 1-3- and some 12-month-old mice has been done for CDK5 and p25/p35 colocalisation with gephyrin and other markers of inhibitory synapses (e.g. VGAT, GABAAR-g2). Confocal images has been acquired and digital image analysis has been performed.
The expression pattern of proteins indicated has been investigated by Western blot analysis following protein isolation of freshly prepared and frozen hippocampus of APPPS1- and WT-mice.
Cell culture experiments have been performed using rat hippocampal neurons: neurons were transfected with WT- and mutated hAPPswe and the effects on proteins of inhibitory synapses were evaluated by immunofluorescence and Western blot analysis.
A part of the innovative results has been presented (POSTER PRESENTATION) at the 13th Göttingen Meeting of the German Neuroscience Society 20‐23 March 2019 (Abstract: T11-15B) and at the 114th Annual Meeting of the German Anatomical Society 25-27 September in Würzburg, Germany (Poster 133).
A second manuscript comprising a part of the obtained results has been edited and submitted for publication (September 2019).
Main focus 2: a) Analysis of a common pattern of specific phenotypic changes in gephyrin and other specific proteins of inhibitory synapses in APPPS1 mice and in humans – for this purpose the acquisition and processing of human post mortem material is ongoing. b) Analysis of the protein composition of the cerebrospinal fluid (CSF) in APPPS1 mice and humans and proof of the potential of gephyrin as a possible disease- and stage-specific biomarker – for this purpose the acquisition of CSF is ongoing.
2019/2
A part of the innovative results has been presented (POSTER PRESENTATION) at the Düsseldorf-Jülich Symposium on Neurodegenerative Diseases: “Aggregation, Fibrils, Autophagy, Prions and Biomarkers” in Düsseldorf, Germany, on November 12-14, 2019 (Poster is attached)
A second manuscript comprising a part of the obtained results submitted for publication with the title: “Increased p35/CDK5 signaling contributes to changes of inhibitory synapses in early stages of cerebral amyloidosis” in September 2019 (see 2019/1) is in revision process (November-December 2019).
Continuation and completion of the experiments from „2019/1“