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1

The role of hippocampal PML in transgenic mouse circadian rhythm

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Olech-Kochanczyk G., Kiryk-Jaskiewicz A., Domanskyi A., Konopka W., Wilczynski G. M.
Acta Neurobiologiae Experimentalis
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2017
|
tom 77
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nr Suppl.1
INTRODUCTION: Circadian clock is an evolutionarily well-conserved mechanisms in higher organisms. PML protein implicated in many important biological processes (response to DNA damage, cell division control) influences also circadian rhythm by regulating nuclear localization of Per2, a significant positive regulator of the clock transcriptional mechanisms. AIM(S): Our aim is to explain how overexpression or knock‑out of PML affect the oscillations of levels of proteins involved in circadian rhythm in hippocampus. METHOD(S): We generated transgenic mouse models with overexpression or knock-out of PML gene that are induced in specific time and location in brain. The project consists of two tasks: 1) generation of transgenic animals with overexpression of PML gene using AAV vectors encoding PML and mCherry reporter gene, and 2) generation of transgenic animals with knock-out of PML gene using AAV vectors encoding PML gene-targeting gRNAs and Cas9 (CRISPR/Cas9 system). RESULTS: We generated AAV vectors with PML-mCherry and microinjected them into the hippocampus of mice that were subject to behavioral tests (in the IntelliCage system). AAV PML-mCherry mice showed impairment in circadian activity 45 days after surgery and displayed proper spatial learning of cage corners with appetitive reinforcement and slower re-learning process. Next, we designed gRNAs for PML exon I and II and generated plasmid vectors containing gRNAs for the PML gene and tdTomato reporter. Using both these vectors and vectors containing Cas9 endonuclease fused with GFP reporter we transfected NIH 3T3 cells to induce PML gene knock-out in vitro. Next, using T7E1 endonuclease we confirmed that knock-out of PML works. Afterwards, we generated AAV vectors encoding PML gene-targeting gRNAs and Cas9 endonuclease. Both vectors were injected into the hippocampus. CONCLUSIONS: Thus far research on PML function in the circadian rhythm was usually performed in vitro. Our tools enable us to study the role of PML in mammalian molecular clock mechanisms in vivo.
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Neurogenesis and behavioral strategies in ICER overexpressing rats

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Klejman A., Janusz A., Kiryk-Jaskiewicz A., Gorlewicz A., Bieganska K., Jedynak P., Kaczmarek L., Konopka W.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr Suppl.1
INTRODUCTION: ICER (Inducible cAMP Early Repressor) is an effective endogenous repressor of CREB/ CREM/ATF transcription factors family, including its own expression. We have developed a Syn‑Flag‑ICER II transgenic rat line. In transgenic animals, we have surprisingly detected increased levels of mRNA for CREB or CREM transcription factors. We have also detected upregulation of CREB dependent miR‑132. Nerogenesis is a process of generation and maturation of newborn neurons into neuronal networks in the developing brain. We have found that ICER II overexpressing rats showed reduced hippocampal adult neurogenesis. The number of the SGZ BrdU positive cells was similar, but in the mature granular neurons layer, the number of BrdU positive cells was decreased when compared to control animals. One of the crucial elements enabling the incorporation of newborn neurons into neuronal network of the brain is the active reorganization of the extracellular matrix mostly by action of metalloproteinases. The most known for its activity in the brain is matrix metalloproteinase 9 (MMP-9), which is also one of the known targets of miR‑132. We have examined MMP-9 activity in the ICER overexpressing rat brain lysates, and we observed decreased activity of MMP‑9 in ICER mutants as compared to WT controls. RESULTS: We have also found that ICER rats with affected neurogenesis employ different learning strategies than their control littermates in the Morris Water Maze learning paradigm. The results of this behavioral tests indicate that transgenic rats didn’t differ from controls in their learning and memory capabilities, but they showed differences in strategies of finding the hidden platform. Male ICER rats more often were choosing imprecise strategies to find the platform than control males. CONCLUSIONS: Those results demonstrate that disruption of CREB dependent gene expression in neurons by overexpression of ICER affects adult neurogenesis and causes changes that affect discrete aspects of animal cognitive behavior.
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Manipulation of CREB related gene expression in neurons influences adult neurogenesis and morris water maze strategies

72%
Janusz A., Klejman A., Kiryk-Jaskiewicz A., Gorlewicz A., Bieganska K., Jedynak P., Kaczmarek L., Konopka W.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Active reorganization of extracellular matrix in the brain allows for growth of neuronal dendrites and axons which guarantees successful incorporation of new born neurons into neuronal network during adult neurogenesis in the hippocampus. Activity of surrounding neurons may affect adult neurogenesis. AIM(S): In order to test whether manipulation of CREB dependent gene expression in neurons and hence their activity will influence adult neurogenesis we have developed the Syn-Flag-ICER II transgenic rat line. The ICER (Inducible cAMP Early Repressor) is effective endogenous repressor of CREB/CREM/ATF transcription factors family. METHOD(S): BrdU labeling to asses a level of adult neurogenesis in the hippocampus qPCR for changes in transcription of CREB/CREM and related genes gelatin zymography to measure MMP9 activity Morris Water Maze spatial learning tests Patch Clamp RESULTS: ICER II overexpressing rats showed diminished hippocampal neurogenesis. We have observed a reduced number of mature BrdU positive cells in granular zone of hippocampus of transgenic rats, in comparison to control group. We have observed also that neurons of dentate gyrus demonstrate increased excitability. Paradoxically, we have detected increased levels of mRNA for CREB or CREM factors. Also CREB dependent miR-132 expression was upregulated in transgenic rats, which regulates expression of MMP-9 – extracellular matrix metalloproteinase. We have found the decreased activity of MMP9 in ICER overexpressing rats. Morris Water Maze tests didn’t show overall differences in rats learning and memory capabilities, however male ICER rats chose more often imprecise strategies to find hidden platform than control males. CONCLUSIONS: Obtained results indicate that CREB dependent gene expression in neurons regulates a set of genes e.g. miR-132 that may in turn regulate translation of proteins involved in remodeling of extracellular matrix and affect adult neurogenesis, what changes discrete aspects of animal cognitive behavior.
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