Page 51 - AN-1-1

Advanced Neurologyurology
Advanced Ne EPAC2 null leads to tauopathy

AD than SPs. Therefore, it is important to understand the purchased from Vital River Laboratory Animal Technology
mechanisms underlying the formation of NFTs in AD [2,3] . (Beijing, China). Wild-type C57/BL6J was purchased
Cyclic adenosine monophosphate (cAMP)-dependent from the Experimental Animal Center of Tongji Medical
signals have been implicated in the regulation of the College, Huazhong University of Science and Technology,
expression of numerous genes and are essential for Hubei, China.
normal neuronal function and memory. There are two Tg2576 mice were purchased from Jackson Laboratory
ubiquitously expressed intracellular cAMP receptors in (Bar Harbor, USA). The mice were fed in a room under
eukaryotic cells; namely, protein kinase A and cAMP/ a 12/12 h light-dark cycle and a temperature of 25 ±
cAMP-regulated guanine nucleotide exchange factor 2°C, with free access to food and water. All animal care
(EPAC/cAMP-GEF) [4,5] . To date, two EPAC isoforms and experimental procedures complied with local and
have been identified in mammals – EPAC1 and EPAC2. international guidelines on the ethical use of animals,
Although EPAC1 and EPAC2 are present in most tissues, and were approved by The University Animal Welfare
EPAC2 is primarily expressed in the central nervous system. Committee, Tongji Medical College, Huazhong University
A number of studies have implicated EPAC proteins in of Science and Technology.
neurological disorders, including AD [6,7] . For example,
postmortem quantitative polymerase chain reaction (PCR) 2.2. Cell culture and treatments
analyses have shown that AD patients had increased EPAC1 In 6-well plates filled with DMEM and 10% fetal bovine
mRNA and decreased EPAC2 mRNA in the frontal cortex serum, we seeded mouse neuroblastoma 2a (N2a) cells
and hippocampus compared with age-matched healthy (friendly gift of Dr. Huaxi Xu at Xiamen University). Cells
controls [8,9] . EPAC activates, through crosstalk between were cultured in a humidified atmosphere of 5% CO at
2
Ras and Rho small G-protein subfamilies, the α-secretase 37°C. When 60–80% confluent, the culture medium was
pathway to cleave amyloid precursor protein (APP) in its replaced with serum- and antibiotic-free DMEM before
extracellular domain, thereby releasing the soluble APPα treatment. Plasmids used for transfection were amplified
domain into the extracellular space [10-13] . Together, these and purified using EndoFree Plasmid Kits (Qiagen, Hilden,
lines of evidence support a critical role for EPAC proteins Germany), according to the manufacturer’s instructions.
in AD pathogenesis. However, the underlying mechanisms For transfection, N2a cells were seeded in 6-well plates,
remain unclear, and it is not known whether abnormal grown to 60–70% confluence, and then cultured in
EPAC2 signaling contributes to the aberrant tau pathology. serum- and antibiotic-free OPTI-MEM media for 4 h.
In the present study, we report for the 1 time that According to the manufacturer’s instructions, plasmids
st
EPAC2 null mice (EPAC2 ) display abnormal tau were transfected with Lipofectamine 2000 (Invitrogen,
−/−
hyperphosphorylation at multiple sites, and that these Carlsbad, USA). Cells transfected with green fluorescent
changes are also detected in EPAC2 siRNA transfected protein (GFP) constructs were visualized at 48 h after
−/−
neurons. The hyperphosphorylation of tau in EPAC2 mice transfection under an Olympus IX70 microscope with a
was age-dependent and formed aggregates. Moreover, using 209LCPlanF1 lens (Olympus Corporation, Matsue, Japan).
Bielschowsky sliver staining, we found that the intracellular 2.3. Chemicals and antibodies
accumulation of argyrophilic substances was more apparent
in the EPAC2 mice. We also screened upstream tau kinases/ The bicinchoninic acid (BCA) protein assay kit was
−/−
phosphatases and found that the activation of CDK5 might purchased from Pierce Chemical Company (Rockford,
play an important role in the tau hyperphosphorylation [14,15] . IL, USA). The immunohistochemistry kit (Histostain-SP)
Administration of roscovitine, a specific inhibitor of CDK5, was purchased from ZEMED Company (San Francisco,
effectively attenuated the tau hyperphosphorylation and CA, USA), and the DAB kit was from ZSGB-Bio (Beijing,
aggregation in EPAC2 mice [16,17] . We further explored the China). The Odyssey two-color infrared fluorescence
−/−
possible reasons for aberrant CDK5 activation and found imaging system reagent was purchased from LI-COR
that calpain activity was upregulated in EPAC2 mice. Biosciences (Lincoln, NE, USA). The primary antibodies
−/−
Application of a calpain inhibitor, calpain inhibitor IV, also employed in this study are listed below (Table 1). The
reduced tau hyperphosphorylation and aggregation [18,19] . secondary antibodies for Western blot analysis were
purchased from Amersham Pharmacia Biotech (Little
2. Materials and methods Chalfont, England).

2.1. Mice and experimental procedure 2.4. Reverse transcription PCR (RT-PCR)
The EPAC2 mice used in the present study have been Total RNA from brain tissue was extracted using TRIzol
−/−
previously described . The EPAC2 mice (S129sv) were reagent (Invitrogen). A 6 μg aliquot of the RNA was
[6]
+/+
Volume 1 Issue 1 (2022) 2 https://doi.org/10.36922/an.v1i1.8