Inhibiting PDE7A Enhances the Protective Effects of Neural Stem Cells on Neurodegeneration and Memory Deficits in Sevoflurane-Exposed Mice

Abstract Sevoflurane is widely used in general anesthesia, especially for children. However, prolonged exposure to sevoflurane is reported to be associated with adverse effects on the development of brain in infant monkey. Neural stem cells (NSCs), with potent proliferation, differentiation, and renewing ability, provide an encouraging tool for basic research and clinical therapies for neurodegenerative diseases. We aim to explore the functional effects of injecting NSCs with phosphodiesterase 7A (PDE7A) knock-down in infant mice exposed to sevoflurane. The effects of PDE7A in NSCs proliferation and differentiation were determined by cell counting kit-8 (CCK-8) assay and differentiation-related gene expression assay, respectively. The effects of NSCs with modified PDE7A on mice’s long-term memory and learning ability were assessed by behavioral assays. Our data demonstrated that depleting PDE7A promoted, whereas forcing PDE7A suppressed the activation of cAMP/cAMP-response element binding protein (CREB) signaling as well as cell proliferation and neuronal differentiation of NSCs. Inhibition of PDE7A in NSCs exhibited profound improved effects on long-term memory and learning ability of mice exposed to sevoflurane. Our results for the first time show that knock-down of PDE7A improves the neurogenesis of NSCs in vitro and in vivo, and is beneficial for alleviating sevoflurane-induced brain damage in infant mice.

basic research and clinical therapies for neurodegenerative diseases. We aim to explore 23 the functional effects of injecting NSCs with Phosphodiesterase 7A (PDE7A) 24 knockdown in infant mice exposed to sevoflurane. The effects of PDE7A in NSCs 25 proliferation and differentiation were determined by CCK-8 assay and 26 differentiation-related gene expression assay, respectively. The effects of NSCs with 27 modified PDE7A on mice's long-term memory and learning ability were assessed 28 by behavioral assays. Our data demonstrated that depleting PDE7A promoted, 29 whereas forcing PDE7A suppressed the activation of cAMP/CREB signaling as well as 30 cell proliferation and neuronal differentiation of NSCs. Inhibition of PDE7A in NSCs 31 exhibited profound improved effects on long-term memory and learning ability of mice 32 exposed to sevoflurane. Our results for the first time show that knockdown of PDE7A 33

Introduction 45
Millions of people, including newborns, infants, and children, are subjected to general 46 anesthesia for surgical procedures worldwide. Accumulating evidence has 47 7 0.3 m high of water was incorporated in the study. A round platform (0.12 m diameter) 120 was placed in the center of one of the four-quadrant and submerged 1 cm beneath the 121 water surface. The mice were trained for 3 times per day for a consecutive 7 days 122 before the test. The mouse was placed into the water, and its navigation to the 123 platform was recorded by a video camera located above the water tank. The maximum 124 time for a mouse in the water tank was 80 s. If a mouse cannot find the platform with 125 60 s, the mouse was guided to the platform, and the time was recorded as 60 s.

Enzyme-linked immunosorbent assay (ELISA) 149
The cAMP levels were tested by cAMP Assay Kit (ab133051) following the 150 manufactory's instruction.

Statistical analysis 187
Data values were presented as the mean and standard deviation (mean ± SD) and were 188 analyzed using GraphPad Prism 5. The Student's t-test, two-or one-way analysis of 189 variance (ANOVAs) followed by a post hoc test was used to compare the statistical 190 difference. A value of P < 0.05 was considered statistically significant. NSCs were infected with two lentivirally shRNA (shPDE7A-1, and shPDE7A-2) 199 specifically against PDE7A, but not PDE7B. As showed in Figure 1B was confirmed by western blot assay ( Figure 1E and F). We found that forced PDE7A 206 attenuated the cell proliferation of NSCs ( Figure 1G). These data suggested that 207 PDE7A plays a negative role in the cell growth of NSCs. 208

Suppression of PDE7A enhances the cell differentiation of NSCs 209
To study the effect of PDE7A on NSCs differentiation, we applied the same strategies 210 in Figure 1 to knockdown of or overexpression of PDE7A in NSCs. The differentiated 211 NSCs generally present prolonged fusiform, and continuously growing spores. We 212 observed that silencing PDE7A promoted, whereas enhancing PDE7A inhibited cell 213 differentiation of NSCs (Figure 2A and C). The results were further confirmed by 214 detection of microtubule-associated protein 2 (MAP-2) and suppressor of cytokine 215 signaling 2 (SOCS2), two NSCs differentiation markers, in the NSCs. As illustrated in 216 Transplantation of NSCs/shPDE7A enhances the long-term memory of 231 sevoflurane exposed mice 232 To determine whether PDE7A inhibition promotes the cellar function of NSCs in vivo, 233 the two-week aged mice were exposed to sevoflurane, and then received PBS, 234 NSCs/shNC or NSCs/shPDE7A transplantation 14-day post sevoflurane exposure 235 ( Figure 4A). The long-term memory ability of mice was assessed by the Morris water 236 maze test. As revealed in Figure 4B-C, after the first day of training, mice from all 237 groups exhibited comparable time (60 s) to find the platform and length of swimming 238 (2500 cm). On days 2 and 3 after training, the normal control mice spent 38s (day 2) 239 and 20s (day 3), as well as swan 1250 cm (day 2), and 1000 cm (day 3) to find the 240 platform. However, sevoflurane exposed and sevoflurane+PBS mice spent the amount 241 of time (58 s on day 2 and 3) and slightly decreased swimming length (2250 cm on 242 day 2, and 2000 cm on day 3), suggesting sevoflurane exposed significantly impaired 12 long-term memory of mice ( Figure 4B and C). The NSCs/shNC transplanted 244 sevoflurane-exposed mice spent less time and swam less length to find the platform 245 compared to sevoflurane-exposed mice, suggesting NSCs/shNC transplantation 246 partially improved the neural function of sevoflurane exposed mice ( Figure 4B and C). 247 Strikingly, the NSCs/shPDE7A transplanted sevoflurane-exposed mice spent similar 248 time and swam comparable length to find the platform compared to normal control 249 mice ( Figure 4B and C). 250 Furthermore, mice subjected to sevoflurane exposure spent approximately 30% less 251 time in the targeted quadrant compared to normal control mice. Sevoflurane exposed 252 mice with NSCs/shNC transplantation, but not PBS injection, stayed longer (20% 253 more time) in the targeted quadrant than in control mice. Furthermore, 254 NSCs/shPDE7A transplantation further improved the long-term memory healing 255 effect of NSCs/shNC on sevoflurane exposed mice, as evidenced by comparable time 256 in the targeted quadrant between sevoflurane+NSCs/shPDE7A mice, and normal 257 control mice ( Figure 4D). 258 Transplantation of NSCs/shPDE7A promotes the learning ability of sevoflurane 259

exposed mice 260
To further assess the learning and memory in those mice, mice were subjected to the 261 novel object recognition test. As displayed in Figure 5, the normal healthy mice spent 262 80% longer time on the new object than on the old object. Both sevoflurane-and 263 sevoflurane+PBS-mice spent equivalent amounts of time on new and old objects, with 264 the same discrimination index value, which was considerably low compared to normal 265 control ( Figure 5A and 5B). Both NSCs/shNC and NSCs/shPDE7A transplantation 266 resulted in a prolonged time on the new object than on the old one. Of note, the 267 NSCs/shPDE7A transplantation yielded an improved memory rescue effect than 268 sevoflurane+NSCs/shPDE7A mice was higher than that in sevoflurane+NSCs/shNC 270 mice ( Figure 5A and 5B). Furthermore, the mRNA levels of PDE7A, MAP-2, and 271 SOCS2 in brain tissues were measured by RT-qPCR. As manifested in (Figure 5C-5E infected with shNC, shPDE7A-1 or shPDE7A-2 were cultured for two weeks, and 499 then cell-differentiation rate was measured. B. NSCs infected with shNC, shPDE7A-1 500 or shPDE7A-2 were cultured for two weeks, and then cells were prepared for 501 measuring the mRNA expression of cell differentiation-related genes, including 502 MAP-2 and SOCS2. C. NSCs infected with PLVX-PDE7A were cultured for two 503 weeks, and then cell-differentiation rate was measured. D. NSCs infected with 504 PLVX-PDE7A were cultured for two weeks, and then cells were prepared for 505 measuring the mRNA expression of cell differentiation-related genes, including 506 MAP-2 and SOCS2. ** p<0.01. C-E. The mRNA levels of PDE7A, MAP-2 and SOCS2 in brain tissues were 533 measured by RT-qPCR. β-actin was used as an internal control. * p<0.05, ** p<0.01. 534 protein levels of pCREB in brain tissues were measured by Western blot. β-actin was 547 used as a loading control. 548 549