Altered Signaling leads to Increase, Neural Remodeling

Modulating factor  

Details  

Effects on the KER  

References  

Genetic 

Src (regulates the activation of MAPK pathways) knockout 

Src knockout in mice inactivated MAPK and apoptotic signaling and reduced apoptosis in the brain after middle cerebral artery occlusion. 

Tian et al., 2020 

miR-137 (silences Src) knockout 

miR-137 knockout in mice increased MAPK and apoptotic signaling and further increased apoptosis after middle cerebral artery occlusion. 

Tian et al., 2020 

p38 and ERK1/2 knockout 

p38 and ERK1/2 knockout in mice inactivated MAPK and apoptotic signaling and reduced apoptosis in the brain after middle cerebral artery occlusion. 

Tian et al., 2020 

p53 knockout 

Irradiation (1-5 Gy) of p53 knockout mice led to a higher number of neurons and decreased apoptosis compared to irradiation of wild-type mice. 

Chow, Li and Wong, 2000; Limoli et al., 2004 

Drug 

LY367385 (mGluR1 inhibitor). mGluR1 is involved in neuronal differentiation. 

LY367385 (25 M) increased the number of NSCs after 6 Gy radiation of C17.2 neural stem-like cells. 

Eom et al., 2015 

SP600125 (JNK inhibitor) 

SP600125 (5 μM) restored neuronal differentiation after it was reduced by 2 Gy radiation of rat NSCs. 

Kanzawa et al., 2006 

Cyclosporin (CsA, prevents NFATc4/3 nuclear translocation) 

CsA (1 µg/mL) further reduced the levels of dephosphorylated NFATc4/3 as well as total neurite length and branching points after both 2 and 8 Gy irradiation of rat neurons. 

Zhang et al., 2018 

BDNF (induces NFATc4/3 nuclear translocation) 

BDNF (100 ng/mL in vitro, 0.75 µg/1.5 μL in vivo) slightly restored the levels of dephosphorylated NFATc4/3 after 2 Gy irradiation and completely restored neurite length and total branching points both in vitro and in vivo. 

Zhang et al., 2018 

Sex 

Female mice 

Male mice showed many changes in Akt and ERK1/2 activity following acute and chronic irradiation at 0.5 Gy. However, female mice showed only few changes. In addition, male mice showed a trend of fewer immature neurons after 0.5 Gy radiation. 

Silasi et al., 2004 

Exercise 

Forced running in 30-minute intervals twice per day, 5 times per week for 3 weeks. 

Forced running after irradiation completely restored the levels of the signaling molecules in the BDNF-pCREB pathway and slightly restored neurogenesis. 

Ji et al., 2014 

Reference 

Experiment Description 

Result 

Kumar et al., 2005 

In vitro. Hippocampal cornu ammonis (CA)3/CA1 pyramidal neurons extracted from rats were subjected to pharmacological inhibition of various signaling molecules. The level of various signaling molecules was determined with western blot. Confocal microscopy was used to assess hippocampal neuron morphology, including total dendritic branch length, number of terminal tips, soma area, spine density, and filopodia density. 

The PI3K inhibitor LY294002, at a dose of 50 μM, resulted in a 0.4-fold decrease in p-Akt (activated Akt) and a decrease in p-S6 (activated S6, downstream in the PI3K/Akt pathway) to less than 0.1-fold. The mTOR inhibitor rapamycin, at a dose of 1 μM, resulted in a decrease in p-S6 to less than 0.1-fold. The MAPK/ERK kinase (MEK) (kinase upstream of ERK) inhibitor U0126, at a dose of 10 μM, resulted in a decrease in p-ERK to less than 0.1-fold. 

LY294002 decreased total dendritic branch length 0.7-fold, the number of terminal tips 0.7-fold, soma area 0.6-fold, spine density 0.8-fold, and filopodia density 0.7-fold. Rapamycin decreased total dendritic branch length 0.5-fold, the number of terminal tips 0.6-fold, soma area 0.6-fold, spine density 0.7-fold, and filopodia density 0.5-fold. U0126 alone did not show any changes to neural remodeling, but U0126 with overexpression of RasL61 (activates the signaling pathway) decreased the total dendritic branch length 0.8-fold, decreased the number of terminal tips 0.4-fold, increased spine density 1.4-fold, and decreased filopodia density 0.5-fold compared to just RasL61 alone. 

Kanzawa et al., 2006 

In vitro. NSCs isolated from the frontal cortex of embryonic Fisher 344 rats were irradiated with a maximum of 10 Gy 137Cs gamma rays at 3.4 Gy/min. Signaling was determined by western blot. Apoptotic morphology of cells was determined with Hoechst 33258 staining. Apoptosis of just neurons was measured by a TUNEL assay. 

p-JNK (activated) increased a maximum of 60% after 10 Gy. No changes in p38 or ERK1/2 activation were observed. Cytochrome C and BAX were increased, and Bcl-2 was decreased after 10 Gy. The percent of apoptotic cells increased from 40 to 65% after 10 Gy. The percent of apoptotic neurons increased from 15 to 42% after 10 Gy. The percent of TUNEL+ cells that were p-JNK+ increased from 33 to 82% after 10 Gy as well. 

Silasi et al., 2004 

In vivo. Male and female C57/Bl6 mice were whole-body irradiated with either acute (single 0.5 Gy dose) or chronic/fractionated (0.05 Gy/day for 10 days) X-ray radiation, both at 0.002 Gy/s. Western blot was used to assess the levels of proteins in various signaling pathways in the hippocampus. DCx staining (immature neurons) was performed to determine hippocampal neurogenesis. 

After chronic radiation, male mice showed a 1.2-fold increase in Akt, a 0.95-fold decrease in p-Akt, a 0.6-fold decrease in p-ERK1/2, a 0.8-fold decrease in CaMKII, and a 0.9-fold decrease in p-CREB. Female mice showed a 1.3-fold increase in ERK1/2. After chronic radiation, DCx+ cells decreased 0.5-fold in both males and females. After acute radiation, a 0.6-fold decrease in p-ERK1/2 and a 0.7-fold decrease in CaMKII in males was observed, and DCx+ cells decreased 0.8-fold in males (non-significant) and 0.9-fold in females (non-significant). 

Ivanov & Hei, 2014 

In vitro. Human NSCs or neuroblastoma SK-N-SH cells were irradiated with 2.5, 5 or 10 Gy of 137Cs gamma rays (0.82 Gy/min). Western blot analysis was used to assess the levels of various signaling proteins. The percentage of hypodiploid nuclei was analyzed using flow cytometry to quantify apoptotic cells. Survival of differentiated cells was assessed with staining for Nestin (NSCs) and DCx (immature neurons). 

NSCs: p53 and TRAIL were increased at 2.5 and 5 Gy. Akt, p-Akt, p-p38, p-JNK, and pro-caspase-8 and -3 (inactive) were decreased at 5 Gy. The percent of NSCs that were apoptotic increased at 2.5 and 5 Gy, with a 5-fold increase at 10 Gy. At 5 Gy, just 11% of NSCs survived after differentiation compared to the unirradiated control. 

Neuroblastoma cells: p53, BAX, and p-ERK1/2 were increased at 2.5, 5, and 10 Gy. p-p38 was increased at 5 and 10 Gy. p-JNK2 and p-Akt were decreased at 10 Gy. Differentiation of NSCs cultured with non-irradiated SK-N-SH cells led to a survival rate of 19%, while NSCs cultured with 5 Gy irradiated SK-N-SH cells had a survival rate of 5%. 

Zhang et al., 2018 

In vivo and in vitro. Male 1-month-old Sprague-Dawley rats were whole-brain irradiated with electrons (4 MeV) at 2 Gy. Primary cultured hippocampal neurons from 18-day-old Sprague-Dawley rat embryos were irradiated with electrons at 2 or 8 Gy. Western blot was used to assess phosphorylated (inactive) and dephosphorylated (active) NFATc4/3 levels in vitro. Neurite growth in vitro was determined by immunofluorescence of β-tubulin+ neurons. In vivo dendritic growth was determined in the dentate gyrus with a retrovirus labeling newborn neurons with green fluorescent protein. 

In vitro: Dephosphorylated NFATc4/3 decreased by 20% at 2 Gy and 30% at 8 Gy. p-NFATc4/3 increased by 60% at 2 Gy and 90% at 8 Gy. Total neurite length decreased after 3 days by 24% at 2 Gy and 32% at 8 Gy. Branching points decreased by 29% at 2 Gy and 36% at 8 Gy after 3 days.  

In vivo: Total dendritic length in the dentate gyrus decreased about 30% after 2 Gy. 

Chow, Li and Wong, 2000 

In vivo. Female C57BL6/J mice (57 to 123 days old) were irradiated with X-rays to the entire brain (2 Gy). p53 levels were determined through immunohistochemistry. Apoptosis levels were quantified through morphological assessment after hematoxylin and eosin staining. 

In the subependymal region of the brain, 2 Gy resulted in the identification of many p53+ cells while none were found in the control. Specifically in glial cells, 2 Gy in the subependyma increased the p53+ cells from 0.23 to 15.7%. Apoptosis in the subependyma increased from 0.33 to 11.5% at 2 Gy. 

Huang et al., 2021 

In vitro. HT22 hippocampal neuronal cells were irradiated with 10 Gy of X-rays (6 Gy/min). Levels of proteins in the PI3K/Akt, p53, and apoptotic signaling pathways were determined by western blot. Apoptosis was measured by flow cytometry with annexin V (marker for apoptotic cells) and propidium iodide staining. 

The ratio of p-PI3K/PI3K increased 5-fold (non-significant), the ratio of p-Akt/Akt increased 2-fold (non-significant), the ratio of BAX/Bcl-2 increased 5-fold, p53 increased 2-fold, cleaved caspase-3 increased 2.7-fold, and apoptosis increased 9-fold all at 10 Gy. 

El-Missiry et al., 2018 

In vivo. Adult male albino Wistar rats were whole-body irradiated with 137Cs gamma rays at 4 Gy (0.695 cGy/s). Levels of signaling proteins in the hippocampus were determined with respective assay kits. Hematoxylin and eosin staining in the hippocampal dentate gyrus was used for histopathological analysis. Apoptosis levels in the hippocampus were determined by flow cytometry with annexin V (marker for apoptotic cells) and propidium iodide staining. Radiation was delivered to the animal's entire body. 

Radiation at 4 Gy resulted in 2- to 4-fold increases in p53, cytochrome C, BAX, and caspase-3, -8, and -9 levels. Radiation at 4 Gy also resulted in a 0.2-fold decrease in Bcl-2. The percent of live cells decreased 0.6-fold, the frequency of apoptosis increased 3- to 4-fold, and the frequency of necrosis increased 7-fold. In addition, 4 Gy resulted in extensive damage to the dentate gyrus. 

Pius-Sadowska et al., 2016 

In vivo. Female 6- to 8-week-old BALB/c mice were whole-brain irradiated with 60Co gamma rays at 10 Gy. After whole-brain irradiation, the levels of various signaling molecules were assessed with western blot in the brain. Apoptosis was measured with a TUNEL assay in the hippocampus. Nissl staining was used to assess neuron morphology in the hippocampus. 

Irradiation at 10 Gy resulted in a maximum 1.8-fold increase in caspase-3, a 1.7-fold increase in BDNF, a 1.8-fold increase in TrkA, a 3.8-fold increase in TrkB, a 1.7-fold increase in TrkC, a 4.1-fold increase in p-ERK1/2, and a 2.9-fold increase in p-Akt. Without irradiation, no TUNEL+ cells were found in the brain, while 10 Gy resulted in the detection of apoptotic nuclei in the dentate gyrus. Also, degenerative morphological changes were observed in the hippocampus after 10 Gy. 

Ji et al., 2014 

In vivo. Male 1-month-old Sprague-Dawley rats were whole-brain irradiated with electrons (4 MeV) at 20 Gy. Western blot was performed to assess the levels and activity of proteins in the BDNF-pCREB pathway in the hippocampus. Neurogenesis in the dentate gyrus was assessed using DCx (immature neurons) or BrdU/NeuN (new neurons) staining. 

Irradiation at 20 Gy decreased p-ERK1/2 by 15%, p-Akt by 34% BDNF by 38%, p-TrkB by 54%, p-CaMKII by 30%, and p-CREB by 29%. Irradiation at 20 Gy also reduced the number of DCx+ cells by 92% and the number of BrdU+/NeuN+ cells by 82%. 

Reference 

Experiment Description 

Result 

Kanzawa et al., 2006 

In vitro. NSCs isolated from the frontal cortex of embryonic Fisher 344 rats were irradiated with 137Cs gamma rays at 10 Gy (3.4 Gy/min). Signaling was determined by western blot. Apoptotic morphology of cells was determined with Hoechst 33258 staining. Apoptosis of just neurons was measured by a TUNEL assay. 

p-JNK increased 30% after 1h and 60% after 2h post-irradiation. Starting 24h post-irradiation, BAX and cytochrome C were increased, and Bcl-2 was decreased. Apoptosis increased 48h post-irradiation. 

Suman et al., 2013 

In vivo. Female 6- to 8-week-old C57BL/6J mice were irradiated with either 137Cs gamma rays (2 Gy) or 1 GeV/n 56Fe ions (1.6 Gy) both at 1 Gy/min. p16, p21, p53, BAX, and Bcl-2 levels were determined in the cerebral cortex with immunoblotting. Hematoxylin and eosin staining was used to measure cerebral cortex thickness, and a TUNEL assay was used to measure apoptosis. 

Altered signaling through p16, p21, p53, BAX, and Bcl-2 protein levels was observed as early as 2 months post-irradiation, while increased apoptosis and decreased cortical thickness were only shown at 12 months. 

Limoli et al., 2004 

In vivo and in vitro. Male 2-month-old C57BL/J6 mice (1.75 Gy/min) and neural precursor cells (4.5 Gy/min) isolated from rats were irradiated with X-rays at 5 Gy. Mice were irradiated cranially. Western blot analysis was done in vitro to measure levels of p53 and p21 proteins. An antibody against DCx was used to detect immature neurons in the dentate gyrus in vivo. FACS analysis of propidium iodide fluorescence was used to assess apoptosis in vitro. 

In vitro: At 2h post-irradiation, p53 protein levels increased 2-fold compared to unirradiated controls. At 6h post-irradiation, p53 protein levels increased 4-fold compared to unirradiated controls. At the same timepoints increases in p21 and p-p53 were observed. Apoptosis showed a maximum increase 12h post-irradiation. 

In vivo: DCx+ cells were decreased at 24h post-irradiation. 

Ivanov & Hei, 2014 

In vitro. Human NSCs or neuroblastoma SK-N-SH cells were irradiated with 5 Gy of 137Cs gamma rays (0.82 Gy/min). Western blot analysis was used to assess the levels of various signaling proteins. The percentage of hypodiploid nuclei was analyzed using flow cytometry to quantify apoptotic cells. Survival of differentiated cells was assessed with staining for Nestin (neuroprogenitors) and DCx (immature neurons). 

Altered signaling in both cell types was observed 6h post-irradiation. Apoptosis of NSCs was observed only 24h post-irradiation. NSC differentiation was reduced 10-12 days post-irradiation. 

Zhang et al., 2018 

In vivo and in vitro. Male 1-month-old Sprague-Dawley rats were whole-brain irradiated with electrons (4 MeV) at 2 Gy. Primary cultured hippocampal neurons from 18-day-old Sprague-Dawley rat embryos were irradiated with electrons at 2 or 8 Gy. Western blot was used to assess phosphorylated (inactive) and dephosphorylated (active) NFATc4/3 levels in vitro. Neurite growth in vitro was determined by immunofluorescence of β-tubulin+ neurons. In vivo dendritic growth was determined with a retrovirus labeling newborn neurons with green fluorescent protein. 

In vitro: Dephosphorylated NFATc4/3 was decreased and p-NFATc4/3 was increased at both 1- and 3-days post-irradiation. Although total neurite length was decreased at both 1 and 3 days as well, the number of branching points was only decreased at 3 days post-irradiation. 

In vivo: Total dendritic length decreased at both 14- and 28-days post-irradiation. 

Pius-Sadowska et al., 2016 

In vivo. Female 6- to 8-week-old BALB/c mice were whole-brain irradiated with 60Co gamma rays at 10 Gy. The levels of various signaling molecules were assessed with western blot in the brain. Apoptosis was measured with a TUNEL assay in the hippocampus. Nissl staining was used to assess neuron morphology in the hippocampus. 

Following 10 Gy, the various signaling molecules were increased as early as 3h, while apoptosis and morphological changes were found 48h post-irradiation. 

Reference 

Experimental Description 

Results 

Tian et al., 2020 

In vivo. Male 8- to 10-week-old C57BL/6J mice were subjected to middle cerebral artery occlusion to simulate an ischemic stroke. MAPK signaling molecules and BAX/Bcl-2 apoptotic markers were measured with western blotting in ischemic brain tissues. Apoptosis was determined using a TUNEL assay in the ischemic cerebral cortex. Endpoints were measured 7 days after surgery. 

After surgery, ERK1/2, p38 and JNK mRNA increased 2- to 3- fold. The ratios of phosphorylated to total ERK1/2, p38 and JNK increased 2- to 3- fold as well. BAX increased 2.5-fold, Bcl-2 decreased 0.15-fold, and cleaved caspase-3 increased 1.5-fold. The % of TUNEL+ cells increased 2-fold. 

Eom et al., 2015 

In vitro. C17.2 mouse neural stem-like cells were irradiated with 6 Gy of 137Cs gamma rays at 0.95 Gy/min. Protein levels in signaling pathways were determined by western blot. The number of cells expressing nestin (NSC marker) were quantified with immunocytochemistry. Endpoints were measured 72h post-irradiation. 

Radiation at 6 Gy led to increased p-Akt, p-p53, p-STAT3, and mGluR1 at least 2-fold. NSCs decreased 0.4-fold. 

Suman et al., 2013 

In vivo. Female 6- to 8-week-old C57BL/6J mice were irradiated with either 137Cs gamma rays (2 Gy) or 1 GeV/n 56Fe ions (1.6 Gy) both at 1 Gy/min. p16, p21, p53, BAX, and Bcl-2 levels were determined in the cerebral cortex with immunoblotting. Hematoxylin and eosin staining was used to measure cerebral cortex thickness, and a TUNEL assay was used to measure apoptosis. 

p16 increased a maximum of 3.4-fold after gamma rays and 5-fold after 56Fe radiation. p21 increased a maximum of 1.5-fold after gamma rays and 3-fold after 56Fe radiation. p53 increased a maximum of 8.4-fold after gamma rays and 9-fold after 56Fe radiation. BAX increased a maximum of 2.3-fold after gamma rays and 6.7-fold after 56Fe radiation. Bcl-2 decreased a maximum of 0.6-fold after gamma rays and 0.4-fold after 56Fe radiation. Gamma rays increased apoptosis 1.8-fold and 56Fe ions increased apoptosis 3.6-fold. Gamma rays decreased cortical thickness 0.9-fold and 56Fe ions decreased cortical thickness 0.7-fold. 

Limoli et al., 2004 

In vivo and in vitro. Male 2-month-old C57BL/J6 mice (1.75 Gy/min) and neural precursor cells (4.5 Gy/min) isolated from rats were irradiated with X-rays. Mice were irradiated cranially. Western blot analysis was done in vitro to measure levels of p53 and p21 proteins. An antibody against DCx was used to detect immature neurons in the dentate gyrus in vivo. FACS analysis of propidium iodide fluorescence was used to assess apoptosis in vitro. 

In vitro: At 5 Gy, p53 levels increased a maximum of 4-fold, while p-p53 and p21 were also increased at this dose. Apoptosis was increased a maximum of 1.4-fold after 5 Gy. 

In vivo: The number of DCx+ cells decreased 0.4-fold after 5 Gy.