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Relationship: 2840

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Altered Signaling leads to Abnormal Neural Remodeling

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Altered Signaling

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Abnormal Neural Remodeling

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	Low	NCBI
mouse	Mus musculus	Moderate	NCBI
rat	Rattus norvegicus	Low	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Male	Moderate
Female	Low
Unspecific	Moderate

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Juvenile	Moderate
Adult	Low

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Alterations in signaling pathways can trigger disruption to neuronal structures, which can lead to altered morphology, changes in neurogenesis, neurodegeneration, apoptotic activity and synaptic activity, collectively known as neural remodeling (Cekanaviciute et al., 2018; Chakraborti et al., 2012; Hladik & Tapio, 2016). These intracellular pathways are key processes to control various cell functions such as cell growth, death or communication. Within the neuron, multiple signaling pathways influence its structure and function. For example, the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) family pathways are involved in neuronal survival, proliferation, morphology, and synaptic plasticity (Davis and Laroche, 2006; Long et al., 2021; Mazzucchelli and Brambilla, 2000). The senescence pathway induces cell cycle arrest and can restrict neurogenesis (McHugh and Gil, 2018). The apoptotic pathway can be initiated within the mitochondria due to dysfunction within the respiratory chain and induces various signaling proteins such as p53, BAX, caspases and cytochrome C (Betlazar et al., 2016; Mielke and Herdegen, 2000; Wang et al., 2020). Apoptosis of neurons results in a reduction in neuron numbers, demonstrating neural remodeling. A few studies also measure high apoptosis levels over time, indicating sustained neuron loss contributing to reduced neural activity (Chow, Li, and Wong, 2000; Limoli et al., 2004; Pius-Sadowska et al., 2016). Additionally, the brain-derived neurotrophic factor (BDNF)-cAMP-calcium response element binding protein (CREB) pathway is involved in the regulation of excitatory transmission as CREB-dependent transcription allows for persistent pre- and post-synaptic neurotransmitter release at excitatory synapses (Ran et al., 2012).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

The strategy for collating the evidence to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Overall Weight of Evidence: Moderate

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Neural remodeling can be induced by changes in multiple signaling pathways, including MAPK signaling, PI3K/Akt signaling, senescence signaling, and apoptotic signaling. These pathways are involved in the homeostatic regulation of neuron numbers, morphology, proliferation, differentiation, and synaptic activity.

Like many signaling pathways, MAPK pathways help maintain the biological functions in neurons, and changes to the expression or activity of signaling molecules in MAPK pathways can result in neural remodeling. The extracellular signal-regulated protein kinase (ERK)1/2 MAPK pathway is crucial for modulating synaptic function and alteration in expression of critical proteins in this pathway will result in long-term potentiation (LTP) deficits (Davis and Laroche, 2006; Mazzucchelli and Brambilla, 2000). Research shows that modulations in ERK1/2 expression and activity can decrease cell proliferation in the hippocampus (Betlazar et al., 2016). The p38 MAPK pathway is also involved in maintaining neuronal plasticity and synaptic function, by inducing metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) in hippocampal neurons (Falcicchia et al., 2020). However, p38 demonstrates variable effects in neuronal survival and proliferation. Although p38 signaling is required for the survival of developing neurons, p38 can also be involved in the induction of apoptosis, and subsequent inhibition of p38 promotes cell survival (Mielke and Herdegen, 2000; Nebreda and Porras, 2000). The role of p38 is often dependant on the cell type and stimulus and will determine whether p38 has a positive or negative role on neural cell proliferation (Nebreda and Porras, 2000). The c-Jun NH2-terminal kinase (JNK) MAPK pathway plays a similar role to the p38 pathway, and its function is also dependant on the cell type and context of the cellular environment. JNK can induce apoptosis as well as regulate proteins like tau and microtubule-associated protein (MAP)2 involved in altering cytoskeletal dynamics and cell morphology (Mielke and Herdegen, 2000; Sherrin, Blank, and Todorovic, 2011). JNK is also involved in both pre- and post-synaptic function through the phosphorylation of AMPA receptors and postsynaptic density protein (PSD)95 (Sherrin, Blank, and Todorovic, 2011).

The PI3K/Akt pathway is involved in many neuronal functions. Activation of the PI3K/Akt pathway promotes transcription of survival genes and inhibits death genes, while also regulating the activity of various death pathways (Long et al., 2021; Rai et al., 2019). Alterations in Akt expression and activity can decrease cell proliferation in the hippocampus (Betlazar et al., 2016). The pathway can also regulate neuron morphology, as neurite outgrowth can be induced by activation of the pathway (Rodgers and Theibert, 2002). Synaptic plasticity and LTP, which is induced by the activation of NMDA receptors and the subsequent insertion of AMPA receptors to the membrane, can additionally be regulated by the PI3K/Akt pathway. It has been shown that the mammalian target of rapamycin (mTOR), downstream of Akt, increases the expression of LTP-related proteins while PI3K guides AMPA insertion on the membrane (Long et al., 2021). Therefore, maintaining the appropriate expression levels of signaling molecules is critical for proper neural development and function.

The signaling molecules p53/p21 and p16 as part of the cellular senescence pathway can induce cell cycle arrest. For example in neural stem cells (NSCs), reduced functionality and limited neurogenesis is associated with increased senescence markers (McHugh and Gil, 2018).

The apoptosis pathway, consisting of the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, caspases, cytochrome C, and Bcl-2-associated X protein (BAX) as well as the anti-apoptotic B-cell lymphoma (Bcl)-2 protein, contributes to a reduction in neuron numbers through cell death when activated (Betlazar et al., 2016; Wang et al., 2020). This pathway may be induced by perturbations to other signaling pathways, including MAPK, PI3K/Akt, and senescent pathways (Hladik and Tapio, 2016; Mielke and Herdegen, 2000). For example, JNK and p53 can both antagonise the anti-apoptotic Bcl-2, while JNK can stabilize p53 and p53 enhances BAX (Mielke and Herdegen, 2000).

Signaling pathways not just in neurons, but also in astrocytes and microglia, can influence neural remodeling. For example, it was previously mentioned that p38 signaling in neurons contribute to hippocampal mGluR-dependent LTD. In astrocytes, p38 signaling is necessary for NMDA-dependent LTD during astrocyte-to-neuron communication (Falcicchia et al., 2020). In addition, BDNF signaling in both neurons and astrocytes prevents cell death in the respective cells through activation of the ERK and PI3K/Akt pathways. Neuronal death can be prevented by BDNF signaling in both cell types because astrocytes release factors that prevent neuronal death (Rai et al., 2019).

Synergistic and antagonistic interactions between signaling pathways can also occur, contributing to the complexity and context-dependence of the neural remodeling response to various signaling pathways. For example, nuclear factor of activated T-cells (NFATc) nuclear translocation and transcriptional activation can be encouraged by the PI3K/Akt pathway and the ERK pathway and inhibited by p38 and JNK (Macian, 2005; Mielke and Herdegen, 2000). Activation of NFATc promotes neuronal survival, synaptic plasticity, and neurite outgrowth through the transcription of multiple target genes (Zhang et al., 2018). BDNF activation of the ERK, PI3K/Akt, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways through tropomyosin receptor kinase (Trk) activation results in the activation of CREB transcriptional activity (Cunha, Brambilla, and Thomas, 2010). CREB is essential for the regulation of excitatory transmission, and exogenous stressors can induce hippocampal neuronal damage through the inhibition of this pathway (Hladik and Tapio, 2016; Wang et al., 2020).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

The empirical evidence for this KER comes from in vivo mouse and rat models as well as in vitro rat-, mouse-, and human-derived cell models. Stressors used included gamma ray radiation (El-Missiry et al., 2018; Eom et al., 2015; Ivanov and Hei, 2014; Kanzawa et al., 2006; Pius-Sadowska et al., 2016; Suman et al., 2013), X-ray radiation (Chow, Li and Wong, 2000; Huang et al., 2021; Limoli et al., 2004; Silasi et al., 2004), electron radiation (Ji et al., 2014; Zhang et al., 2018), iron-56 ion radiation (Suman et al., 2013), simulated ischemic stroke (Tian et al., 2020), and pharmacological modulation of signaling molecules (Kumar et al., 2005). Neural remodeling can be determined through various endpoints, including neuronal apoptosis, morphology, proliferation, differentiation, and synaptic activity.

Dose Concordance

Several studies demonstrate dose concordance for this relationship. Studies have observed that altered signaling can occur at the same stressor doses as neural remodelling. X-irradiation at doses ranging from 0.5 Gy to 10 Gy resulted in changes in protein levels and phosphorylated proteins in key signaling pathways, as well as increased apoptotic activity and decreased number of neurons. Silasi et al. (2004) irradiated male and female mice with either acute (single 0.5 Gy dose) or chronic (0.05 Gy/day for 10 days) X-ray radiation at 0.002 Gy/s and found that chronic radiation decreased the levels of phosphorylated Akt, ERK1/2, CREB and CaMKII in males. Female mice showed a 1.3-fold increase in ERK1/2. A decrease in DCx+ cells was also observed in both sexes. At 2 Gy, Chow, Li and Wong (2000) reported an increase in p53+ cells and apoptosis within the brain. 10 Gy of X-irradiation resulted in an increase in BAX/Bcl-2 ratio, p53 and cleaved caspase 3 levels, as well as apoptosis within hippocampal neurons (Huang et al., 2021). Another radiation source is electrons, and doses from 2 to 20 Gy also affect protein levels and phosphorylated proteins in key signaling pathways, as well as neuronal structure and number. Zhang et al. (2018) reported a dose-dependent decrease in dephosphorylated (active) NFATc4/3 after 2 and 8 Gy, as well as an increase in phosphorylated (inactive) NFATc4/3 at these doses within the brains of rats. Total neurite length and branching points dose-dependently decreased at 2 and 8 Gy, whereas total dendritic length decreased at only 2 Gy. Whole-brain irradiation of rats at 20 Gy decreased p-ERK1/2, p-Akt, BDNF, p-TrkB, p-CaMKII, and p-CREB. Irradiation at 20 Gy also reduced the number of DCx+ cells and the number of BrdU+/NeuN+ cells (Ji et al., 2014).

Furthermore, studies using gamma irradiation as a stressor have also shown changes within this KER. Doses ranged from 2-10 Gy and resulted in altered levels of phosphorylated and dephosphorylated proteins in the MAPK family, apoptotic pathway, senescence pathway and BDNF-pCREB pathway (El-Missiry et al., 2018; Ivanov & Hei, 2014; Kanzawa et al., 2006; Pius-Sadowska et al., 2016; Suman et al., 2013). In another study, Kumar et al. (2005) utilized pharmacological inhibition of various signaling proteins such as those associated with the PI3K-Akt and MAPK family pathway. The results elicited changes in hippocampal neuron morphology as defined by a reduction in dendritic branch length, number of terminal tips, soma area, spine density and filopodia density.

Time Concordance

Multiple studies demonstrate that signaling pathways are altered before neural remodeling is observed in a time-course. Altered signaling pathways are often found as early as hours post-irradiation, while neural remodeling is first measured after days. NSCs isolated from rats showed an increase in p-JNK as early as 1h post-irradiation (10 Gy of gamma rays), while apoptosis was measured 48h post-irradiation (Kanzawa et al., 2006). After 5 Gy X-ray irradiation of rat neural precursor cells, p53 and p21 signaling was increased after 2h, while the first significant increase in apoptosis was observed after 24h (Limoli et al., 2004). In mice exposed to gamma ray irradiation at 10 Gy, multiple signaling molecules were increased as early as 3h post-irradiation, while measurements of apoptosis and neuronal morphological changes occurred 48h post-irradiation (Pius-Sadowska et al., 2016). Neuron levels were found decreased 24h post-irradiation in this study, although this was in an in vivo mouse model. Human NSCs irradiated with 5 Gy of gamma rays showed many signaling molecules were dysregulated after 6h, apoptosis was first increased after 24h, and the differentiation of NSCs was decreased after 10-12 days (Ivanov and Hei, 2014).

A few studies also demonstrated time concordance when altered signaling was measured after days or longer. Rat neurons irradiated with electrons at 2 Gy showed higher inactive and lower active levels of NFATc4/3 after both 1 and 3 days (Zhang et al., 2018). The number of branching points were subsequently decreased after only 3 days. However, total neurite length was decreased after 1 day. Furthermore, total dendritic length decreased after both 14 and 28 days, although this was in an in vivo rat model (Zhang et al., 2018). In a longer-term study using both gamma ray (2 Gy) and 56Fe ion (1.6 Gy) irradiation of mice, Suman et al. (2013) found changes in apoptotic and senescent signaling pathways at both 2 and 12 months post-irradiation, while increased apoptosis and decreased cortical thickness were only measured after 12 months.

Incidence Concordance

Several studies reported an incident-concordant relationship between altered signaling pathways and neural remodeling. In the study conducted by Suman et al. (2013), female mice were irradiated with either gamma rays (2 Gy) or 1 GeV/n 56Fe ions (1.6 Gy) and at both doses, an increase in p16, p21, p53, BAX and apoptosis was observed. A decrease in Bcl-2 and cortical thickness was also seen after both gamma and 56Fe irradiation. Another study irradiated neural stem cells with 6 Gy of gamma rays and found increased p-Akt, p-p53, p-STAT3, and mGluR1, with decreased neural stem cells (Eom et al., 2015). At 5 Gy of X-irradiation, Limoli et al. (2004) also found increased levels of p53 and phosphorylated p53 with increased apoptosis and decreased DCx+ cells.

Through the use of middle cerebral artery occlusion, a technique to simulate an ischemic stroke, alteration in signaling pathways were shown. Following the administration of this procedure in male mice, the ratios of phosphorylated to total ERK1/2, p38 and JNK increased, as well as levels of BAX, cleaved caspase-3 and the percent of apoptosis. Anti-apoptotic marker Bcl-2 also decreased after surgery (Tian et al., 2020).

Essentiality

Attenuation of altered signaling consistently results in a reduction in neural remodeling. Multiple studies showed this using genetic knockout of signaling molecules. For example, genetic knockout of Src, an upstream activator of multiple signaling pathways, or a combination of p38 and ERK1/2 in mice with a simulated ischemic stroke led to the inhibition of signaling in the MAPK pathways, decreased apoptosis, and increased neuron levels compared to wild-type mice (Tian et al., 2020). In accordance, activation of Src led to increased MAPK signaling and apoptosis compared to wild-type mice after simulated ischemic stroke (Tian et al., 2020). Two studies showing that neurons decreased following 1-5 Gy irradiation of mice found that knockout of p53 decreased apoptosis and slightly restored neuron numbers (Chow, Li and Wong, 2000; Limoli et al., 2004).

Inhibition of various signaling molecules after irradiation also led to reduced neural remodeling. The mGluR1 inhibitor LY367385 restored NSC numbers after irradiation of neural-like stem cells with 6 Gy of gamma rays (Eom et al., 2015). Similarly, the JNK inhibitor SP600125 restored neuronal differentiation after 2 Gy gamma ray irradiation of NSCs (Kanzawa et al., 2006). Zhang et al. (2018) found that activation of NFATc4/3 nuclear translocation with BDNF was able to restore neurite length and the number of total branching points in rats and rat-derived neurons after 2 Gy electron irradiation, while inactivation of NFATc4/3 nuclear translocation with CsA produced the opposite effect in rat-derived neurons after irradiation.

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

The changes to a signaling pathway may provide inconsistent outcomes in neural remodeling. For example, the p38 pathway is involved in many, often opposing, biological processes (Nebreda and Porras, 2000). Different cell types and exposures can be associated with the expression of different receptors of the p38 pathway, resulting in different biological changes. In addition, signaling pathways that synergize or antagonize with each other may be influenced at the same time resulting in cumulative effects across different pathways (Nebreda and Porras, 2000).
Eom et al., 2015: Irradiation of C17.2 mouse neural stem-like cells with 6 Gy of gamma rays resulted in an increase in β-III tubulin expression, indicating a rise in neurons post-irradiation. However, all other studies observed a decrease in neuron numbers post-irradiation.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

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

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

The table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data is statistically significant unless otherwise stated.

Dose Concordance

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%.

Time Concordance

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.

Incidence Concordance

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.

Response-response Relationship

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Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Evidence for this relationship comes from human-derived cells, rat, and mouse models, with most of the evidence in mice. There is in vivo evidence in both male and female animals, with more evidence in males. Animal age is occasionally not indicated in studies, but most evidence is in adolescent rodent models with a few studies using adult animals.

References

List of the literature that was cited for this KER description. More help

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