Inhibition of necroptosis mitigates paclitaxel-induced neuronal damage and cognitive impairment

doi:10.21203/rs.3.rs-3841052/v1

Download PDF

Research Article

Inhibition of necroptosis mitigates paclitaxel-induced neuronal damage and cognitive impairment

https://doi.org/10.21203/rs.3.rs-3841052/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Paclitaxel (PTX) is a first-line chemotherapy agent for treating many types of cancers, induces cognitive impairment and neuronal damage. However, PTX-induced a limited apoptosis of neurons is not consistent with a wide range of neuroinflammation. Here, we demonstrated that in addition to inducing apoptosis in hippocampal neurons (HT22 cells), PTX causes necroptosis, a programmed cell death, via activation of the RIPK1-RIPK3-MLKL signaling pathway. Annexin V/PI dual labeling, flow cytometric analysis, image-based PI staining, and western blot techniques were used to evaluate PTX-induced necroptosis. Cell viability was determined using the CCK8 assay, whereas Ca²⁺ levels were measured using the Fluo-4 AM fluorescent probe. The number of cells that were positive for both Annexin V and PI staining was considerably higher in PTX-treated HT22 cells compared to those treated with the vehicle. Additionally, the nuclei of PTX-treated cells showed more diffuse necrotic staining with PI compared to the vehicle-treated cells. The Western blot study demonstrated a considerable increase in the expression of necroptotic proteins, including RIPK1, RIPK3, MLKL, and p-MLKL, following PTX treatment. The compound Necrotatin-1 (Nec-1), which specifically inhibits the protein RIPK1, effectively decreased the occurrence of necroptosis in HT22 cells triggered by PTX by lowering the excessive accumulation of intracellular Ca²⁺ overload. In addition, administration of Nec-1 in vivo rescued cognitive impairments in novel object recognition and Morris Water Maze tests in PTX-treated mice. These data suggest that PTX induces cognitive impairments through RIPK1-mediated necroptosis. Inhibition of necroptosis provided a potential therapeutic approach to reduce PTX-induced cognitive deficits.

Paclitaxel

Cognitive deficits

Necroptosis

Necrostatin-1

RIPK1-RIPK3-MLKL signaling

Chemotherapy is commonly used to treat malignant tumors; however, its neurotoxicity significantly affects the quality of life of cancer survivor and hampers the efficacy of the treatment. Paclitaxel (PTX) is a widely used chemotherapy medicine that is known to cause cognitive problems as a significant adverse effect. Within clinical settings, a range of 15–75% of breast cancer patients who have chemotherapy with PTX experience mild to severe cognitive impairment. Among these patients, 35% continue to exhibit symptoms for an extended period of time, lasting from months to years following the treatment. This condition is commonly referred to as "post-chemotherapy cognitive impairment" [1; 2]. The occurrence of attention impairment can reach up to 48% and memory impairment can reach up to 53% as a result of post-chemotherapy cognitive impairment, which might further worsen the functional deterioration of the central nervous system [3]. Although several attempts have been made to mitigate the cognitive damage caused by PTX, the fundamental mechanism behind it remains mostly unclear.

It has been shown that a small amount of systemically administrated radioactivity-labeled PTX cross the blood-brain barrier to enter the brain [4; 5]. The PTX that enters the brain is mainly distributed in the hippocampus tissue [6], a brain region that is crucial for learning and memory and cognitive function. It has been shown that PTX includes neuron apoptosis and autophagy cell death in the hippocampus and neuroinflammation in multiple brain regions [7; 8; 9; 10]. However, inconsistence of limited neuron apoptosis and autophagy cell death in the hippocampus and wide range of neuroinflammation in multiple brain regions indicates that other mechanisms are involved in PTX-induced neuronal damage. Necroptosis is one form of necrosis that largely depending on receptor-interacting protein kinase 1 (RIPK1)-receptor-interacting protein kinase 3 (RIPK3)-and mixed lineage kinase domain-like (MLKL) signaling [11; 12]. Necroptosis is a type of programmed cell death that occurs independently of caspase activity and is initiated by the activation of death receptors [13]. When the phosphorylated MLKL accumulates in the cell membrane and alters membrane permeability, membrane is ruptured to release cellular contents [14; 15; 16; 17] and damage-associated molecular patterns (DAMPs), which trigger immune response and inflammation [18]. It has been shown that RIPK1 regulates cell death and promotes neuroinflammation in several neurodegenerative diseases [19; 20; 21]. A RIPK1 kinase specific inhibitor, necrostatin-1 (Nec-1), is able to inhibit necroptosis process [16]. Thus, inhibition of RIPK1 prevents necroptosis and is a potential approach to differentiate necroptosis and necrosis [22].

Several studies have shown that Nec-1 inhibits necroptosis to induce neuroprotection in several animal models of neurological disorders [23; 24; 25; 26]. Furthermore, the RIPK1-RIPK3 pathway is involved in neurodegenerative diseases [27]. However, it is unclear if necroptosis plays a role in the pathophysiology of PTX-induced cognitive deficits. In this study, we detected cell damage and the expression of necroptosis-related proteins in HT22 cells exposed to PTX. Furthermore, application of Nec-1 mitigated PTX-induced cell damage and expression of necroptosis-related proteins. More importantly, application of Nec-1 reduced cognitive deficits in PTX-treated mice. To our knowledge, this is the first study showing that Nec-1 reduced PTX-induced cell death of HT22 cells and cognitive deficits. These findings are important because identification of PTX-induced necroptosis will help to identify new targets for therapeutics to treat post-chemotherapy cognitive impairment.

2.1 Culture of HT22 cell line and PTX treatment

The HT22 cell line, derived from immortalized mouse hippocampal neurons, was acquired from Bluefbio Biology Technology Development Co., Ltd (cat. no. BNF60808571, Shanghai, China). The cells were cultured in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin-amphotericin B at 37°C in a 5% CO₂ incubator [28]. When the HT22 cells in culture reached the logarithmic phase with a confluence of 60–80%, the culture media was fully changed with new DMEM/F12 mixture containing 10% FBS. PTX (P1632, TCI, Shanghai, China) was solubilized in phosphate-buffered saline (PBS) with 0.1% dimethyl sulfoxide (DMSO) to provide a 1 mM stock solution. The PTX was diluted in Dulbecco's modified Eagle medium to the designated concentrations employed in each individual experiment. The control group was administered with a solution of PBS containing 0.1% DMSO. The compound Nec-1 (11658, Cayman Chemical Company, Kentucky, USA) was dissolved in PBS and made into a 1mM stock solution. This stock solution was then diluted into Dulbecco’s modified Eagle medium to achieve the desired final concentrations for each experiment. In the studies, the cells in the experimental group were subjected to pretreatment with Nec-1 for a duration of 2 hours. Following this, the cells were treated with or without PTX (10 µM) for a period of 24 hours.

2.2 Cell viability assay

HT22 cells treated with PTX were seeded onto 24-well culture plates at a density of 1 × 105 cells/1 mL/well. Various doses of PTX ranging from 1 to 20 µM were used [29]. The HT22 cell shape and growth status were examined using a TS100 inverted microscope (model BH2-RFCA, manufactured by OLYMPUS in Japan) at intervals of 2–4 hours following treatment with PTX. Cell proliferation and survival were assessed using the Cell Counting Kit-8 (CCK-8, cat. no. K009, ZETA, USA), following the manufacturer's instructions. The cells were placed in 96-well plates and incubated for 24 hours. Afterwards, PTX was introduced into the growth medium at the specified doses. The cells were subsequently incubated for an additional 24 hours. Subsequently, 10 µl of CCK-8 working solution were introduced into each well and subjected to incubation at a temperature of 37°C for a duration of 0.5 hours. Absorbance at 450 nm was measured using a Spectra Max Absorbance Reader (Molecular Devices, China).

2.3 Lactate Dehydrogenase Assay

The levels of the cytoplasmic enzyme lactate dehydrogenase (LDH) were measured using the Cytotoxicity LDH test kits (JL-T1070, Shanghai Jianglai Industrial Limited By Share Ltd) following the instructions provided by the manufacturer. Concisely, the cells were ruptured and the liquid above them, as well as the liquid in which they were growing, were gathered to measure LDH concentrations. The absorbance readings were monitored using a SpectraMax absorbance reader (Molecular Devices, China) at a wavelength of 450 nm. Subsequently, the percentage of LDH release was determined.

2.4 Flow cytometry analysis

The technique of flow cytometry was employed to ascertain the alterations in the morphological characteristics of cells undergoing apoptosis and necrosis. In summary, the cells were placed in the 12-well plate with a confluency of 40% and exposed to certain treatments. Subsequently, the cells were collected, rinsed with PBS on two occasions, and subjected to staining using the FITC Annexin V apoptosis detection kit (AT101-100-kit, MULTISCIENCES), following the guidelines provided by the manufacturer. The stained cells were examined by flow cytometry (E97501093, BD Biosciences) with the use of the Flow jo software. Apoptotic cells were detected using Annexin V labeling, whereas necrotic cells were detected with PI staining.

2.5 Microscopic imaging of cell death

In order to evaluate cell death, HT22 cells were introduced onto a 24-well plate with a concentration of 1x10⁵ cells per well. The assessment of cell necrosis was conducted using Hoechst 33258/PI staining, following the manufacturer's procedure. Cells were washed twice with PBS before being incubated with Hoechst 33258/PI reagents (94403, Sigma, Germany) at 37°C for 5 minutes in the dark. The cell pictures were obtained using fluorescent microscopy (BH2-RFCA, OLYMPUS, Japan).

2.6 Western blot analysis

A Western blot was conducted using established protocols. HT22 cells were lysed using ice-cold RIPA buffer (Solarbio, Beijing, China). The protein produced from the lysate was divided into equal quantities and subjected to 10% SDS-polyacrylamide gel electrophoresis. Subsequently, the protein was transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were exposed to primary antibodies specific to several proteins, including anti-RIPK1 (Arigo, Shanghai, China), anti-RIPK3 (Arigo, Shanghai, China), anti-MLKL (Abcam, Shanghai, China), anti-phosphorylation MLKL (Abcam, Shanghai, China), and anti-GAPDH (Abcam, Shanghai, China). Subsequently, the appropriate alkaline phosphatase-conjugated secondary antibodies were used to see the bands. The bands were scanned and shown utilizing the Epson Perfection V39 Scanner system (EPSON, Japan). The band signals' strength was assessed utilizing the Image J program. The internal control utilized in this study was GAPDH.

2.7 Transmission electron microscopy (TEM)

The TEM (Fan and Fan, 2017) was used to view and study the ultrastructure of HT22 cells. The cells were grown in a 6-well plate, treated with 10 µM PTX, and collected at 24 hr following treatment. Subsequently, the cells were immobilized using a PBS solution containing 4% glutaraldehyde and 1% osmium tetroxide. They were then dried, embedded in epoxy resin, and visualized using a H700 TEM (Hitachi, Tokyo, Japan).

2.8 Estimation of intracellular Ca²⁺ concentration

The Fluo-4 AM Kit (KGAF024, Keygen Biotech) was used to measure the concentration of Ca²⁺ inside the cells, following the instructions provided by the manufacturer. In summary, HT22 cells were placed in a 6-well plate and left to incubate overnight. Subsequently, the manufacturer's loading buffer was used to replace the culture medium. Following a 1-hour incubation at a temperature of 37°C, the buffer solution was substituted with a recording buffer that included Fluo-4 AM. The fluorescence intensity was evaluated by measuring the absorbance at the peak excitation wavelength of 340/380 nm and the peak emission wavelength of 510 nm.

2.9 Animals and behavioral tests

The behavioral tests were conducted in accordance with the standards and regulations set out by the National Institute of Health. The experimental procedures were authorized by the Experimental Animal Welfare Ethics Committee of Hebei Medical University (Approval No. IACUC-Hebmu-2020009). Male C57BL/6N mice, aged six to eight weeks and weighing 20–25 grams, were acquired from Beijing Huafukang Biotechnology Co., LTD in Beijing, China. The mice were kept in a controlled laboratory environment. Thirty mice were randomized to three groups using random assignment (n = 30). (1) Control: The mice in this group were given the same amount of Nec-1 vehicle (DMSO) and Sodium carboxymethyl cellulose (1:10), as well as PTX vehicle (anhydrous ethanol) and Cremophor EL (1:1). (2) PTX: The mice in this group were given the same amount of Nec-1 vehicle (DMSO) and Sodium carboxymethyl cellulose (1:10), and then injected with PTX 10 mg/kg, 2 hours later [30]. (3) PTX + Nec-1: The mice in this group were injected with Nec-1 6.5 mg/kg and PTX 10 mg/kg, with a 2-hour interval, every other day [31]. Figure 5A illustrates the experimental procedure. The weight of the mice was measured and recorded daily.

2.10 Open field test (OFT)

The OFT is a behavioral test that evaluates general exploration by monitoring locomotor and anxiety-like behaviors [32]. The OFT apparatus, manufactured by Anhui Zheng-Hua Instrument in China, has four chambers, each measuring 50 × 50 × 50 cm. It is equipped with an automated data gathering system and a camera for monitoring the chambers. A mouse was positioned at the middle of the room, and the locomotion of the mice was observed for a duration of 5 minutes following a 1-hour period of adaptation, as described previously [33]. The analytical program autonomously partitions each chamber into nine squares and monitors the mouse motions. Each test, the instrument was cleaned with ethanol for removing any odor. The mice's average velocity and overall distance covered were calculated and then compared.

2.11 Novel Object recognition test (NOR)

The novel object recognition (NOR) task was conducted using a 45 x 45 cm open-field apparatus, following the method reported by Rosa et al.[34]. Prior to the trial, the mice were provided with a familiarity period during which they were allowed to freely explore the open space without any items for 10 minutes, 24 hr before the trial. Following that, several items were employed, including A1, A2, B, and C. Notably, A1 and A2 were indistinguishable blue rectangles, whereas the object labeled "B" was a yellow cube, and the object labeled "C" was a red cylinder. Following a 24-hour period of habituation, a training trial was carried out by placing each mouse in an open field containing A1 and A2 items. These objects were positioned in two neighboring corners, 10 cm apart from the walls. The assessment of short-term memory involved observing the exploratory preference towards a familiar (A) and a novel (B) object 1.5 hours following the train track. Long-term memory was assessed by substituting the "B" item with the "C" object using the identical approach 24 hours after conducting short-term memory tests. The duration of exploration was measured for a period of 5 minutes and was defined as the act of smelling or touching the object using the nose and/or forepaws. After each testing, the plastic items were cleaned using a 40% ethanol solution. The exploratory preference was determined by dividing the time spent with the new item by the sum of the time spent with the familiar object and the time spent with the novel object.

2.12 The Morris Water Maze (MWM)

The MWM test was employed to evaluate spatial learning and memory [35]. The apparatus consisted of a circular tank with a diameter of 136 cm and a height of 60 cm. The tank was painted black on the inside. It contained water that was 30 cm deep and had a temperature of 24 ± 1°C. The circular tank was partitioned into four identical quadrants, each occupying one-fourth of the tank's area. A concealed circular platform with a diameter of 10 cm was positioned in the middle of a single quadrant, buried 1.5 cm beneath the surface of the water. Visual markers were strategically placed at many areas throughout the tank. A video tracking system was used to capture the mouse's movement in order to assess the distance it traveled and the time it took to discover the platform. The mice underwent four daily trials for six consecutive days, with the disguised circular platform consistently positioned at the same location. Each day, the release points were allocated to each animal in a random manner. The trial concluded either when the mouse successfully reached the secret platform or after 60 seconds had passed. The mice were permitted to remain on the platform for a duration of 15 seconds. Subsequently, the subsequent experiment commenced following a 20-minute interval. If the mice failed to locate the platform within a 60-second timeframe, they were manually placed on the platform and given a score of 60 seconds. Following the conclusion of the last trial, the mice were provided with a warm and dry environment for one hour before being returned to their original cage. The computer software was used to determine the traveling distance and escape delay of a mouse in reaching the hiding platform. The daily score per mouse was calculated as the mean of the 4 trials conducted on that day. On the seventh day, a 60-second probe trial was conducted without the presence of the platform in the tank. The computer program was used to calculate the amount of time spent in the target quadrant during the probe trial.

2.13 Statistical Analysis

The statistical analyses were conducted on data collected from three separate studies using the SPSS 20.0 statistical program (SPSS Inc., Chicago, IL, United States). The data was presented as means ± SEM. One-way analysis of variance (ANOVA) with post hoc Tukey’s tests was used to compare data between multiple groups.The data between two groups was compared using the Student's t-test. Statistical differences were deemed significant with a level of confidence (alpha value) of P < 0.05.

3.1 PTX induced programmed cell death of HT22 cells

Apoptosis and necrosis exhibit distinct morphological features [36; 37; 38; 39; 40; 41]. In order to assess the impact of PTX on the viability of HT22 cells, we conducted a dose-dependent response study using the CCK8 assay. The cells were utilized for the tests following a 24-hour period of cultivation. Various quantities of PTX (ranging from 0 to 20 µM) were introduced into the growth media of HT22 cells for a duration of 24 hours. This allowed for the acquisition of dose-dependent response curves (Fig. 1A). Subsequently, we conducted Annexin V/PI double labeling and employed flow cytometry to ascertain the level of apoptosis in HT22 cells. Figure 1B and 1C demonstrate that PTX triggered apoptosis, as evidenced by a gradual rise in Annexin V positive staining without concurrent PI uptake (from 6.91–16.1%, bottom right quadrant). Moreover, the treatment of PTX resulted in a substantial rise in the number of cells that tested positive for both Annexin V and PI (from 17.4–46.2%, namely in the upper right quadrant).

The IC50 value, determined from the dose-response curve after 24 hours, was 9.568 µM (Fig. 1D). Thus, we determined that a concentration of 10 µM PTX at 24 hours is the most suitable dosage and treatment duration for the subsequent trials. The traditional flow cytometry technique was unable to differentiate between late apoptotic and necrotic cells when utilizing Annexin V and PI double labeling. Therefore, we differentiated between late apoptotic and necrotic cells by employing image-based analysis of Annexin V/PI staining. As depicted in Fig. 1E, the HT22 cells treated with PTX displayed a widespread staining of PI, resulting in a highly intense fluorescence signal throughout the enlarged necrotic nuclei. This contrasted with the limited regions of intense PI staining caused by the condensed chromatin found during the advanced apoptotic stage. In addition, treatment with PTX at a concentration of 10 µM resulted in the release of LDH from HT22 cells, indicating damage to the plasma membrane (P < 0.05, Fig. 1F). The findings indicate that PTX triggers many forms of programmed cell death in HT22 cells, including apoptosis and necrosis.

3.2 PTX induced necroptosis in HT22 cells

Necroptosis, a form of designed cell death, can exhibit comparable changes in cell shape to those observed in cell necrosis. In order to ascertain if PTX triggered necroptosis, we evaluated the presence of p-MLKL, an essential mediator of necroptosis, in HT22 cells by western blot analysis. The levels of p-MLKL were significantly elevated following PTX treatment in comparison to the cells treated with the vehicle (P < 0.05, Fig. 2E). The expression levels of p-MLKL and MLKL were significantly enhanced by 1.7-fold and 1.3-fold, respectively, in cells treated with PTX, as shown in Fig. 2E and 2F. Subsequently, we exposed HT22 cells to varying doses of Nec-1, a selective inhibitor of RIPK1. The survival rate of HT22 cells was not significantly affected by the administration of Nec-1 (P > 0.05, Fig. 2G). Afterwards, we cultivated varying doses of Nec-1 together with 10 µM PTX. The greatest concentration of Nec-1, which was 80 µM, exhibited the most significant ability to improve cell survival in cells treated with PTX (P < 0.05, Fig. 2H). Consequently, we employed a concentration of 80 µM Nec-1 for the subsequent experiment. The protein levels of RIPK1, RIPK3, MLKL, and p-MLKL, which were increased by PTX, were drastically suppressed by Nec-1 (P < 0.05, Fig. 2A-2F). Nec-1 effectively inhibited the PTX-induced rise in the number of PI-positive cells (P < 0.05, Fig. 3A and 3B), as well as the number of cells positive for both PI and Annexin V–FITC (P < 0.05, Fig. 3C and 3D). In addition, Nec-1 effectively decreased the release of LDH in cells treated with PTX (P < 0.05, Fig. 3E). The reduction in cell viability caused by PTX was dramatically prevented by Nec-1 (P < 0.05, Fig. 3F).

Given the absence of distinct markers for necroptosis, a process characterized by particular structural alterations, we employed transmission electron microscopy (TEM) to examine the morphological changes in neurons (Fig. 3G). The Control group demonstrated that HT22 cells had mitochondria and endoplasmic reticulum (ER) with typical shape. On the other hand, the cells treated with PTX exhibited distinct physical features of necroptosis, such as condensed chromatin, significant vacuolation, early disruption of the plasma membrane, and many autophagosomes [13; 42; 43; 44]. The administration of Nec-1 reduced the occurrence of ER vacuolation and the structural alterations of mitochondria caused by PTX therapy. The data suggest that PTX triggered necroptosis in HT22 cells, besides apoptosis.

3.3 Nec-1 reduced necroptosis by inhibiting intracellular calcium overload

Previous research has demonstrated a strong correlation between PIPK1 and the accumulation of Ca²⁺ within cells [45]. Nec-1 suppresses the release of cytoplasmic Ca²⁺ to provide neuroprotection in many neurodegenerative disorders [46]. Fluo-4 AM was employed as a Ca²⁺ indicator for the purpose of evaluating intracellular calcium levels. Initially, we examined the impact of PTX on the levels of intracellular Ca²⁺ by employing Fluo-4 AM fluorescence labeling (Fig. 4A). Our findings revealed a noteworthy enhancement in the fluorescence intensity of Fluo-4 AM in HT22 cells treated with PTX. In addition, following the incubation of Fluo-4 AM, the flow cytometry experiment revealed a substantial increase in the number of cells exhibiting fluorescence (P < 0.05, Fig. 4B and C) in response to PTX treatment. Furthermore, our investigation revealed that pre-treating HT22 cells with Nec-1 (80 µM) resulted in a considerable reduction in both the PTX-induced Ca²⁺ level and the number of fluorescent cells (Fig. 4A-C). Together, our findings indicate that intracellular Ca²⁺ have a substantial impact on the necroptosis of HT22 cells caused by PTX.

3.4 Nec-1 attenuated PTX-induced cognitive impairment

Initially, we evaluated the mice's body weight and locomotor activity after administering vehicle, PTX, and PTX with Nec-1. The body-weight growth index exhibited a substantial reduction in mice treated with PTX (n = 10) compared to the mice treated with the vehicle (n = 10). However, the body weight rose in mice treated with PTX plus Nec-1 (n = 10, Fig. 5B). Furthermore, the body weight of mice treated with PTX alone did not exhibit a significant difference compared to animals treated with PTX in combination with Nec-1. However, it was lower than the body weight of mice treated with the vehicle (P > 0.05, Fig. 5B). The locomotor activity was evaluated using the Open Field Test (OFT). The three groups did not exhibit any notable disparities in terms of total distance and average speed (P > 0.05, Fig. 5C and D).

In our previous research, we showed that giving male C57BL/6N mice PTX at a dose of 10mg/kg per day for 7 days in a row caused cognitive impairments [13, 27]. In order to assess the impact of inhibiting RIPK1 on PTX-induced cognitive abnormalities, we investigated the influence of Nec-1 on such deficits. The NOR test was employed to evaluate the memory capacity of the mice. During the familiarization phase, there was no statistically significant difference in object choices among the three groups (P > 0.05, Fig. 5E). The preference index exhibited a substantial reduction in mice treated with PTX as compared to animals treated with the vehicle. Treatment with Nec-1 reversed the decline in preference index observed in mice treated with PTX during both short-term and long-term memory tests (P < 0.05, Fig. 5F and G). These findings indicate that the administration of PTX in mice enhances cognitive performance, while Nec-1 reverses the cognitive damage caused by PTX counseling.

In addition, we employed the Morris Water Maze (MWM) test to assess the impact of Nec-1 on the spatial learning and memory impairment resulting from PTX therapy. The mean escape delay time among the three groups on the first day of testing did not show any statistically significant differences (p > 0.05, Fig. 5H). Following a second day of training, the PTX-treated mice exhibited a significantly longer average escape delay compared to the vehicle-treated rats (p < 0.05, Fig. 5H). The mice treated with PTX + Nec-1 exhibited a significantly reduced average escape delay compared to those treated with PTX alone (p < 0.05, Fig. 5H). In addition, mice that received PTX treatment exhibited a decrease in the number of times they crossed the platform area and in the duration of time they spent in the target quadrant, as compared to mice who received the vehicle treatment (p < 0.05, Fig. 5I to K). Nevertheless, mice who received both PTX and Nec-1 exhibited a significant improvement in the frequency of crossing the platform area and the amount of time spent in the target quadrant, as compared to mice treated with PTX alone (p < 0.05, Fig. 5I to K).

This is the first study to demonstrate that PTX can induce necroptosis, a programmed cell death, of HT22 cells in addition to inducing apoptosis, Furthermore, blocking pathways involved in necroptosis with Nec-1 significantly reduced PTX-induced necroptosis and intracellular Ca²⁺ overload and improved in cognitive behaviors in PTX-treated mice.

Evidence demonstrates that a little quantity of PTX, when administered systemically, can penetrate the blood-brain barrier (BBB) and reach the brain. The PTX compound exhibits a tendency to collect mostly in the hippocampal tissue of the brain, resulting in neuronal loss throughout various brain areas [6; 47]. The hippocampus, situated beneath the cerebral cortex, has a crucial function in spatial memory, navigation, learning, and other cognitive functions [48; 49]. Therefore, we hypothesize that cognitive impairment caused by PTX may be linked to the disruption of the structure and function of the hippocampus. The brain consists of several cell types, such as neurons and glial cells [50]. The HT22 cell line is a mouse hippocampus neuron cell line that originated from HT4. This cell line has been extensively utilized to investigate several neurodegenerative disorders, including Alzheimer's Disease (AD) and Parkinson's Disease (PD). Previous research has demonstrated that PTX has the ability to trigger apoptosis in primary hippocampus neurons [29]. Apoptosis is an active process of cell death, with a complete cell membrane structure that does not induce inflammatory reactions [51]. PTX exhibited cytotoxic effects in the CCK8 experiment, leading to a reduction in cell viability. Additionally, PTX caused an elevation in LDH levels, which serve as an indicator of membrane damage. The flow cytometry data demonstrated that cell apoptosis was promoted by PTX in a concentration-dependent manner. Further, imaging with PI/Annexin V demonstrated that at this concentration, PTX induced necrosis in the upper right quadrant. Necrosis is traditionally regarded as an unintentional and non-gene-regulated form of cell death characterized by the disruption of membrane integrity, release of cellular contents, and the production of pro-inflammatory molecules, such as IL-1β and TNF-α [52]. In the chemotherapeutic brain model generated by PTX, many areas including the hippocampus, striatum, spinal cord, and DRG experience the release of a significant number of inflammatory factors [29; 53]. These findings indicate that necrosis may play a role in the inflammation caused by PTX, but further research is required to understand its specific mechanism. In this study, we found that systemic administration of PTX causes other types of programmed cell death in hippocampal neurons in addition to apoptosis.

Necroptosis is a newly identified form of cell death that is defined by the appearance of necrotic cell death morphology and the activation of autophagy [13]. Necroptosis is clearly implicated in several disorders, including those involving the cardiovascular system, ischemic brain damage, and neurodegenerative conditions [36]. In order to ascertain the involvement of necroptosis in the harm inflicted upon hippocampal neurons by PTX, we employed a method to assess the presence of necroptosis-related proteins. Our findings revealed a substantial elevation in the expression levels of RIPK1, RIPK3, MLKL, and p-MLKL upon PTX exposure. Nec-1 specifically inhibits necroptosis and is now employed as a practical criterion for defining necroptosis [13; 24; 54]. Nec-1 is the first drug used to study necroptosis, and has a strong protective effect in a variety of neurodegenerative diseases and ischemic or hemorrhagic brain injury [13; 55]. Whether it has a protective effect in PTX-induced post-chemotherapy cognitive impairment remains unclear. Therefore, we introduced Nec-1 as a neuroprotective drug in this experiment. We found that Nec-1 can significantly improve cell survival while reducing the number of PI (+) cells and the concentration of LDH. At the same time, Nec-1 can significantly reduce the occurrence of PTX-induced cognitive dysfunction in vivo, which provides evidence to develop novel treatment for post-chemotherapy cognitive impairment.

Intracellular Ca²⁺ triggers many events including necroptosis [56; 57]. An noteworthy discovery in the current investigation is that Nec-1 effectively prevented the PTX-induced elevation of intracellular Ca²⁺ levels. The degree of inhibition of necroptosis by Nec-1 and the reduction in intracellular Ca²⁺ levels were comparable. This discovery clearly indicates that the intracellular Ca²⁺ signal is one of the mechanisms that cause PTX-necroptosis. While earlier research suggested a connection between necroptosis and death-domain receptors (DRs) [13], the specific underlying processes have yet to be fully understood. Our work showed that an increase in intracellular Ca²⁺ might be one of the possible pathways that initiate necroptosis.

In conclusion, our study confirms that hippocampal neurons treated with PTX undergo necroptosis, in which increase in intracellular Ca²⁺ was critically involved. As an inhibitor of necroptosis, Nec-1 has a protective effect on PTX-induced damage to hippocampal neurons and improves PTX-induced cognitive dysfunction in mice. Our research results showed a new mechanism for PTX-induced post-chemotherapy cognitive impairment. These data provide a new target to develop new treatment for PTX-induced post-chemotherapy cognitive impairment patients. To our knowledge, this is the first study determining the role of Nec-1 in the treatment of PTX-induced cognitive deficits. Future translational studies are warranted to examine the effect of Nec-1 on cognitive deficits in chemobrain patients.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval

This experimental procedures in this study were approved by the Experimental Animal Welfare Ethics Committee of Hebei Medical University (Approval No. IACUC-Hebmu-2020009).

Funding

The funding for this work was provided by the National Natural Science Foundation of China (81971001), the Natural Science Foundation of Hebei Province (H2021206109, H2021206149), and the Scholarship Program for Introducing Overseas Students of Hebei Province (C20230511).

Author Contribution

All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by L-L. L., S. Z., Z.L, H-Z.L, X.L, and X-L.W. The first draft of the manuscript was written by L-L.L and X-L.W. All authors commented on the manuscript, read it, and approved the final manuscript.

Data Availability

The data presented in this study are available on request from the corresponding author.

Schagen SB, van Dam FS, Muller MJ, Boogerd W, Lindeboom J, Bruning PF (1999) Cognitive deficits after postoperative adjuvant chemotherapy for breast carcinoma. Cancer 85:640–650
Tchen N, Juffs HG, Downie FP, Yi QL, Hu H, Chemerynsky I, Clemons M, Crump M, Goss PE, Warr D, Tweedale ME, Tannock IF (2003) Cognitive function, fatigue, and menopausal symptoms in women receiving adjuvant chemotherapy for breast cancer. J Clin Oncol 21:4175–4183
Kohli S, Griggs JJ, Roscoe JA, Jean-Pierre P, Bole C, Mustian KM, Hill R, Smith K, Gross H, Morrow GR (2007) Self-reported cognitive impairment in patients with cancer. J Oncol Pract 3:54–59
Gangloff A, Hsueh WA, Kesner AL, Kiesewetter DO, Pio BS, Pegram MD, Beryt M, Townsend A, Czernin J, Phelps ME, Silverman DH (2005) Estimation of paclitaxel biodistribution and uptake in human-derived xenografts in vivo with (18)F-fluoropaclitaxel. J Nucl Med 46:1866–1871
van der Veldt AA, Hendrikse NH, Smit EF, Mooijer MP, Rijnders AY, Gerritsen WR, van der Hoeven JJ, Windhorst AD, Lammertsma AA, Lubberink M (2010) Biodistribution and radiation dosimetry of 11C-labelled docetaxel in cancer patients. Eur J Nucl Med Mol Imaging 37:1950–1958
Huehnchen P, Boehmerle W, Springer A, Freyer D, Endres M (2017) A novel preventive therapy for paclitaxel-induced cognitive deficits: preclinical evidence from C57BL/6 mice. Transl Psychiatry 7:e1185
Grant CV, Sullivan KA, Wentworth KM, Otto LD, Strehle LD, Otero JJ, Pyter LM (2023) Microglia are implicated in the development of paclitaxel chemotherapy-associated cognitive impairment in female mice. Brain behav immun 108:221–232
Klein I, Isensee J, Wiesen MHJ, Imhof T, Wassermann MK, Müller C, Hucho T, Koch M, Lehmann HC (2023) Glycyrrhizic Acid Prevents Paclitaxel-Induced Neuropathy via Inhibition of OATP-Mediated Neuronal Uptake. Cells 12 null
Li Z, Liu P, Zhang H, Zhao S, Jin Z, Li R, Guo Y, Wang X (2017) Role of GABAB receptors and p38MAPK/NF-κB pathway in paclitaxel-induced apoptosis of hippocampal neurons. Pharm biol 55:2188–2195
Sung PS, Chen PW, Yen CJ, Shen MR, Chen CH, Tsai KJ, Lin CK (2021) Memantine Protects against Paclitaxel-Induced Cognitive Impairment through Modulation of Neurogenesis and Inflammation in Mice. Cancers (Basel) 13 null
Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G (2010) Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol 11:700–714
Moriwaki K, Bertin J, Gough PJ, Orlowski GM, Chan FK (2015) Differential roles of RIPK1 and RIPK3 in TNF-induced necroptosis and chemotherapeutic agent-induced cell death. Cell Death Dis 6:e1636
Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J (2005) Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1:112–119
Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, Ward Y, Wu LG, Liu ZG (2014) Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 16:55–65
Cho Y, Challa S, Chan FK (2011) A RNA interference screen identifies RIP3 as an essential inducer of TNF-induced programmed necrosis. Adv Exp Med Biol 691:589–593
Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, Chan FK (2009) Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137:1112–1123
Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, Dong MQ, Han J (2009) RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325:332–336
Pasparakis M, Vandenabeele P (2015) Necroptosis and its role in inflammation. Nature 517:311–320
Yuan J, Amin P, Ofengeim D (2019) Necroptosis and RIPK1-mediated neuroinflammation in CNS diseases. Nat Rev Neurosci 20:19–33
Zhu K, Liang W, Ma Z, Xu D, Cao S, Lu X, Liu N, Shan B, Qian L, Yuan J (2018) Necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression. Cell Death Dis 9:500
Ito Y, Ofengeim D, Najafov A, Das S, Saberi S, Li Y, Hitomi J, Zhu H, Chen H, Mayo L, Geng J, Amin P, DeWitt JP, Mookhtiar AK, Florez M, Ouchida AT, Fan JB, Pasparakis M, Kelliher MA, Ravits J, Yuan J (2016) RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS. Science 353:603–608
Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nuñez G, Peter ME, Tschopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G (2009) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 16:3–11
Smith CC, Davidson SM, Lim SY, Simpkin JC, Hothersall JS, Yellon DM (2007) Necrostatin: a potentially novel cardioprotective agent? Cardiovasc Drugs Ther 21:227–233
Degterev A, Hitomi J, Germscheid M, Ch' ILen, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber SA, Lugovskoy A, Yuan J (2008) Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 4:313–321
Linkermann A, Bräsen JH, Himmerkus N, Liu S, Huber TB, Kunzendorf U, Krautwald S (2012) Rip1 (receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury. Kidney Int 81:751–761
Oerlemans MI, Liu J, Arslan F, den Ouden K, van Middelaar BJ, Doevendans PA, Sluijter JP (2012) Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia-reperfusion in vivo. Basic Res Cardiol 107:270
Le Verche V (2014) [Familial and sporadic ALS astrocytes activate necroptosis in motor neurons]. Med Sci (Paris) 30:748–750
Zhang M, Li J, Geng R, Ge W, Zhou Y, Zhang C, Cheng Y, Geng D (2013) The inhibition of ERK activation mediates the protection of necrostatin-1 on glutamate toxicity in HT-22 cells. Neurotox Res 24:64–70
Li Z, Liu P, Zhang H, Zhao S, Jin Z, Li R, Guo Y, Wang X (2017) Role of GABA(B) receptors and p38MAPK/NF-κB pathway in paclitaxel-induced apoptosis of hippocampal neurons. Pharm Biol 55:2188–2195
Tang M, Zhao S, Liu JX, Liu X, Guo YX, Wang GY, Wang XL (2022) Paclitaxel induces cognitive impairment via necroptosis, decreased synaptic plasticity and M1 polarisation of microglia. Pharm Biol 60:1556–1565
Dong Y, Yu H, Li X, Bian K, Zheng Y, Dai M, Feng X, Sun Y, He Y, Yu B, Zhang H, Wu J, Yu X, Wu H, Kong W (2022) Hyperphosphorylated tau mediates neuronal death by inducing necroptosis and inflammation in Alzheimer's disease. J Neuroinflammation 19:205
Seibenhener ML, Wooten MC (2015) Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp e52434
Jiao H, Fan Y, Gong A, Li T, Fu X, Yan Z (2024) Xiaoyaosan ameliorates CUMS-induced depressive-like and anorexia behaviors in mice via necroptosis related cellular senescence in hypothalamus. J ethnopharmacol 318:116938
Rosa RM, Flores DG, Appelt HR, Braga AL, Henriques JA, Roesler R (2003) Facilitation of long-term object recognition memory by pretraining administration of diphenyl diselenide in mice. Neurosci Lett 341:217–220
Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11:47–60
Vandenabeele P, Declercq W, Vanden Berghe T (2008) Necrotic cell death and 'necrostatins': now we can control cellular explosion. Trends Biochem Sci 33:352–355
Ueda N, Shah SV (2000) Tubular cell damage in acute renal failure-apoptosis, necrosis, or both. Nephrol Dial Transplant 15:318–323
Teng X, Degterev A, Jagtap P, Xing X, Choi S, Denu R, Yuan J, Cuny GD (2005) Structure-activity relationship study of novel necroptosis inhibitors. Bioorg Med Chem Lett 15:5039–5044
Cummings BS, Schnellmann RG (2002) Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways. J Pharmacol Exp Ther 302:8–17
Sanz AB, Santamaría B, Ruiz-Ortega M, Egido J, Ortiz A (2008) Mechanisms of renal apoptosis in health and disease. J Am Soc Nephrol 19:1634–1642
Galluzzi L, Kroemer G (2008) Necroptosis: a specialized pathway of programmed necrosis. Cell 135:1161–1163
Han W, Xie J, Li L, Liu Z, Hu X (2009) Necrostatin-1 reverts shikonin-induced necroptosis to apoptosis. Apoptosis 14:674–686
Hitomi J, Christofferson DE, Ng A, Yao J, Degterev A, Xavier RJ, Yuan J (2008) Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135:1311–1323
Han W, Li L, Qiu S, Lu Q, Pan Q, Gu Y, Luo J, Hu X (2007) Shikonin circumvents cancer drug resistance by induction of a necroptotic death. Mol Cancer Ther 6:1641–1649
Nomura M, Ueno A, Saga K, Fukuzawa M, Kaneda Y (2014) Accumulation of cytosolic calcium induces necroptotic cell death in human neuroblastoma. Cancer Res 74:1056–1066
Li Y, Yang X, Ma C, Qiao J, Zhang C (2008) Necroptosis contributes to the NMDA-induced excitotoxicity in rat's cultured cortical neurons. Neurosci Lett 447:120–123
Karavelioglu E, Gonul Y, Aksit H, Boyaci MG, Karademir M, Simsek N, Guven M, Atalay T, Rakip U (2016) Cabazitaxel causes a dose-dependent central nervous system toxicity in rats. J Neurol Sci 360:66–71
Czerniawski J, Miyashita T, Lewandowski G, Guzowski JF (2015) Systemic lipopolysaccharide administration impairs retrieval of context-object discrimination, but not spatial, memory: Evidence for selective disruption of specific hippocampus-dependent memory functions during acute neuroinflammation. Brain Behav Immun 44:159–166
Kanoski SE, Grill HJ (2017) Hippocampus Contributions to Food Intake Control: Mnemonic, Neuroanatomical, and Endocrine Mechanisms. Biol Psychiatry 81:748–756
Chang C, Zuo H, Li Y (2023) Recent advances in deciphering hippocampus complexity using single-cell transcriptomics. Neurobiol dis 179:106062
Lu D, Bai XC, Liu B, Li XM, Deng F, Li M, Cheng BL, Luo SQ (2004) [Hydrogen peroxide inhibits arsenic trioxide-induced apoptosis of Burkitt lymphoma cells]. Di Yi Jun Yi Da Xue Xue Bao 24:375–378
Moriwaki K, Chan FK (2017) The Inflammatory Signal Adaptor RIPK3: Functions Beyond Necroptosis. Int Rev Cell Mol Biol 328:253–275
Li X, Yang S, Wang L, Liu P, Zhao S, Li H, Jiang Y, Guo Y, Wang X (2019) Resveratrol inhibits paclitaxel-induced neuropathic pain by the activation of PI3K/Akt and SIRT1/PGC1α pathway. J Pain Res 12:879–890
He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X (2009) Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 137:1100–1111
You Z, Savitz SI, Yang J, Degterev A, Yuan J, Cuny GD, Moskowitz MA, Whalen MJ (2008) Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice. J Cereb Blood Flow Metab 28:1564–1573
Zhong W, Cheng J, Yang X, Liu W, Li Y (2023) Heliox Preconditioning Exerts Neuroprotective Effects on Neonatal Ischemia/Hypoxia Injury by Inhibiting Necroptosis Induced by Ca2 + Elevation. Transl stroke res 14:409–424
Zhang J, Cao J, Qian J, Gu X, Zhang W, Chen X (2023) Regulatory mechanism of CaMKII δ mediated by RIPK3 on myocardial fibrosis and reversal effects of RIPK3 inhibitor GSK'872. Biomed pharmacother 166:115380

No competing interests reported.

WB.tif

Download PDF

Version 1

posted

You are reading this latest preprint version

Inhibition of necroptosis mitigates paclitaxel-induced neuronal damage and cognitive impairment

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Culture of HT22 cell line and PTX treatment

2.2 Cell viability assay

2.3 Lactate Dehydrogenase Assay

2.4 Flow cytometry analysis

2.5 Microscopic imaging of cell death

2.6 Western blot analysis

2.7 Transmission electron microscopy (TEM)

2.8 Estimation of intracellular Ca²⁺ concentration

2.9 Animals and behavioral tests

2.10 Open field test (OFT)

2.11 Novel Object recognition test (NOR)

2.12 The Morris Water Maze (MWM)

2.13 Statistical Analysis

3. Results

3.1 PTX induced programmed cell death of HT22 cells

3.2 PTX induced necroptosis in HT22 cells

3.3 Nec-1 reduced necroptosis by inhibiting intracellular calcium overload

3.4 Nec-1 attenuated PTX-induced cognitive impairment

4. Discussion

Declarations

Competing Interests

Ethics approval

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Inhibition of necroptosis mitigates paclitaxel-induced neuronal damage and cognitive impairment

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Culture of HT22 cell line and PTX treatment

2.2 Cell viability assay

2.3 Lactate Dehydrogenase Assay

2.4 Flow cytometry analysis

2.5 Microscopic imaging of cell death

2.6 Western blot analysis

2.7 Transmission electron microscopy (TEM)

2.8 Estimation of intracellular Ca2+ concentration

2.9 Animals and behavioral tests

2.10 Open field test (OFT)

2.11 Novel Object recognition test (NOR)

2.12 The Morris Water Maze (MWM)

2.13 Statistical Analysis

3. Results

3.1 PTX induced programmed cell death of HT22 cells

3.2 PTX induced necroptosis in HT22 cells

3.3 Nec-1 reduced necroptosis by inhibiting intracellular calcium overload

3.4 Nec-1 attenuated PTX-induced cognitive impairment

4. Discussion

Declarations

Competing Interests

Ethics approval

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

2.8 Estimation of intracellular Ca²⁺ concentration