Neuroprotective effects of candesartan in 3-nitropropionic acid-induced Huntington’s disease: modulation of angiotensin and CREB/BDNF/PGC1-α signaling

Huntington’s disease (HD) is a progressive neurodegenerative disorder primarily affecting the basal ganglia. It is characterized by motor dysfunction (notably involuntary choreiform movements), cognitive decline, and psychiatric disturbances (Vinther-Jensen et al. 2014). The pathogenesis of HD involves mitochondrial dysfunction, impaired energy metabolism, oxidative stress, excitotoxicity, transcriptional dysregulation, and disrupted synaptic transmission (Ferguson et al. 2022a). Although the exact mechanisms remain incompletely understood, HD is caused by an unstable expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene, leading to the production of mutant huntingtin protein. This mutant protein aggregates predominantly in the striatum, particularly in the putamen and caudate nucleus, causing progressive neuronal loss (Ramaswamy et al. 2007; Ross and Tabrizi 2011; McColgan and Tabrizi 2018).

The expression of the mutant huntingtin gene is widely recognized in the cardiomyocytes of Huntington patients, resulting in cardiac failure and hypertension, which is the primary reason for death in those patients. Meanwhile, cardiovascular diseases (CVS) such as cardiac failure and hypertension (HTN) can negatively impact the function and the structure of the brain, hastening the progression of neurodegenerative diseases such as HD (Burg et al. 2009; Steventon et al. 2020; Schultz et al. 2020). In this context, the Renin–Angiotensin System (RAS) which exists in the central and peripheral nervous system has been implicated in neuropsychiatric and neurodegenerative diseases as HD besides CVS diseases as HTN and cardiac failure (Mello et al. 2017a; Pugliese et al. 2020; Machado et al. 2020; Villapol 2023; Ahmadi and Khaledi 2023).

The RAS consists of two primary axes: the first axis is composed of angiotensin-converting enzyme (ACE), which transforms angiotensin I (Ang I) into angiotensin II (Ang II). In turn, Ang II binds to the Angiotensin II Type 1 receptor (AT1R) or Angiotensin II Type 2 receptor (AT2R). Depending on the receptor that Ang II binds to, several physiological responses might be observed. Nonetheless, the AT1 receptor is the main Ang II mediator (Machado et al. 2020; Fyhrquist and Saijonmaa 2008; Miranda and Teixeira 2023). In the CNS, the ACE/Ang II/AT1 receptor axis is often linked to neurodegenerative disease, because it stimulates the accumulation of inflammatory markers and free radicals, and decreases neuronal survival (Gironacci, et al. 2018; Rocha et al. 2016; Almeida-Santos et al. 2017).

On the other hand, the components of the second axis, which is the counter-regulatory axis formed of ACE [Angiotensin-Converting Enzyme 2 (ACE2)], angiotensin-(1-7) [Ang-(1-7)], and Mas receptor (Jankowski et al. 2007; Lautner et al. 2013; Santos, et al. 2019). Ang-(1-7) can be formed directly or indirectly from Ang I by angiotensin-converting enzyme 2 (ACE2) (Jiang et al. 2014). Interestingly, the activation of the ACE2/Ang-(1-7)/Mas receptor was reported to develop a neuroprotective action. For instance, it sustained neuronal survival, reduced oxidative stress and neuroinflammation, and improved cognitive function (Gironacci, et al. 2018; Rocha et al. 2016; Jiang et al. 2016). Furthermore, AT2 receptor activation by Ang II has comparable functions to Mas receptor activation by Ang-(1-7). It opposes the major response produced by the activation of the AT1 receptor, exerting mostly neuroprotective benefits (Almeida-Santos et al. 2017; Kangussu et al. 2022). Moreover, a recent publication noted a marked reduction in the beneficial axis ACE2/Ang-(1-7)/Mas receptor in the striatum of HD brain tissues. On the contrary, the Ang II/AT1R axis was significantly activated in brain tissues (Kangussu et al. 2022).

Emerging evidence supports a pivotal role for RAS in HD. For example, human post-mortem studies have revealed reduced ACE activity in HD-affected brain regions, including the striatum and substantia nigra. Additionally, striatal ACE2 activity and the levels of Angiotensin-(1-7) are decreased in HD mouse models, while AT1 receptor expression is increased. These findings demonstrate a shift in RAS balance toward the pathological, pro-inflammatory classical axis and provide a strong biological rationale for targeting RAS modulation as a therapeutic strategy in HD (Kangussu et al. 2022).

In addition, further stimulation of the AT2 receptor leads to the phosphorylation of CREB via the activation of the PI3K/Akt axis (Guimond and Gallo-Payet 2012). Consequently, this leads to the activation of the BDNF protein downstream (Hashikawa-hobara et al. 2012; Umschweif et al. 2014; Guimond, et al. 2013; McFall et al. 2020). The CREB and BDNF proteins both play a crucial role in providing neuroprotection and cell survival against neurodegenerative diseases by enhancing cellular proliferation besides suppressing inflammatory biomarkers and apoptosis, eventually resulting in expanded cell survival (Jiang et al. 2013; Ribeiro et al. 2014; Heras-Sandoval et al. 2014; Zuo et al. 2016). Moreover, CREB can trigger the expression of PGC-1α, a transcriptional coactivator that plays a major role in brain energy homeostasis, therefore, hindering the mitochondrial dysfunction associated with neurodegenerative diseases (Singulani et al. 2020). Correspondingly, CREB could halt the transcription of the JNK/C-Jun axis, which has been implicated in chronic neurological disorders (Perrin et al. 2009; Zhang et al. 2019).

For a model of HD to be deemed reliable, it must possess neuropathological characteristics and symptomatic manifestations associated with the illness. 3-Nitropropionic acid (3NP) is an environmental toxin that mimics the pathology and motor abnormalities associated with Huntington’s disease (HD). 3NP irreversibly inhibits succinate dehydrogenase (SDH), disrupting the Krebs cycle and consequently reducing adenosine triphosphate (ATP) levels in the brain. Additionally, 3NP induces free radical production, impairs the brain’s antioxidant defense mechanisms, and promotes the release of pro-inflammatory cytokines, including TNF-α, NF-κB, and interleukin-1β (Haddadi et al. 2024; Jamwal and Kumar 2016). These factors contribute to the generation of excess free radicals, forthcoming impending neuronal damage (Upadhayay et al. 2023). Furthermore, it has been documented that striatal lesions induced by 3NP exhibit a high degree of resemblance to the neurochemical, histological, and morphological pathology observed in HD (Ramaswamy et al. 2007).

Currently, there is a continuous lack of appropriate curative therapy for Huntington’s disease (Ferguson et al. 2022b). There is a necessity for the pinpointing of novel targets to facilitate the advancement of therapeutic techniques. The possible neuroprotective impact of Candesartan, an AT1R blocker, has been postulated in the context of neural inflammation and neurological diseases. In particular, prior investigations have demonstrated a significant neuroprotective potential of Candesartan in AD and PD (Elkahloun et al. 2016; Gaur and Kumar 2011; Wu et al. 2013).

Accordingly, the following study was designed to elucidate the potential neuroprotective properties of Candesartan for the first time, in mitigating neuronal degeneration caused by 3NP induced HD through investigating the prospective role of Ang II/AT2R/Ang-(1-7)/Mas receptor, CREB/BDNF/PGC1-α besides JNK/c-Jun trajectories. In addition, the anti-apoptotic effect of Survivin was also highlighted in this study.

The current study provides novel insights into the potential biological mechanisms behind the neurotherapeutic efficacy of Candesartan against 3NP induced neurotoxic model of HD. The results indicated that the treatment using Candesartan effectively mitigated the behavioral abnormalities and neuronal degeneration, which was verified by a multitude of incidents: (1) amelioration of cognitive and motor impairments; (2) significant improvement in the histological and immunohistopathological observations; (3) activation of the neuroprotective pathway Ang II/AT2R/Ang-(1-7)/Mas receptor and CREB/BDNF/PGC1-alpha that promote neurogenesis; (4) suppression of mitochondrial dysfunction and oxidative stress in the striatum through the restoration of HO1 and NQO1 activities, as well as inhibition of JNK/c-Jun pathway together with the inflammatory mediators.

The striatum is considered the major region of the basal ganglia that regulates motor coordination. Accordingly, experimental studies involving the administration of 3NP have induced striatal lesions, resulting in motor deficits, cognitive decline, and impaired memory retention (Jang and Cho 2016; Blum et al. 2002; D’Egidio et al. 2023). Likewise, previous studies have indicated that 3NP induces damage to the pyramidal neurons in the CA1 and CA3 regions of the hippocampus, which are known to be associated with cognitive performance (Danduga et al. 2018). The current study employed the utilization of 3NP (10 mg/kg/day for 14 days) as an induction model for HD, which consequently led to several behavioral impairments, and neurochemical and pathological alterations causing cognitive impairments and loss of memory.

While the 3NP model does not reproduce the genetic underpinnings of HD, such as the presence of mutant huntingtin aggregates, it offers a well-characterized, reproducible platform to investigate the neurotoxic cascade driven by mitochondrial dysfunction. Mitochondrial dysfunction is a central feature of HD pathogenesis, contributing to impaired energy metabolism, increased ROS production, and progressive neuronal loss. In HD patients, several enzymes in the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain (ETC) are downregulated, resulting in reduced ATP synthesis and elevated oxidative stress. 3-NP mimics these metabolic defects by irreversibly inhibiting succinate dehydrogenase (complex II of the ETC), disrupting both the TCA cycle and oxidative phosphorylation. This leads to mitochondrial electron leakage, ROS overproduction, and energy failure, which collectively trigger neuronal apoptosis and striatal degeneration features that overlap strongly with the neurobiology of HD (Ramaswamy et al. 2007).

In the current study, the novel object recognition, open field, and Morris water maze tests were used as assessment tools to examine motor, behavioral, and cognitive impairments. 3NP administration significantly decreased the time spent exploring the novel object and the total time exploring both objects as well as the discrimination index in the NORT. Similarly, 3NP administration significantly reduced rearing and ambulation frequency and total time spent in the target quadrant in the open-field test and Morris water maze test, respectively. Interestingly, co-administration of Candesartan treatment resulted in significant enhancement in locomotor activities, memory retention, spatial learning, and cognitive function when compared to rats treated with 3NP, with major prominent effect at the highest dose (5 mg/kg). This suggests a positive impact of Candesartan treatment on these memory, cognitive, and behavioral measures, which comes in line with a previous study (Trofimiuk et al. 2018).

Moreover, histopathological assessments of the striatum in rats injected with 3NP displayed severe perineuronal edema and astrogliosis. Correspondingly, immunoreactivity analysis of GFAP expression in the striatum revealed significant expression of GFAP in the 3NP treated group. These pathological and immunohistochemical changes were ameliorated in the 3NP group treated with Candesartan (2.5 mg/kg/day and 5 mg/kg/day for 14 days). Additionally, the administration of 3NP resulted in a substantial decrease in the ultimate body weight, which is one of the features of HD (Mahdi et al. 2023). Co-treatment with Candesartan restored the body weight in comparison to diseased rats with a more noticeable impact at the greater dose (5 mg/kg).

The constituents of the RAS were implicated in various neuropsychiatric and neurodegenerative conditions, including depression and anxiety (Almeida-Santos et al. 2016; Kangussu et al. 2017), Alzheimer’s disease (Jiang et al. 2016; Tian et al. 2012), Parkinson’s disease (Rocha et al. 2016), and Huntington’s disease (Mello et al. 2017b). Moreover, previous literature has reported that the Ang II/AT2R/Ang-(1-7)/Mas receptor axis possesses neuroprotective properties (Miranda and Teixeira 2023; Gironacci, et al. 2018; Xu et al. 2011). It was previously mentioned that the AT2R activation in the brain displayed several outcomes; it reduced the generation of reactive oxygen species (ROS), promoted the survival of neuronal cells, and decreased the levels of pro-inflammatory cytokines (Shan et al. 2018; Fouda et al. 2017; Dai 2016; Khorooshi et al. 2020). Similarly, the activation of Ang-(1-7)/Mas receptors displayed specific effects on brain cells. These effects included a decrease in nuclear and mitochondrial superoxide production (Costa-Besada et al. 2018), as well as improved survival of neurons accompanied by a decrease in the level of oxidative stress in the brain (Mo et al. 2019). In addition, this signaling reduced microglial activation and astrogliosis, leading to improved neuronal density after traumatic brain injury (Janatpour et al. 2019). Interestingly, the activation of AT2R and Ang-(1-7)/Mas receptors stimulates the PI3K/Akt pathway, which subsequently stimulates the phosphorylation of CREB (Sakamoto et al. 2011; Wu et al. 2016; Mateos et al. 2016). The involvement of phosphorylated CREB is significant in the promotion of neuronal survival through the transcriptional activation of BDNF along with its receptor TrKB (Rabie et al. 2018; Song et al. 2015). It is noteworthy to emphasize that the upregulation of BDNF leads to the stimulation of neurogenesis and the initiation of TrKB phosphorylation, which serves as a positive feedback mechanism, subsequently reactivating Ang-(1-7)/Mas receptor axis, ultimately promoting the survival of neurons (Rabie et al. 2018; Yao et al. 2012). Interestingly, the administration of AT1R blockers resulted in a surge in the concentrations of Ang II; consequently, Ang II is converted to produce Ang-(1-7) which in turn is also reported to stimulate the AT2R (Hashikawa-hobara et al. 2012; Clark and Khalil 2024).

Our study revealed that co-administration of Candesartan induced the expression of Ang-(1-7)/MasR axis, as well as AT2 receptor, with the higher dose (5 mg/kg) exhibiting a significant and remarkable influence. These results were consistent with the previous studies (Widdop et al. 2002; Callera et al. 2016). Based on the previous data, our study displayed a high level of CREB and expression of BDNF with Candesartan treatment, with the higher dose succeeding to normalize CREB level and BDNF expression. These results were in harmony with the previous study (Goel et al. 2018).

PGC-1α, a transcriptional coactivator, carries a significant function in the process of mitochondrial biogenesis and the maintenance of brain energy homeostasis. Its ability to enhance mitochondrial electron transport activity and activate a comprehensive anti-ROS program renders it a highly desirable protein for regulating or mitigating the detrimental effects linked to impaired mitochondrial function, which is prominent in the early stages of neurodegenerative disorders like Parkinson’s, Alzheimer’s, and Huntington’s diseases (St-Pierre et al. 2006; Carmo 2018). In addition, the suppression of PGC-1α expression by 3NP has been reported in previous studies (Weydt et al. 2006; Ahmed et al. 2016). Hence, the diminished expression of the PGC-1α protein may potentially contribute to the manifestation of mitochondrial impairment and striatal deterioration observed in rats treated with 3NP, as reported by Chen et al. (2012). However, herein, treatment using Candesartan improved the mitochondrial function through significant elevation in the protein content of the PGC-1α with a prominent effect at the higher dose of Candesartan (5 mg/kg). The observed expression of PGC-1α is induced via CREB/PGC-1α stimulation, as mentioned before by Kang et al. (2017); Fernandez-Marcos and Auwerx 2011). In the same context, the AT1R blocker Losartan was previously proven to elevate the production of PGC-1α in an insulin resistance model (Liu et al. 2021).

Additionally, NQO1 and HO-1 were analyzed to provide further details regarding the immediate impacts of Candesartan on markers of oxidative stress. According to Calabrese et al. (2008) and Yuhan et al. (2024), it has been demonstrated that NQO1 has neuroprotective properties by mitigating oxidative damage through the conversion of highly reactive quinones into less-reactive hydroquinone. In a recent study conducted by Qi et al. (2014), the involvement of HO-1 in promoting the release of neurotrophic factors was validated in mice that suffered a stroke, in which enhanced recovery was attributed to the overexpression of HO-1 through activation of BDNF-PI3K/Akt signaling in the hippocampus. Furthermore, it has been previously stated that CREB induces the activation of NQO1 and HO-1 through the stimulation of Nrf2 (Zhou et al. 2016). In the present investigation, the administration of 3NP resulted in a noteworthy decrease in the protein levels of the antioxidants NQO-1 and HO-1, consistent with the findings reported in the prior work conducted by Yuan et al. (2020). It is interesting to note that the co-administration of Candesartan resulted in a marked increase in the concentrations of NQO-1 and HO-1, particularly at the higher dosage of (5 mg/kg). The protective effect of Candesartan treatment against striatal damage induced by oxidative stress is elucidated through the activation of the CREB/Nrf2 pathway and its downstream signaling cascade (Goel et al. 2018). These findings are in agreement with a prior study conducted by Kabel and Elkhoely (2017) and Nioi et al. (2003).

The 3NP compound resembles the pathogenesis of Huntington’s disease (HD) by causing the degeneration of GABAergic neurons in the striatum. This degeneration is facilitated by the activation of microglial cells, which leads to the excessive production of cytotoxic agents including free radicals and pro-inflammatory cytokines, such as NF-κB and IL-1β (Bonsi et al. 2006; Ahuja et al. 2008; Napolitano et al. 2008). Moreover, NF-κB has a crucial role as a transcription factor in M1 macrophages, being essential for the activation of numerous inflammatory genes, particularly those responsible for encoding IL-1β (Wang et al. 2014).

In this study, 3NP intoxication caused marked elevation in the levels of NF-κB and IL-1β, which is in line with the previous study (Mustafa, et al. 2021; Benicky et al. 2011). However, co-treatment using Candesartan leads to significant inhibition in NF-κB and IL-1β with a prominent effect at a higher dose (5 mg/kg), which is consistent with previous studies (Benicky et al. 2011; Qie et al. 2020). Such a decline in the levels of NF-κB and IL-1β can be attributed to the activation of CREB, which was in line according to these studies (Dong et al. 2018; Brautigam et al. 2005; Wen et al. 2010).

The involvement of the JNK pathway has been indicated in both in vitro and in vivo experimental models of neuronal cell death in chronic neurodegenerative conditions, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) (de los Reyes Corrales et al. 2021). Moreover, studies conducted on Huntington’s disease (HD) have demonstrated that the mutant huntingtin protein (htt) activates the JNK pathway in cellular models (Yan et al. 2024). The transcription factor c-Jun is the key component of the JNK pathway, and its regulation occurs at both the transcriptional and post-transcriptional stages through the activation of JNK (Davis 2000; Sun et al. 2024). Various internal and extracellular events may contribute to the activation of JNK in neuronal cells. One of these events is the generation of reactive oxygen species by 3NP, which has displayed the occurrence of nuclear translocation of activated JNK and phosphorylation of c-Jun (Perrin et al. 2009; Garcia et al. 2002). Furthermore, a previous study demonstrated that JNK/p-c-Jun activation leads to caspase-3 activation, thereby mediating apoptotic cell death. For example, JNK-driven cytochrome c release activates caspase-3 in neuronal models (Chen, et al. 2021). In the present study, intoxication with 3NP displayed significant increases in the JNK/c-Jun/caspase-3 expression, which is in line with the previous study (Garcia et al. 2002; Verdaguer et al. 2023; Elbaz et al. 2025). However, co-treatment with Candesartan mediated inhibition of the protein expression of JNK/c-Jun/caspase-3 with a prominent effect at a higher dose (5 mg/kg), which is supported by a previous study (Elkahloun et al. 2016; Ahmed and Mohamed 2019). Therefore, the mechanistic pathway behind the inhibition of JNK/c-Jun can be regarded as due to the activation of CREB as mentioned by Zhang et al. 2008a.

Survivin, a recently discovered member of the inhibitor of apoptosis protein (IAP) family, has been implicated in various neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and brain stroke (Tolosa et al. 2008; Zhang et al. 2008b). It has been suggested that reinforcing the signaling pathway of survivin could potentially contribute to the therapeutic management of neurodegenerative disorders and cognitive impairments (Chu et al. 2017). According to Baratchi et al. (2010), prior research has demonstrated that survivin functions to inhibit the activation of Caspase-3 and Caspase-7. For instance, previous studies have demonstrated that survivin exhibits neuroprotective properties in the hippocampus of a mouse model of brain stroke (Sehara et al. 2013). Additionally, it has been observed that survivin enhances antioxidant activity and reduces apoptosis, as reported in studies conducted by Baratchi et al. (2010); Baratchi et al. 2011). In a study conducted by Syahrani et al. (2020), it was observed that the suppression of survivin resulted in an elevation in oxidative stress levels and a reduction in the expression of the superoxide dismutase (SOD) gene. In the present study, the expression of survivin was significantly reduced with the administration of 3NP. However, co-treatment with Candesartan ameliorated the effect of 3NP, leading to a marked elevation in survivin expression with a prominent effect at higher dose (5 mg/kg). Such an increase can be regarded as the stimulation of PI3K/Akt by Candesartan, which causes upregulation of survivin leading to a cytoprotective effect achieved by the transcriptional activation of genes linked with antioxidants or cell survival within the nucleus; this is in line with the previous studies by Zhang et al. (2016) and Hashikawa-hobara et al. (2012).

Moreover, various RAS-targeting strategies, such as ACE inhibitors, AT₂R agonists, and MasR agonists, have demonstrated neuroprotective roles in different models of neurodegeneration. However, AT1 receptor blockade via candesartan provides distinct advantages. Unlike ACE inhibitors, which may reduce both harmful Ang II and protective Ang-(1-7), Candesartan selectively inhibits the deleterious AT1 receptor-mediated effects while preserving or enhancing Ang II availability for AT2 receptor activation and Ang-(1-7) synthesis. This allows for indirect stimulation of the protective RAS arms without reducing overall RAS tone. While direct AT2 receptor or Mas receptor agonists have shown benefit in preclinical studies, they remain clinically unavailable (Rodríguez-Pallares et al. 2025).

In conclusion, the results of the present investigation offer the initial and first empirical support for the neuroprotective impact of Candesartan in mitigating striatal degeneration and mitochondrial dysfunction induced by 3NP, hence suppressing the progression of Huntington’s disease through the activation of AngII/AT2R/Ang-(1-7)/MasR/CREB/BDNF pathway as well as involvement of JNK/c-Jun and survivin, offering new prospective and possibility for the use of AT1 receptor blockers in the treatment of HD.

One limitation of the present study is the lack of direct mechanistic confirmation regarding the effect of candesartan on survivin expression. While survivin is a key anti-apoptotic marker, its regulation by candesartan remains speculative and warrants further investigation, especially considering that most studies on survivin have focused on oncology rather than neurodegeneration.

Additionally, clinical translation requires evaluation of Candesartan’s effects in HD patients with comorbid hypertension, since blood pressure modulation could influence both CNS and cardiovascular outcomes. This is particularly relevant given that mutant huntingtin is expressed in cardiomyocytes, contributing to cardiac dysfunction and hypertension, which are among the leading causes of mortality in HD patients. Moreover, cardiovascular diseases (CVD) can accelerate neurodegenerative progression by impairing cerebral perfusion and promoting systemic inflammation.

Therefore, future studies should explore the dual neuroprotective and cardioprotective potential of candesartan in translational HD models and in clinical settings where neurodegeneration and cardiovascular dysfunction intersect. Investigating candesartan’s role in both central and peripheral RAS modulation may open new therapeutic avenues for HD patients, particularly those at risk for or suffering from cardiovascular complications.