Investigating the Involvement of Cytokines and Neurotrophic Factors in the Advanced Stages of Huntington’s Disease: A BACHD Study

Research Article

Austin Alzheimers J Parkinsons Dis (aapd). 2023; 6(1): 1034.

Investigating the Involvement of Cytokines and Neurotrophic Factors in the Advanced Stages of Huntington’s Disease: A BACHD Study

Priscila Aparecida Costa Valadão¹*; Bruna da Silva Oliveira¹; Caroline Amaral Machado¹; Heliana de Barros Fernandes¹; Thatiane Cristina Machado¹; Kívia Santos Soares¹; Antonio Lucio Teixeira2,3; Cristina Guatimosim¹; Aline Silva de Miranda¹*

¹Departamento de Morfologia, Universidade Federal de Minas Gerais, Belo Horizonte –MG, Brazil

²Neuropsychiatry Program, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, University of Texas Health Science Center at Houston, USA

³Faculdade Santa Casa BH, Belo Horizonte, MG, Brazil

*Corresponding author: Aline Silva de MirandaDepartamento de Morfologia, ICB Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901 Brazil. Email: drapriscilavaladao@gmail.com; mirandaas@icb.ufmg.br; mirandas.aline@gmail.com

Received: February 04, 2023 Accepted: March 27, 2023 Published: April 03, 2023

Abstract

Neuroinflammation seems to be involved in the pathophysiology of Huntington’s Disease (HD), but its specific role on different stages of the disease, especially in later stages, remains to be understood. Here in, we investigated the concentrations of cytokines, chemokines and neurotrophic factors in striatum and frontal cortex of 24-month-old BACHD mice, a murine model of that displays several behavioral and pathological features of human HD. Our results revealed increased concentrations of the chemokine MCP-1 and the neurotrophin NGF in the striatum of BACHD mice alongside a reduction in the levels of the cytokine IL-6 and of the neurotrophin BDNF. In the frontal cortex, we found decreased levels of BDNF and MCP-1. We provide the first evidence that cytokines and neurotrophic factors may contribute to the pathophysiology of advanced HD.

Keywords: Huntington’s disease; Neuroinflammation; Neurotrophic factors; Cytokines; Chemokines

Introduction

Huntington's Disease (HD) is an autosomal-dominant inherited neurodegenerative disease caused by abnormal expansion of CAG trinucleotide repeats in the Huntingtin gene (HTT) located on chromosome 4 (The Huntington's Disease Collaborative Research Group, 1993; [2]. This mutation promotes huntingtin misfolding and formation of insoluble toxic aggregates in the nucleus and cytoplasm of neurons. In the Central Nervous System (CNS), the expression of mutant huntingtin proteins (mHTT) has also been described in microglia and astrocytes (Van der Burg et al., 2009; Jansen et al., 2017). Accumulations of mHTT with subsequent atrophy of brain areas such as striatum, cerebral cortex, hippocampus and brain stem, contribute to the constellation of motor, cognitive and psychiatric symptoms observed in HD [26,27,33]. As the progression of HD symptoms occurs in parallel with the neuropathological changes, HD seems to be a unique model to investigate neurobiological mechanisms involved in neurodegeneration [7,8,17].

Apart from the well-known role of mHTT proteins in HD development, the cellular and molecular mechanisms underlying disease progression remain to be fully understood. Accumulating evidence has supported a pivotal role of neuroinflammation in HD pathophysiology (Björkqvist et al., 2008) [7,9,35]. For instance, post-mortem studies demonstrated higher levels of inflammatory mediators such as IL-6, IL-8, tumor necrosis factor-α (TNF-α), monocyte chemoattractive protein-1 (MCP-1)/CCL2 and the anti-inflammatory cytokine IL-10 in the striatum, cortex and cerebellum of HD patients compared to controls (Björkqvist et al., 2008) [32]. Similar findings have been also reported in pre-clinical studies with rodent models of HD [14,15,23,29].

Neurotrophic factors, such as BDNF, NGF and GDNF, are a group of growth factors that play important roles in neuronal survival, differentiation and plasticity [20]. In parallel with HD-associated neurodegeneration, BDNF deficits have been reported in cell lines expressing mHTT and in brains of HD murine models and patients (FERRER et al., 2000; SEO et al., 2004;) [11,37]. Up-regulation of BDNF and GDNF in the striatum leads to neuroprotective effects in the YAC72 mice, a transgenic model of HD with over expression of mutant full-length huntingtin [37], as well as in a toxic model of HD induced by quinolinic acid (Martınez-Serrano & Bjorklund, 1996; Perez-Navarro et al., 1996; Alberch, 1999) [37].

Due to ethical concerns associated with human brain studies, animal models, especially genetic modified murine models, have been valuable tools to investigate HD-related pathophysiological mechanisms [24,35]. The BACHD transgenic mouse model carries the full-length human HTT gene in bacterial artificial chromosome and displays behavioral and neuropathological features of the human disease (Gray et al., 2008). Compared with other transgenic models of HD like the R6/2 mice, the BACHD mice live more than 24 months and exhibit a slow progression of the disease, which allows the study of pathophysiological mechanisms implicated in HD advanced stages (Gray et al., 2008; Yang, 1997) [19].

We hypothesize that an imbalance between inflammatory cytokines and neurotrophic factors may underlie HD progression. Here, using the BACHD murine model, we investigated the expression of cytokines and neurotrophic factors in 24-month-old mice. To the best of our knowledge, this is the first study addressing the involvement of these mediators in advanced HD. Our findings may shed lights in potential mechanisms involved in HD progression opening new venues for the identification of novel therapeutic targets.

Materials and Methods

BACHD mice

All experiments were carried out in accordance with the rules applicable by the local authorities (Ethics Committee on Animal Experiments of the Universidade Federal de Minas Gerais-CEUA/UFMG; approved protocol #036/2013). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Male FVB/NJ (wild-type or WT) and FVB/N-Tg (HTT*97Q)IXwy/J (BACHD) transgenic mice were purchased from Jackson Laboratory (Barl Harbor, ME, USA) (JAX stock #008197) and used to establish a colony. Food and water were offered ad libitum and the mice were kept in a temperature-controlled place (23°C) in a 12 to 12 hour light-dark cycle. All animals used in this study were genotyped ten days after birth using multiplex Polymerase Chain Reaction (PCR) (HTT-Forward: CCGCTCAGGTTCTGCTTTTA/HTT-Reverse: GTCGGTGCAGCGGCTCCTC; Actin-Forward: TGGAATCGTGTGGCATCCATCA/Actin-Reverse: AATGCCTGGGTACATGGGGTA). The animals were identified by numbers according to their genotype (WT or BACHD) and subsequently separated in mini-isolation cages with a maximum of 4 animals per cage.

All experiments were conducted with 24-month-old WT and BACHD mice as this age corresponds to older age in humans. Accordingly, the 24-month-old BACHD animals have established neurodegeneration, allowing the investigation of cytokine and neurotrophic factors alterations in the main brain areas affected by HD (Flurkey, Currer, and Harrison, 2007; Gray et al., 2008). Our research group has conducted several studies with male BACHD mice [16,35], as HD-associated behavioral phenotypes do not seem to be influenced by sex in this model [19].

Assessment of Cytokines, Chemokines and Growth Factors in Brain Regions

Brain regions (striatum and prefrontal cortex) of WT and BACHD mice (n=5 per group) were carefully removed and homogenized in an extraction solution (100mg of tissue per milliliter), containing 0.4MNaCl, 0.05%Tween 20, 0.5%BSA, 0.1mM phenyl methyl sulphonyl fluoride, 0.1mM benzethonium chloride, 10mM EDTA, and 20KIU aprotinin, using Ultra-Turrax. The lysates were centrifuged at 13.000×g for 10 min at 4°C and supernatants were collected and stored −70°C until use. The brain concentrations of the cytokines IL-6, IL-12p70, TNF-α and of the chemokine MCP-1/CCL2 were measured using an inflammatory CBA kit (BD Biosciences, San Diego, CA) and acquired on a FACS CANTO II flow cytometer (Becton Dickinson, San Jose, CA). These cytokines have been implicated in human HD development and/or progression (Björkqvist et al., 2008; [10,32]. The CBA results were analyzed by employing the software FCAP Array version 3.0 (Soft Flow Inc. Pecs, Hungary). The concentration of the neurotrophic factors, Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Glial cell-Derived Neurotrophic Factor (GDNF) and of the chemokine Fractalkine/CX3CL1 was determined by ELISA (R&D Systems, Minneapolis, MN) in accordance to the manufacturer’s instructions. Results are expressed as picogram per 100mg of tissue. The detection limit of the CBA and ELISA assays was 0.2 and 5pg/mL, respectively.

Statistical Analysis

All results are presented as mean±SD. The Shapiro-Wilk test was used in all data to determine the normality. For variables normally distributed differences were compared by Student's t-test. In case of variables not normally distributed differences were analyzed by Mann–Whitney U test. The significance was set at p<0.05. Statistical analyses were performed using Prism 6 software (GraphPad, La Jolla, CA, USA).

Results

At 24-month-old, has already been demonstrated in work previously published by our research group that BACHD mice display significant motor deficits as revealed by a decrease in the total distance traveled in the open field (p=0.0003) and in the latency to fall in both wire-hang (p=0.0073) and rotarod (p=0.0043) compared with controls [16].

In the striatum, BACHD mice presented decreased concentration of IL-6 (BACHD: 5.618±0.4367 Vs. WT: 6.833±0.1653; **p=0.04; Figure 1E) and BDNF (BACHD: 148.6±9.075 Vs. WT: 218.6±18.66; **p=0.008; (Figure 1G) and increased concentration of MCP-1 (BACHD: 59.91±4.762 Vs. WT: 36.23±7.436; *p=0.03; (Figure 1D) and NGF (BACHD: 4900±530.8 Vs. WT: 2934±416.9; *p=0.02; (Figure 1H) compared with WT animals. No significant differences were found in the concentrations of IL-12p70(p=0.60), TNF-α(p=0.91), GDNF(p=0.93) and CX3CL1(p=0.95).