Advances in Treatment of Huntington's Disease

#### **Chapter 5**

## Endocannabinoid System as a New Therapeutic Avenue for the Treatment of Huntington's Disease

*Kamila Saramak and Natalia Szejko*

#### **Abstract**

Huntington's disease (HD) is a progressive, neurodegenerative disorder manifested by chorea as well as a variety of psychiatric abnormalities. Up to this date, only symptomatic treatment exists. Therefore, there is an urgent need for further therapies. Several neuroanatomical circuits are involved in the pathophysiology of HD, mainly the dopaminergic system. Animal studies and limited studies in humans have shown that abnormalities in the endocannabinoid system could also play an important role in the pathophysiology of HD. These findings have important clinical implications since cannabis-based medicines could potentially be used in the treatment of HD. The aim of this chapter is to summarize the current state of the research regarding the involvement of the endocannabinoid system in HD.

**Keywords:** chorea, Huntington's disease, experimental therapies, cannabis-based medicine, dronabinol

#### **1. Introduction**

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor dysfunction, cognitive decline as well as psychiatric disturbance [1, 2]. The prevalence of HD is estimated to be between 0.4 and 5.70 per 100,000. Since HD is a genetic disorder, the prevalence depends strongly on the study population and it is higher in Europe, North America, and Australia than in Asia [3]. HD is caused by a dominantly inherited CAG repeat expansion in the huntingtin gene (HTT). The disease develops in individuals bearing a number of repetitions greater than 40, whereby greater CAG repeats found in the huntingtin gene are associated with early-onset forms of the disorder, fast rate of disease progression, and the most severe neurological deficits [4].

The mean age of HD onset is around 40 years, meanwhile the Juvenile Onset Huntington's Disease (JOHD), occurs in individuals bearing more than 60 CAG repeats, which usually starts at the age of 21. HD eventually leads to death 15–20 years after the symptomatic onset [5]. It is believed that mutant huntingtin (mHTT) affects many cellular functions and leads to cell death, preferentially subpopulations of GABAergic medium spiny projection neurons and neurons in the cerebral cortex [1, 6]. This leads to imbalances in diverse neurotransmission, including the dopaminergic

(DA) and glutaminergic systems. In the early stages of HD, DA neurotransmission is increased, whereas expression of DA receptors is reduced. However, in the course of the disease DA neurotransmission decreases. In turn, time-dependent abnormal DA neurotransmission affects glutamate receptor modulation, which may cause excitotoxicity [7, 8]. As DA plays a crucial role in the control of coordinated movements, motivation, and reward as well as cognitive function, alterations in DA balance in the striatum and provoke neurological and psychiatric symptoms of HD. The early stages of the disease are often characterized by chorea, followed by akinesia, while dystonia is more typical for the late stages [9]. Major non-motor symptoms include apathy and depression, anxiety, irritability, or aggressive behavior [9]. Impairment in cognitive functioning eventually ending in dementia, which has been mentioned by George Huntington in his first report, is another integral part of the disease [10]. Until today, there is no cure for HD, and treatment is only symptomatic, targeting mainly dopaminergic and glutaminergic systems [11].

#### **2. Possible role of the endocannabinoid system in Huntington's disease**

Over the last 30 years, the endocannabinoid system (ECS) has emerged as an important neuromodulatory system, which could be efficiently targeted in a number of neurological diseases, including HD [8, 12, 13]. The primary cannabinoid receptor subtypes are cannabinoid receptors type 1 (CB1) and type 2 (CB2). The CB1 receptor is a protein-coupled receptor, highly expressed in the central nervous system (CNS), particularly in the neocortex, hippocampus, basal ganglia, cerebellum, and brainstem. In addition to its CNS location, CB1 has also been identified in numerous peripheral tissues and cell types [14]. On the other hand, the CB2 receptor is expressed mainly outside CNS, predominantly in the immune system. However, it has also been identified in the CNS, especially in the glial cells and brainstem neurons [15, 16]. The abovementioned high distribution of the CB1 receptor in basal ganglia indicates an indispensable role of the ECS in the control of movements by inhibitory modulation of other neurotransmitter systems [16]. Moreover, the CB1 receptors regulate glutamatergic neurotransmission under both physiological and pathological conditions and thus are able to downregulate excitotoxic glutamate release [17].

#### **3. Studies in animal models**

Studies in animal models suggest that the pathogenesis of HD may be related to an early and widespread reduction in the ECS, particularly to the loss of CB1 receptors [16, 18, 19] and decreased endocannabinoid levels in the striatum, which in turn may lead to hyperkinesia [19]. The administration of substances, which increase endocannabinoid activity led to a significant improvement of motor disturbances in a rat model of HD [16, 20]. In particular, Lastres-Becker et al. [17] hypothesized that substances that increase the endocannabinoid activity could be applied for the treatment of hyperkinetic symptoms. To test this hypothesis the authors created a rat model of HD through bilateral striatal injections of 3-nitropionic acid that leads to impaired striatal GABAergic neurotransmission. As a result, these rats started suffering from abnormal movements followed by motor depression. In addition, they demonstrated that the severity of motor hyperkinesias was correlated with decreased concentration

#### *Endocannabinoid System as a New Therapeutic Avenue for the Treatment of Huntington's Disease DOI: http://dx.doi.org/10.5772/intechopen.104566*

of several neurotransmitters, such as GABA, dopamine, and their metabolites. Moreover, mRNA levels for the CB1 receptor were depleted in the caudate-putamen of 3-nitropropionic acid (3-NP) injected rats. In addition, the authors demonstrated a reduction in CB1 receptor binding in the caudate-putamen, the globus pallidus, and also substantia nigra. Finally, the administration of AM404, an inhibitor of endocannabinoid uptake, led to the alleviation of motor disturbances. The same group from Madrid [21] explored the status of CB1 receptors in the HD94 transgenic mouse model of HD. To investigate this problem, the authors analyzed mRNA levels of the CB1 receptor and the number of specific binding sites, and the activation of GTPbinding proteins by the CB1 receptor agonist. As a result, they have demonstrated that mRNA transcripts of the CB1 receptor were significantly decreased in selected regions of the brain, such as caudate in the HD transgenic mice compared to controls. This depletion was correlated with a marked reduction of reception density in the caudate, globus pallidus, and substantia nigra pars reticulata. In addition, the efficacy of CB1 receptor activation was depleted in the globus pallidus and there was a trend toward a decrease in substantia nigra.

Another significant contribution was done by the group from the Autonomous University in Madrid led by Isabel Lastres-Becker [22]. The scientists used a previously mentioned rat model of HD for this purpose created via bilateral intrastriatal injections of 3-NP. As a result, CB1 receptor binding and activation of GTP-binding proteins were also reduced in the basal ganglia. In parallel, the authors demonstrated a significant decrease of two endocannabinoids, anandamide and 2-arachidonoylglycerol in the striatum of affected rats, while there was an increase in anandamide concentration in the substantia nigra. Importantly, both CB1 receptors concentration, as well as endocannabinoid levels, were not changed in the cerebral cortex. Another study by the same group [23] has shown that compounds acting at the endocannabinoid systems reduce hyperkinesia in a rat model of HD. In particular, they applied AM404, an inhibitor of the endocannabinoid reuptake, which was able to reduce hyperkinesia and provoke recovery from neurochemical deficits.

As for exocannabinoids used in the treatment of neurological and psychiatric disorders, in one study [24], delta9-tetrahydrocannabinol (THC), a nonselective cannabinoid receptor agonist, and SR141716, a selective antagonist for the CB1 receptor, were tested in an animal model of HD. Surprisingly enough, the administration of THC increased malonate-induced striatal lesions, but SR141716 enhanced the same effect to an even greater extent. Another study examined the long-term effects of exocannabinoid exposure in animal models of HD. In this case, they used transgenic mice R6/1 of HD and administered THC for 8 weeks. This chronic treatment preserved CB1 receptors in the R6/1 striatum, suggesting that the manipulation of endocannabinoid levels warrants further exploration.

Similarly, Sagredo et al. [25] examined the neuroprotective effect of cannabinoids in rats with 3NP striatal lesions. To tackle this question, the authors used the CB1 agonist arachidonyl-2-chloroethylamide (ACEA), the CB2 agonist HU-308, and cannabidiol (CBD). Interestingly enough, the application of CBD, but not ACEA or HU-308 reversed the effects of 3NP. In particular, CBD reversed 3NP-induced reductions in GABA contents and mRNA levels of substance P (SP), neuronal-specific enolase (NSE), and superoxide dismutase-2 (SOD-2). The authors concluded that CBD has neuroprotective values, but mainly on striatal neurons projecting to substantia nigra. This neuroprotective effect was not reversed by the CB1 receptor antagonist SR141716. Pintor et al. [26] demonstrated that the cannabinoid receptor agonist, WIN 55,212–2, attenuates the effects induced by quinolinic acid (QA) in the rat striatum. In this study,


*Endocannabinoid System as a New Therapeutic Avenue for the Treatment of Huntington's Disease DOI: http://dx.doi.org/10.5772/intechopen.104566*


*HD: Huntington disease; CB1R: cannabinoid receptor type 1; CB2R: cannabinoid receptor type 2; 3 NP mice: 3-nitropropionic acid; eCBRI: endocannabinoid re-uptake inhibitor; TRPV1: the transient receptor potential cation channel subfamily V member 1 (TrpV1); GABA: γ-aminobutyric acid; THC: tetrahydrocannabinol; CBD: cannabidiol; and ACEA: arachidonyl-2-chloroethylamide.*

#### **Table 1.**

*Studies investigating the relevance of endocannabinoid system in HD pathogenesis in animal models. Studies are presented in chronological order.*

QA was introduced in the rat striatum and this, in turn, led to the reproduction of clinical features typical for HD. The administration of WIN 55,212–2 blocked the increase in extracellular glutamate induced by QA. During *in vivo* experiment, WIN 55,212–2 significantly improved the striatal damage induced by QA, but no effect was observed on a behavioral ground. Valdeolivas et al. [27] also explored the neuroprotective potential of cannabinoids in an experimental model of HD. In particular, they investigated *Sativex*®, a combination of tetrahydrocannabinol (THC) and CBD at a ratio of 1:1, to monitor the potential neuroprotective effects of cannabinoids. The authors applied both histological and biochemical markers. As a result, the application of malonate in the striatum led to an increase in edema, while *Sativex*® reduced it. Moreover, *Sativex*® led to a reduction in neurodegeneration and glial activation. Furthermore, the authors found that both CB1 and CB2 receptors are involved in the positive effects of cannabinoids on HD symptoms. Similar findings were reported by Sagredo et al. [28], who used an animal model of HD to examine the potential neuroprotective effects of compounds influencing the endocannabinoid system. Interestingly enough, only compounds activating CB2 receptors had neuroprotective effects. The authors confirmed this statement by using the selective CB2 receptor antagonist, SR144528, which, in turn, led to increased vulnerability to malonate. What is more, the activation of CB2 receptors reduced the levels of tumor necrosis factor-alpha (TNF-alpha) that had been increased in the malonate-induced model of HD.

Another study by de Lago et al. [29] examined whether arvanil, an endocannabinoid "hybrid," could lead to symptom reduction in the rat model of HD. It was demonstrated that arvanil reduced ambulation and stereotypic movements. The same group [30] demonstrated that UCM707, an inhibitor of the anandamide uptake, could be used as a symptom control agent in an animal model of HD and multiple sclerosis (MS), but failed to delay the disease progression.

Furthermore, a number of other studies have suggested that therapies with CB-activating compounds might lead to neuroprotective effects against excitotoxic striatal toxicity through both CB receptor-mediated and independent effects [21, 31–35]. However, in several studies, no benefit or even exacerbation of neurotoxicity could be observed [22, 25, 29].

An overview of studies investigating the relevance of the endocannabinoid system in HD pathogenesis in animal models is shown in **Table 1**.

#### **4. Clinical research**

The post-mortem examination of brain tissue in individuals with HD as well as PET imaging studies *in vivo* showed that CB1 receptors are severely reduced in all regions of the basal ganglia in comparison to other receptor changes in HD, which strengthens the hypothesis of a possible role of cannabinoids in the progression of neurodegeneration in HD [38, 39].

First reports of using cannabinoids in patients with HD were contradictory [24, 28, 30]. In 1991, Consroe et al. conducted the first double-blind randomized cross-over study to evaluate the efficacy and safety of oral CBD (10 mg/kg/ day for 6 weeks) in 15 neuroleptic-free patients with HD [28]. The therapeutic response was evaluated with the use of the Marsden and Quinn chorea severity scale [40]. In this study, no statistically significant improvement has been shown. There was also no significant difference between the CBD and placebo groups in terms of side effects. In 1999 Müller-Vahl et al. published a case of a 58-year-old male with HD who was treated with a single dose of 1.5 mg of a CB1 agonist, nabilone. In this individual, a severe deterioration of chorea was observed [24]. In 2006, Curtis et al. described a case of a 43-year-old female, whose chorea and irritability improved after medication with 1 mg of nabilone [30]. A double-blind placebo-controlled randomized cross-over trial using nabilone was conducted in 2009 by the same author. This time 37 patients were treated with 1 mg or 2 mg of nabilone daily for 5 weeks. For primary measures, the patients were assessed with Unified Huntington's Disease Rating Scale (UHDRS) total motor score and


*CBM: cannabis based medicine; HD: Huntington disease; SAE: severe adverse events; CBD: cannabidiol; UHDRS: United Huntington Disease Rating Scale; and SAE: serious adverse events.*

#### **Table 2.**

*An overview of studies investigating efficacy and safety of CBM in HD.*

*Endocannabinoid System as a New Therapeutic Avenue for the Treatment of Huntington's Disease DOI: http://dx.doi.org/10.5772/intechopen.104566*

UHDRS subsections for chorea, cognition and behavior, and neuropsychiatric inventory (NPI) for secondary measures. There were no statistically significant differences in total UHDRS between the groups. However, statistically, significant improvements were noted for the UHDRS chorea scale and the neuropsychiatric inventory. There were no statistical differences reported between the 1 and 2 mg. Adverse effects were reported for placebo and nabilone similarly. There was one Serious Adverse Event (SAE) related to nabilone—one of the patients withdrew due to severe sedation. Importantly, no psychoses were reported [23]. In 2016, the results of a study conducted by Moreno et al. using nabiximols in the treatment of HD were published [36]. Nabiximols (tradename *Sativex*®) is an oromucosal spray, containing 2.7 mg THC and 2.5 mg cannabidiol (CBD) per puff licensed in most European countries for symptomatic treatment of multiple sclerosis [35]. Both Sativex and placebo were handed to 25 patients in a form of an oral spray, to be administered up to 12 sprays/day for 12 weeks. The main aim of the study was to investigate the safety of nabiximols in HD patients, assessed by the absence of SAE and lack of impairment of motor, cognitive, behavioral, and functional scales during the active treatment. The secondary objective was a clinical improvement of Unified Huntington Disease Rating Scale scores. As a result, safety and tolerability were confirmed. No statistically significant improvement in UHDRS in the nabiximols group was noted with respect to the placebo group. Moreover, no significant changes in the biomarkers could be observed [35].

An overview of all available studies investigating the efficacy and safety of CBM in HD is provided in **Table 2**.

#### **5. Safety profile of cannabis-based medicines in patients with HD**

Even today, very little is known about the safety of CBM in patients with HD due to the limited number of studies exploring this issue. However, the available preliminary results suggest that the safety profile of CBM in HD is similar to that in other groups of patients. A recently conducted meta-analysis, including diverse populations of patients treated with CBM, showed that administration of cannabinoids can be associated with a greater risk of adverse events (AE), including serious adverse events (SAE) [46]. The most common short-term AEs included dizziness, dry mouth, nausea or vomiting, fatigue, somnolence, euphoria, vomiting, disorientation, drowsiness, confusion, loss of balance, and hallucinations. So far, there has been no study evaluating the long-term AEs of cannabinoids [46]. Up to this point, only two CBM-related SAEs in HD have been reported and both occurred after the treatment with nabilone. A 58-year-old male described by Müller-Vahl experienced an exacerbation of chorea. Moreover, the patient noticed the deterioration of short-term memory [42]. During the study performed by Curtis et al. [44], one of the patients experienced severe sedation and had to withdraw from the trial. Importantly, none of the patients enrolled in this study suffered from exacerbation of chorea or psychosis. The most frequent AE was drowsiness and forgetfulness. In the recent study conducted by Moreno et al. [45], dizziness or disturbance in attention were the two most common AEs. No serious alterations in psychiatric or neurological conditions of the participants were noted [45].

#### **6. Conclusions**

There is increasing evidence that the endocannabinoid system is a new promising therapeutical target in patients with HD. However, larger well-designed controlled studies are urgently needed to confirm the efficacy and safety of this treatment.

### **Author details**

Kamila Saramak1 and Natalia Szejko2,3\*

1 Department of Neurology, Hochzirl Hospital, Zirl, Austria

2 Department of Bioethics, Medical University of Warsaw, Poland

3 Department of Neurology, Medical University of Warsaw, Warsaw, Poland

\*Address all correspondence to: natalia.szejko@wum.edu.pl

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Endocannabinoid System as a New Therapeutic Avenue for the Treatment of Huntington's Disease DOI: http://dx.doi.org/10.5772/intechopen.104566*

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#### **Chapter 6**

## Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease Treatment

*Irina Kerkis, Rodrigo Pinheiro Araldi, Cristiane Valverde Wenceslau and Thais Biude Mendes*

#### **Abstract**

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin gene. The disease causes the progressive degeneration of neurons affecting particularly the medium spiny neurons (MSNs) within the striatum. The mHtt inclusions promote neurodegeneration. However, the mHtt can spread to different brain areas through exosomes. For this reason, it is not surprising that HD causes motor, cognitive and neuropsychiatric dysfunctions. To date there is no treatment able to modify the natural history of the disease. In this sense, the advanced cellular therapy, based on the therapeutic use of mesenchymal stem cells (MSCs) emerges as a potential candidate for HD treatment. This is because, the MSCs produce many critical therapeutic molecules which act in multiple cellular and molecular targets. Moreover, in addition, advanced cell therapy is a unique approach that could provides neuroprotection and neuroregeneration. However, the current discovery that the MSC mechanism of action is mediated by exosomes, have encouraged scientist to explore the therapeutic potential of the cell-free therapy. Based on this, we revisited the HD pathophysiology, areas. Providing evidence that MSC and MSC-derived exosomes can be used to change the natural history of HD.

**Keywords:** Huntington's disease, stem cells, therapeutic cells, exosomes, cell-free products

#### **1. Introduction**

Huntington's disease (HD, OMIM 143100) is a rare and incurable hereditary autosomal dominant neurodegenerative disorder, affecting 5–10 individuals per 100,000 in the Caucasian population. In certain regions, such as Australia, North America, and Western Europe, including the United Kingdom, the prevalence of the disease has increased over the past 50 years [1, 2].

HD is characterized by the loss of specific neurons within the striatum. The most sensitive cell population is the gamma-aminobutyric acid (GABA)ergic medium spiny neurons (MSNs). Neuropathologically, the disease leads to about 57% loss of

cross-sectional area from the caudate nucleus and about 65% loss of the putamen (in *postmortem* samples). The progression of HD also leads to loss of cortical volume (particularly in cases with more advanced disease), affecting the large pyramidal neurons predominantly in layers III, V, and VI. In these cases, the loss of neurons in the thalamus, substantia nigra pars reticulata, and the subthalamic nucleus can also be observed. The neurodegeneration of these brain areas results in progressive motor (chorea, saccadic eye movement abnormalities, ataxia of speech, dysphagia, etc.), cognitive (dementia), and neuropsychiatric disturbances (depression, anxiety, apathy, etc.). These HD symptoms usually develop between ages 30 and 50 (adult-onset Huntington's disease—AOHD, which is verified in 95% of cases), but they can appear as early as age 20 (juvenile-onset Huntington's disease—JOHD). However, neurological loss and metabolic alterations generally occur in the adult HD brain before symptoms, but the precise timetable for the neuronal degeneration remains unknown [3].

HD is caused by the expansion of trinucleotide Cytosine-Adenine-Guanine (CAG) repeat, located in the first exon of the HD gene, also known as HTT or IT15 gene (locus 4p16.3, OMIM 613004), which encodes the huntingtin protein (Htt). Since the discovery of HTT gene mutation (in 1993), it has been recognized that larger CAG expansions are associated with early-onset in HD, especially for AOHD. Generally, unaffected individuals have less than 35 CAG repeats (common rage in humans: 17–25), while affected individuals have 36–250 CAG repeats. The CAG repeat range of 36–39 might be found in affected individuals and asymptomatic individuals (reduced penetrance alleles), whereas individuals with over 40 CAG repeats always develop the disease (fully penetrance alleles) [4].

The wild-type allele of the HTT gene (i.e., <35 CAG repeats) typically segregates and stably as a polymorphic locus. However, the allele carrying higher-normal CAG repeats (27–35 repeats) has increased instability. For this reason, individuals with 27–35 CAG repeats have a high risk of passing on repeats in the affected size range to their offspring. HTT gene encodes the huntingtin protein (Htt), a sizeable soluble protein (350 kDa), consisting of 3114 amino acids, which is expressed in all metazoans, is highly conserved among vertebrates. Although, all tissues ubiquitously express the HTT gene, Htt protein is found higher expressed in the brain, represented by all neurons and glial cells [4].

#### **1.1 Htt protein characterization and function in HD**

The Htt protein is crucial for developing and maintaining central nervous systems (CNS) homeostasis since the protein is engaged in many cellular and biological functions, including transcription, transport, vesicular trafficking, and coordination of cell division, energy metabolism, and antiapoptotic activity. For this reason, it is not surprised that Htt co-localizes with many organelles, such as the nucleus, endoplasmic reticulum, Golgi complex, endosomes, mitochondria, and synaptic vesicles. Furthermore, cells expressing mRNA of the HTT gene were described by in situ hybridization in the usual human 20 to 23-week fetal brain, suggesting that huntingtin protein is crucial for the development of the CNS. Studies also demonstrated that the deletion of the mouse homolog of the HTT gene is lethal in the embryo before the brain is formed. By contrast, heterozygote mice for the HTT gene usually develop but exhibit motor deficits and cell loss in basal ganglia. Altogether, these data confirm that the Htt protein is mandatory for CNS development and function [5].

The Htt protein is characterized by the presence of (i) the N-terminal 17 amino acids (or N17 region), which is followed by (ii) the polyglutamine (poly Q ) tract

#### *Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease… DOI: http://dx.doi.org/10.5772/intechopen.102539*

(encoded by the CAG repeats), (iii) a proline-rich region (PRR), (iii) clusters of Huntingtin, Elongation factor 3, PR65/A regulatory subunit of PP2A and target of rapamycin 1 (HEAT) repeats (α-helix-loop-α-helix motif), and (iv) caspase and calpain cleavage sites (in higher vertebrates). The N17 region has been identified as a critical region that plays a role in Htt localization, aggregation, and toxicity. It is subject to several post-translational modifications, including acetylation, SUMOylation, phosphorylation, and ubiquitination. The polyQ tract is encoded by the CAG trinucleotide repeats, which code for the glutamine (Q ) amino acid. PRP region is exclusively found in mammals and is essential for the Htt interactions with proteins containing tryptophans or Src homology 3 domains. In addition, PRP encodes the polyproline (polyP) region, which interacts with polyQ, increasing the Htt protein stability and solubility. HEAT repeats consist of around 50 amino acids and contains two antiparallel α-helices forming a hairpin, which acts as a scaffold for various protein complexes and mediates inter and intramolecular interactions. Sixteen HEAT repeats organized into four clusters were identified in the Htt protein. Htt protein also has several proteolytic cleavage sites, including proline, glutamic acid, serine, and threonine domains. These domains are found in both Htt and mHtt proteins. Thus, these proteins can be cleaved by caspase 3 at amino acid 513 and 552, caspase 1 at amino acid 572, caspase 2 at amino acid 552, and caspase 6 at position 586. In addition, two calpain cleavage sites are located at amino acid 469 and 536, and the metalloproteinase (MMP)-10 cleaves Htt our mHtt at amino acid 402 [5].

The Htt protein interacts with over 200 other proteins, many of them involved in microtubule-mediated axon traffickings, such as the Huntingtin-associated protein 1 (HAP1), which mediates the interaction between Htt protein with microtubule motor proteins and their co-factors (kinesin, dynactin subunit p150, and dynein). Htt protein also mediates long- and short-range axonal transport and vesicle trafficking. This is because the Htt protein binds to the endocytic pathway-related proteins (clathrin and dynamin), as well as endocytic organelle trafficking proteins (α-adaptin, Hip1, Hip14, HAP40, PACSIN1, SH3GL3/endophilin 3). Htt protein is enriched at synaptic terminals and interacts with cytoskeletal and synaptic vesicle proteins to regulate synaptic activity in neurons. However, by exhibiting a C-terminus containing a nuclear export signal (NES), Htt protein can traffics between cytoplasm and nucleus. In addition, the N17 region also interacts with a nuclear pore protein (TRP), which has nuclear translocation activity. The N-terminal domain also forms an amphipathic alpha-helical membrane-binding domain that reversibly mediates association with the endoplasmic reticulum (ER), endosomes, and autophagic vesicles. Thus, it is not surprising that Htt protein also interacts with various transcription factors and transcriptional regulatory proteins, acting as a positive regulator of brain-derived neurotrophic factor (BDNF) transcription (a protein in which expression levels are found reduced in individuals with HD), stimulating the BDNF vesicular trafficking in neurons.

However, by increasing the number of glutamine residues in poliQ, the CAG trinucleotide expansion, verified in HD, reduces the solubility of mutated huntingtin protein (mHtt), resulting in intracellular aggregates (inclusions) in the brain, particularly in GABAergic medium spiny neurons (MSNs), located within the striatum. This event occurs because the expanded polyQ sequence in mHtt protein undergoes conformational changes to form a Β-pleated sheet prone to aggregation. In addition, the early phases of aggregate formation appear to accelerate the hydrophobic interactions with an amphipathic α-helical structure of N17. Under physiological conditions, proteostasis balances protein synthesis, folding, trafficking, and degradation. The impairment of the proteostasis systems aggravates the aggregation of the misfolded

mHtt. In addition, posttranslational modifications influence the mHtt toxicity, aggregation propensity, and intracellular localization. For example, proteolytic cleavage of mHtt generates N-terminal fragments with an increased tendency to aggregate. Furthermore, the mHtt inclusions can block the axonal transport between the cell body and the synaptic cleft and recruit other polyQ-containing proteins, which interact with mHtt, leading to loss of biological function, therefore, cell death. In addition, mHtt also silences the activity of RE1-Silencing Transcription Factor (REST), increasing the binding of REST to RE1/neuron restrictive silencer element, producing transcriptional dysfunction [6, 7].

#### **1.2 Htt protein and mitochondrial dysfunction**

The mHtt inclusions promote mitochondrial dysfunction, decreasing the activity of mitochondrial respiratory complexes II, III, and IV, which was already verified in *postmortem* brain samples of HD patients. Furthermore, the activity decrease of these mitochondrial complexes was also reported in asymptomatic HD carriers, indicating that mitochondrial defects may initiate disease onset. Experimental results also demonstrated that the ectopic expression of mitochondrial complex II subunit in striatal neurons expressing mHtt exon 1 restores complex II respiratory activity and protects against cell death. Confirming this data, it was extensively demonstrated that the rats treated with the neurotoxin 3 -nitropropionic acid (3-NP)—a selective inhibitor of succinate dehydrogenase and complex II—recapitulates the loss of MSNs in the substantia nigra, resulting in HD-like symptoms. In addition, studies showed that humans exposed to 3-NP exhibit motor dysfunction similar to HD patients [8–11].

Moreover, the mHtt can be cleaved by caspase 6. The fragments of cleaved mHtt protein bind to several transcription regulators, including the tumor suppressor, p53, thus regulating genes involved in mitochondrial function. Therefore, the mHtt increased the levels of p53, which in turn increased Bax and Puma expression, resulting in mitochondrial dysfunction and neuronal loss. These actions increase the reactive oxygen species (ROS) production, justifying the oxidative damage commonly observed in the plasma of HD patients, HD *postmortem* brain tissue, lymphoblasts, and cerebrospinal fluid. In addition, markers of oxidative damage, including heme oxygenase (an inducible isoform that occurs in response to oxidative stress), 3-nitrotyrosine (a marker for peroxynitrite-mediated protein nitration), and malondialdehyde (MDA), are elevated in human HD striatum and cortex as compared with age-matched control brain specimens. Consistent with these data, an increase in 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, in both plasma and urine of HD patients is observed [8–11].

Cumulative evidence has also demonstrated that mHtt protein causes a reduction in TORC1, the most potent transcriptional activator of (peroxisome proliferatoractivated receptor (PPAR)-γ coactivator-1 (PCG-1α) [12–14]. In addition, the mHtt protein also increases transglutaminase (Tgase) activity, which impairs the transcription of PCG-1α. Thus, mHtt downregulates the expression levels of PCG-1α [14]. The last is recognized as a critical transcriptional coactivator, which interacts with a broad range of transcription factors within a variety of biological processes. In addition, PCG-1α is involved in the regulation of mitochondrial biogenesis, OXPHOS, antioxidant defense, adaptive thermogenesis, and glucose/fatty acid metabolism. Under physiological conditions, the PGC-1α forms heteromeric complexes with nuclear respiratory factors (NRF-1 and NRF-2), and with the nuclear

receptors (PPARα, PPARδ, PPARγ and estrogen-related receptor α (ERRα)). These heterodimers regulate the expression of many nuclear-encoded mitochondrial genes, including cytochrome c, complexes I–V, and the mitochondrial transcription factor A (Tfam), as well as antioxidant genes, including superoxide dismutase (SOD) and glutathione peroxidase (GPX). Thus, the mitochondrial dysfunctions promoted by the mHtt-mediated PCG-1α downregulation lead to an increase in oxidative stress [12, 13, 15].

#### **1.3 Htt protein and neuroinflammation**

In addition, the mHtt accumulation in neurons promotes microglial activation, increasing oxidative stress. In addition, microglial cells that express mHtt show significant elevations in nuclear factor kappa B (NF-κB). This elevation occurs because the mHtt interacts with the IκB kinase (IKK) γ subunit, promoting the assembly and activation of the IKK complex (comprised by IKKα and IKKβ subunits). The IKKβ kinase phosphorylates IκBα causes the liberation of NF-κB, promotes the gene expression of the pro-inflammatory cytokine, including interleukin (IL)-6, resulting in neuroinflammation [16, 17]. The neuroinflammatory cytokines produced in response to mHtt protein accumulation leading to the activation of microglial cells considered the brain's resident immune cells. Under physiological conditions, i.e., in the absence of inflammatory stimulus, microglia are in a surveilling state, being responsible for maintaining synapses and synaptic plasticity. In addition, Microglia also facilitates the growth and development of surrounding neural networks by secreting neurotrophic factors, such as BDNF, nerve growth factor (NGF), and insulin-like growth factor (IGF-1) [18]. Moreover, significant evidence suggests the microglia promotes neurogenesis by phagocytosing apoptotic neural cells, facilitating the migration and differentiation of neural progenitor cells, and secreting soluble factors related to neurogenesis. However, microglia become activated upon detecting inflammatory stimuli, such as the increase in ROS or cytokine production [19]. When activated, microglia can adopt different polarization states, such as M1 and M2. Interestingly, microglia can alternate between these states. For this reason, recently, studies have suggested using M1/M2 terminology to categorize activated microglial cells. M1 microglia exhibit a proinflammatory phenotype, the significant initiators of innate and adaptive immunity in the brain. In addition, these cells elicit a phagocytic function and release cytotoxic factors, including nitric oxide and ROS. M2 microglia also carry out phagocytosis, but contrary to the role of M1 microglia, M2 microglia exhibit an anti-inflammatory phenotype, releasing anti-inflammatory cytokines such as interleukin (IL)-4, IL-13, and transforming growth factor-beta (TGF-β), which suppress inflammatory responses. The continued activation of microglia, stimulated by the inclusions of mHtt, prolonged the production of inflammatory mediators, resulting in chronic inflammation. The last is implicated in further tissue damage, justifying the microglia activation in striatal GABAergic neurons verified by Positron Emission Tomography (PET) in HD patients. Interestingly, studies based on PET also reported the presence of microglia activation in striatal GABAergic neurons in presymptomatic HD gene carriers, suggesting that microglial activation is an early characteristic of HD before symptom onset. However, the activation of microglia increases oxidative stress, resulting in both nuclear and mitochondrial DNA oxidative damages and protein and lipid oxidation. These damages lead to progressive cell death, particularly of MSN's [20, 21].

#### **1.4 Subventricular zone involvement in HD physiopathology**

Studies based on animal models of HD demonstrate that cell death in the striatum serves as a potent stimulator of progenitor cell proliferation (which are resident into the subventricular zone – SVZ), neuroblast migration, and neurogenesis. This is because, in the transgenic mouse model of HD (in which there is minimal cell loss in the striatum), the SVZ is unaltered, while in rat striatal-lesion models of HD (in which there is a cell loss in the striatum), there is a marked increase in SVZ progenitor cell proliferation and neurogenesis. The SVZ of the lateral ventricle is the resident niche of stem cells. These stem cells give rise to proliferative progenitor cells during brain development, which migrates to the cortex or the basal ganglia, where they differentiate into neurons. SVZ preserves its critical developmental characteristics in the adult brain, responsible for the continuous generation of migrating neuroblasts destined for the olfactory bulb or other areas of cell death in the brain. Thus, the maintenance of SVZ is crucial for neuron replacement along adulthood [22–24].

Supporting the involvement of SVZ with the physiopathology of HD, several studies revealed that the SVZ of HD patients is enriched in endogenous factors and receptors that actively regulate the cell cycle and the differentiation of precursors, such as the neuropeptide Y. Furthermore, studies already showed a significant increase of GABAA, receptor subunit γ2 (involved in the desensitization of the receptor complex to GABA) in SVZ in HD. GABA is an essential trophic factor for neurons during development. High levels of GABA are found in the normal SVZ and the SVZ of HD patients, suggesting that the SVZ maintains a germinal capacity for proliferation and neurogenesis in response to neurodegenerative cell death in adult life. However, it was proved that, while the Htt protein interacts with cAMP response element-binding protein (CREB) and specificity proetin1 (Sp1), conferring anti-apoptotic action, the mHtt protein triggers a pathogenic cascade involving Sp1 transcription factor activation, which leads to repressor element-1 silencing transcription factor (REST) upregulation, repressing neuronal genes [22–24].

#### **2. Exosomes**

With the progression of HD, others brain areas, besides the substantia nigra, are subjected to neuronal loss, leading to cognitive and neuropsychiatric dysfunctions. This occurs because the mHtt (as the Htt) is widespread to different brain areas through extracellular vesicles (EVs). The EVs comprise a heterogeneous group of phospholipid bilayer-enveloped particles that are naturally produced and secreted into the extracellular environment by almost all cell types. According to their size, biogenesis, and content, these vesicles are classified as (i) microvesicles, (ii) exosomes, and (iii) apoptotic bodies. Among these vesicles, exosomes are the most investigated. This is because, due to the repertoire of bioactive molecules carried by these vesicles (coding and non-coding RNA, proteins, lipids, and metabolites), the exosomes play an important role in cell-to-cell communication and intercellular signaling, regulating both physiological and pathophysiological processes. Moreover, in the function of their nanosize (30–200 nm), exosomes easily cross the blood-brain barrier [1, 25].

The growing interest in this class of EV has been reflected in the creation of distinct databases that compile data on exosome content, such as Exocarta (http:// www.exocarta.org/), EVpedia (http://bigd.big.ac.cn/databasecommons/database/

*Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease… DOI: http://dx.doi.org/10.5772/intechopen.102539*

id/4354) and Vesiclepedia (http://microvesicles.org/), which are constantly updated with released studies.

Exosomes are formed by endocytosis and released by exocytosis. During the biogenesis of these vesicles, the inward budding of the plasma membrane results in small intracellular vesicles. These small vesicles fuse, forming early endosomes. The invagination of the early endosome membrane results in the formation of intraluminal vesicles (ILVs) within large multivesicular bodies (MVBs). In contrast, cytoplasmic molecules such as coding and non-coding RNA, proteins, lipids, and metabolites are engulfed and enclosed into the ILV lumen. Along with the maturation of early endosomes to late endosomes, some proteins are directly integrated into the invaginating membrane. However, this process depends on the endosomal sorting complexes required for transport (ESCRTs), which are comprised of four proteins (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) that work cooperatively to facilitate the MVB formation, vesicle budding, and protein cargo sorting [1, 25]. The exosomes biogenesis also occurs through an ESCRT-independent pathway mediated by tetraspanins and ceramide-enriched lipid rafts. Tetraspanins are recruited at early steps to endosome membranes before ILV formation, and at least CD9, CD63, CD81, and CD82 are found in endosome and exosome membranes [1, 25].

Ceramides and their derived metabolites are organized in raft-based microdomains that interact with proteins, such as flotillins. The lipid-enriched structures are involved not only in endosomal membrane invagination for ILV formation but also in cargo loading. The selective cargo loading occurs during exosome biogenesis through tetraspaninsdependent and/or ESCRT-dependent mechanisms [1, 25]. Although, the biological cargo of exosomes varies widely according to their cell type of origin, they mainly consist of proteins, nucleic acids (particularly RNA) and lipids. More than 2400 different RNAs and, 4000 proteins were already identified and characterized in exosomes [1, 25].

Due to their endosomal origin, the exosomes are enriched in several proteins engaged in the biogenesis of MVBs, including clathrin, which can bind to hunting protein. Moreover, exosomes contain CD9, CD63, CD81, CD82, CD54, and CD11b tetraspanins, which serve as specific molecules. In addition, the exosomes contain heat shock proteins (HSP90, HSP70, and HSP60), which act as chaperones and play an essential role in cellular responses related to environmental stress. Besides this, exosomes also carry mRNA and a multitude of long non-coding (lnc) RNA and small RNA (particularly miRNA) that can be transferred into recipient cells, inducing cellular responses [1, 25–27].

The interaction of MVBs with actin and microtubules is essential for their transport to the plasma membrane. The translocation of MVB toward the plasma membrane depends on several molecules via the cytoskeleton. Rab GTPases such as RAB11, RAB27A/B, and RAB35 are mediators of selective sorting of MVB to the plasma membrane and exosome release. The MVBs are decorated with tethering protein complexes, such as HOPS and SNAREs, that mediate the fusion of these vesicles with the plasma membrane, The presence of tetraspanins and lysosomal-associated membrane proteins LAMP1 and LAMP2 in late endosomes also facilitate the fusion of MVB with the plasma membrane [1, 25].

After secretion, the exosomes will dock into the membrane of the target cells and activate signaling events or be internalized through specific receptor-ligand interactions. The transmembrane proteins present in the surface of exosomes (tetraspanins) can be recognized by signaling receptors in the target cells, resulting in activation of transduction pathways and modulation of the intracellular process without entering the target cells. Exosomes can merge with the target cells' plasma membrane, releasing its

cargo directly into the cytosol by a low pH-dependent mechanism. However, the main route for exosome uptake can occur by clathrin-mediated or caveolin-dependent endocytosis, and the presence of lipid rafts in the membrane facilitates the process [1, 25].

After internalization, exosomes are sorted into MVB with two possible fates: (i) to be released again to neighboring cells or (ii) to be degraded after fusion of LE/MVB with lysosomes [1, 25]. The uptake of exosomes by brain cells seems to be cell type-dependent. For instance, neurons and glial cells seem to uptake exosomes by clathrin-mediated endocytosis. Some neurons can also use specific receptors from the SNARE family, such as SNAP25, for exosome uptake. Interestingly, the uptake of exosomes seems to be a selective pathway. Exosomes derived from cortical neurons were primarily internalized by hippocampal neurons, whereas astrocytes and oligodendrocytes took up exosomes released by neuroblastoma cell line N2A. Exosomes derived from oligodendrocytes are mainly internalized by microglia but not by neurons or astrocytes. In addition, the uptake of exosomes was also more active in pre-synaptic regions, which might indicate that these vesicles use constitutive endocytosis processes at these regions for neuronal cell entrance [1, 25].

#### **2.1 Exosomes as a key mediator of HD pathophysiology**

Initially, exosomes were considered vehicles for the elimination of cellular components. However, current studies have provided evidence that exosomes play multiple physiological roles in the nervous system. Exosomes are released by neural cells, including neurons, astrocytes, microglia, and oligodendrocytes, playing essential physiological roles in neurogenesis, synaptic activity and plasticity, myelination, and protection and regeneration neurons after injury and disease. Thus, it is not surprising that exosomes mediate the pathogenesis of neurodegenerative disorders, such as HD. This is because the misfolded proteins related to these disorders can be selectively integrated into ILVs of MVBs, and subsequently released into the extracellular environment within exosomes [28].

In HD, cumulative evidence has demonstrated that exosomes are implicated in the physiopathology of HD, serving as a vehicle for the expanded polyglutamine tract of HTT RNA and protein (mHtt), as well as mHtt aggregates transport to different brain areas. Supporting this evidence, it was verified that exosomes could deliver expanded trinucleotide repeat RNAs among cells and facilitate the propagation of mHtt protein [29–32]. It was shown that the injection of fibroblast-derived exosomes from an HD patient into a newborn mouse brain ventricles triggered the manifestation of HD-related behavior and pathology [31]. Moreover, it is known that the Htt protein regulates anterograde and retrograde transport of endocytic vesicles by interacting with several mediators, such as α-adaptin, Hip1, Hip14, HAP1, HAP40, SH3GL3, clathrin, and dynamin [29, 30]. This process is coordinated by the phosphorylation of Htt, which serves as a molecular decision marker for the anterograde or retrograde direction of vesicle transport. Thus, while the Htt promotes axonal BDNF vesicle trafficking, mHtt interacts with HIP1 and dynactin, leading to de-railing of molecular motors from microtubules tracks and cessation of transport [33].

#### **3. Animal models for Huntington's disease**

Animal models for HD have been successfully used for more than three decades to identify pathways involved in HD pathology or for preclinical testing *Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease… DOI: http://dx.doi.org/10.5772/intechopen.102539*

of therapeutic strategies. These models are divided into (i) monogenetic and (ii) genetic murine models. However, none of these models can mimic the main feature of HD since no rodent model develops the chorea. For this reason, herein, we summarize the pros and cons of each animal model, considering their utility for preclinical test purposes [34–36].

#### **3.1 Monogenetic models**

Historically, monogenetic models have dominated the field of HD disease. These models are based on the use of toxins that typically induce cell death either by excitotoxic mechanism or by disruption of mitochondrial machinery. Among the excitotoxicity toxins used to obtain murine models for HD are quinolinic acid (QA) and kainic acid (KA). These neurotoxins induce cell death by binding to their cognate receptors, N-methyl-d-aspartic acid (NMDA) and non-NMDA, respectively, on striatal neurons. The QA or KA rat models exhibits motors (hyperkinesia, apomorphine-induced dystonia, and dyskinesia) and cognitive symptoms of HD (visuospatial deficits, procedural memory deficits, and poor memory recall). However, for various reasons, QA became the preferred excitotoxin for use in HD studies. The QA is formed from the metabolism of tryptophan via the kynurenine pathway, which is found in high quantities in the urine of rats that received a diet high in tryptophan. Interestingly, the tryptophan crosses the blood-brain barrier (BBB) using transporters shared by other neutral amino acids. In the brain, tryptophan is taken up by astrocytes, macrophages, microglia, and dendric cells and converted into kynurenine. In the presence of the enzymatic 3-hydroxyanthranilic acid oxygenase, a series of enzymatic reactions converts kynurenine to QA. Thus, the expected level of QA does not cause damage, but only small increases in QA levels cause toxicity. Moreover, it was verified that the administration of QA in the mouse models promotes the upregulation of Htt protein, linking the levels of this neurotoxin with HD pathogenesis. However, the QA is incapable of crossing the BBB. For this reason, the QA has been administrated directly within the brain [37–39].

Unlike the QA, the mitochondrial toxin 3-nitropropionic acid (3-NP) crosses the BBB and could be systemically administrated through intraperitoneal or subcutaneous injection [40–42].

The 3-NP is a plant (Indigofera endecapylla) and fungal (Aspergillus flavus, Astragalus, Arthrinium) toxin, which acts as an irreversible inhibitor of succinate dehydrogenase. It inhibits both the Krebs cycle and the mitochondrial complex II of the electron transport chain. The toxin also induces caspase-9 activation, which in turn requires the simultaneous presence of Apaf-1, cytochrome c, and ATP, suggesting that neuronal death may occur in the presence of intense ATP depletion. Moreover, the 3-NP induces oxidative and nitrate stress due to excessive ROS/RNS production and lack of the antioxidant system [40, 43–45]. Interesting, numerous studies demonstrated that the chronic systemic administration of 3-NP in rats impairs energy metabolism and results in striatal lesions, inducing a spectrum of HD-like pathology in rat striatum. In addition, in 1993, Beal et al. [41] showed that the 3-NP model causes selective striatal lesions characterized by the loss of medium spiny neurons (MSNs) and astroglial proliferation, replicating the histological and neurochemical features of HD. Although, the loss of MSNs in 3-NP rat models causes motor and cognitive symptoms analogous to those verified in HD, this model does not exhibit chorea.

However, the 3-NP model is capable of mimicking both hyperkinetic and hypokinetic symptoms of HD depending on the time course of administration. Thus, while the administration of 3-NP in two individual doses causes hyperkinetic movements analogous to those observed in early to mid-stage HD, the administration of more than four injections of 3-NP causes hypokinetic movements similar to those that appear in late-stage HD [40, 43–45]. Nevertheless, the response to the 3-NP changes according to the murine (CD1, C57BL/6, BALB/c, Sebster/Swiss and 129sEMS) or rat strain (Fischer, Lewis, and Wistar). In this sense, it is recognized that rats are most vulnerable to the toxic action of 3-NP treatment than mice. Fisher rats are the most susceptible to the 3-NP toxin but display significant variability in response to the toxin due to the difficulty of controlling damage caused by this toxin. In contrast, Lewis rats are less susceptible to 3-NP but respond more stably and consistently to 3-NP in behavioral alterations and lesions. Wistar and Sprague-Dawley rats are also sensitive to the 3-NP, developing lesions and behavioral modifications of extraordinary value for studying possible routes involved in HD and testing new therapeutic strategies. Although, the 3-NP model leads to a (i) massive cell death induced by the toxin, (ii) serving as a helpful model for (ii) analyzing and studying neuroprotective and (iii) neurorestorative therapies for HD patients, (iv) allowing to study the mechanisms involved in HD pathogenesis, including energy deregulations and ROS production, this model does not express the mHtt protein.

#### **3.2 Genetic models**

The genetic or transgenic animal models emerge as an alternative to nongenetic models since they express the mHtt protein [46, 47]. Transgenic models are divided into (i) those expressing transgenes with a truncated section of human HTT carrying the CAG repeats or full-length human HTT gene, and (ii) those with long CAG repeats replacing mouse Htt. Instability of the CAG repeat has been observed in many of the mouse models and was noted in the first HD model (R6 series). Although, different rodent models have been used to understand the biology of HD or employed in preclinical trials to investigate the therapeutic potential of products candidates to alleviate HD symptoms, they are limited in their ability to provide evidence of the effects of genetic modifiers of disease. In addition, there are many differences among the transgenic rodent models that can lead to different results, especially for preclinical trials.

In this sense, in two independent studies, it was demonstrated that a version of the R6/2 mouse with 90 CAG repeats (R6/2(CAG)90) shows earlier mHtt nuclear aggregation when compared to the R6/2 mouse with 200 CAG repeats (R6/2(CAG)200). Moreover, the R6/2(CAG)90 brains contain nuclear aggregates with a diffuse punctate appearance which remained partly detergent soluble, which correlated with the onset of transcriptional changes. In contrast, the R6/2(CAG)200 brains contain cytoplasmic aggregates that gave larger inclusion bodies related to behavioral changes. These data indicate that CAG length gives different phenotypes [48–50].

Several models encoding glutamine but using a mixed CAACAG rather than a pure CAG tract were developed to prevent germline and somatic expansion of CAG trinucleotide. An example of these models is the BACHD models with 97 glutamines encoded by a diverse CAACAG tract. These mice have five copies of the transgene integrated into their genome and express BACHD HTT, an estimated three-fold level of the transcript, and 1.5 to 2-fold protein level (mHtt).

*Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease… DOI: http://dx.doi.org/10.5772/intechopen.102539*

BACHD rats show string impairment in muscle endurance at 2 months of age. Altered circadian rhythmic and locomotor activity are also observed in these animals [51–53]. However, the BACHD model is not commercially available, difficult to access this model.

#### **4. Therapeutic cells: perspectives of HD treatment**

When discovered, stem cells—therapeutic cells gain exceptional attention due to their capacity to produce precursors and differentiated cells. Propose, therefore, was to use stem cells in tissue regeneration [26, 27]. Stem cells showed differentiation potential *in vitro* and *in vivo* (animal models). Thus, we know two principal types of stem cells: adult and pluripotent. Pluripotent cells are embryonic stem cells or induced pluripotent stem cells, which are adult stem cells reversed in stem cells similar to embryonic [28]. However, this chapter will focus on adult stem cells isolated from different tissues like bone marrow, adipose tissue, umbilical cord, and dental pulp. Adult stem cells, especially mesenchymal stem cells (MSC), differ from their pluripotent counterparts, and being more mature, they cannot differentiate *in vivo* into appropriate tissue. However, these cells present specific characteristics that are of great interest in treating neurodegenerative diseases.

MSC secretes a large number of biologically active molecules, growth factors, hormones, interleukins, etc. [29]. These biomolecules can be found in free form or contained in exosomes, which are recognized as a key component in paracrine regulation [1, 25]. These molecules provide beneficial effects on injured tissues. For example, they induce angiogenesis and tissue regeneration and inhibit fibrosis, apoptosis, and inflammation [30–32]. In addition, which is essential for HD disease, MSCs and MSC's secretomes provide neurogenic, neuroprotective, and synaptogenic effects [33]. They improve the abnormal dopamine transmission and inflammatory reaction in the transgenic HD model [34]. Animal models showed that they produce factors protecting retinal ganglion cells against glutamate excitotoxicity, neurotrophins expressed by MSCs inhibit pro-inflammatory cytokine secretion, MSCs fight oxidative stress and others [35, 36]. Due to the characteristics above, MSCs called medicinal signaling cells or simply therapeutic cells [37].

#### **4.1 Therapeutic cells**

Medicinal signaling cells (MSC) have been used in a variety of preclinical studies, which were focused on behavioral and memory outcomes, reduction of brain damage and minimization of striatal degeneration. "Native" MSC isolated from different adult tissues such as bone marrow, adipose tissue and umbilical cord were used in these studies. Due to their ability to adhere to plastic, MSC can be easily isolated and expanded *in vitro* [38]. They are isolated and cultured using similar protocols and culture medium reagents. However, different research groups usually introduce a few modifications in the protocol, which may affect the MSC quality and efficiency. MSC can differentiate *in vitro*, especially into mesoderm derivatives, but not *in vivo*. After isolation, these cells express similar markers and share similar morphological features. However, different MSC populations can be isolated from the same or different tissues. These populations differ in self-renewal, plasticity and therapeutic potential [29, 39, 40]. To standardize the concept of MSC used in different studies and by various scientific groups the International Society for Gene and Cell Therapy (ISGCT)

established the minimum criteria for defining these cells and populations isolated [41]. Despite MSC populations' heterogeneity, these cells share regenerative, antiapoptotic, anti-fibrotic anti-inflammatory, immunosuppressive, immunomodulatory, and angiogenic properties [42].

#### **4.2 Therapeutic cells in preclinical HD studies**

Stem cell-based therapies are important to reconstruct damaged brain areas in HD patients. These therapies have a dual role: stem cell paracrine action, stimulating local cell survival, and brain tissue regeneration through the production of new neurons from the intrinsic and likely from donor stem cells. Initially, preclinical studies were mainly focused on the neuroprotective function of MSC. Since these cells express a variety of neurotrophins and in particular brain-derived neurotrophic factor (BDNF), which is implicated in the survival of striatal neurons. BDNF expression is reduced in Huntington's disease (HD) contributing to striatal neurodegeneration.

Initially, preclinical studies mainly discussed the neuroprotective function of MSC. Since these cells express BDNF, which is implicated in the survival of striatal neurons, its expression is reduced in Huntington's disease (HD) contributing to striatal neurodegeneration. The regenerative approaches of MSC potentially can cause the (i) promotion of endogenous neuronal growth; (ii) amelioration of the synaptic connection from damaged neurons; (iii) decrease of apoptosis of endogenous neurons; (iv) reduction of the levels of free radicals; (v) immunomodulation. Our group widely discussed these MSC functions in animal models in previous publications (**Figure 1**) [43, 44].

Furthermore, MSC can transfer larger molecules and even organelles, therefore, their use as delivery vehicles for therapeutic RNA inhibition was suggested [45]. MSC can transfer larger molecules and even organelles; therefore, suggesting their use as delivery vehicles for therapeutic RNA inhibition [45]. *In vitro* model systems showed that MSC can transfer RNAi targeting both reporter genes and mutant huntingtin in neural cell lines. Thus, the flow cytometry assay demonstrated that MSC expressing shRNA antisense to GFP decreases expression of GFP when co-cultured with SH-SY5Y cells. Furthermore, these cells, which express shRNA antisense to HTT, decreased the levels of mutant HTT expressed in both U87 and SH-SY5Y target cells

#### **Figure 1.**

*Schematic representation of the paracrine and cellular mechanism of MSC action observed in pre-clinical studies in Huntington and other degenerative diseases. Paracrine action mainly includes neurotrophins and other soluble factors, including growth factors, small molecules, and cytokines, providing signals to cells and resulting in different cell actions such as survival, proliferation, and differentiation. The paracrine factors are secreted directly into the intercellular matrix or included in extracellular vesicles (exosomes) before secretion. The cellular effect includes mitochondria transfer and MSC mediated autophagy. MSC acting by paracrine and cellular mechanisms showed significant therapeutic potential.*

*Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease… DOI: http://dx.doi.org/10.5772/intechopen.102539*

as analyzed by Western blot and densitometry. These results are encouraging for expanding the therapeutic abilities of both RNAi and MSC for future treatments of Huntington's disease [46–48].

In addition, more recent findings suggest the potential therapeutic effect of MSC on different pathophysiological aspects of HD, such as (i) mitochondrial dysfunction; (ii) transcriptional dysregulation [49, 50]; (iii) altered axonal transport of critical factors [51, 52]; (iv) disrupted calcium signaling [53, 54]; (v) abnormal protein interactions [55]; (vi) impaired autophagy [56, 57]. However, here we will focus our review on HD mitochondrial dysfunction and MSC mitochondria transfer.

**Figure 1** combines the well-known paracrine mechanism of MSC action and a novel cellular mechanism mediated by mitochondria transfer and autophagy. Both, paracrine and cellular, mechanisms provide clinical, cellular and molecular benefits in HD [43, 44, 49–59]. The complex mechanisms of MSC action and her multi-target orientation are the unique biological tool that could act on multiple pathophysiological aspects of HD cited above.

#### *4.2.1 MSC and mitochondrial dysfunction in HD*

Mitochondria roles in neurons differ from only a cell power source. Mitochondria are also dynamic organelles that fragment and fuse to achieve a maximal bioenergetics action. They are transported along microtubules, regulated intracellular calcium homeostasis through the interaction with the endoplasmic reticulum. In addition, they produce free radicals and participate in cell apoptosis [60]. These activities have been demonstrated to be changed in HD, potentially contributing to neuronal dysfunction in early pre-symptomatic HD phases. Thus, a polyglutamine-expansion disorder that primarily affects the striatum and the cerebral cortex has been described as mitochondrial dysfunction, an early pathological mechanism presenting selective HD neurodegeneration [61, 62]. One of the hallmarks of HD is an altered mitochondrial morphology that can be seen in different cell types and neurons, which are characterized by increased mitochondrial fragmentation [63]. The cells with altered mitochondrial morphology in HD cells showed a decrease in electron transport chain activity, oxygen consumption, Ca2+ buffering, and decreased ATP and NAD+ production [64]. It has been suggested that mitochondrial abnormalities can significantly affect MSNs due to the high-energy demand of this neuronal subtype [65]. Therefore, the mitochondria are a central regulatory organelle in HD-affected neurons.

In addition, mitochondria act as a reservoir for pro-apoptotic factors, thus regulating cell death. The mitochondrial permeability transition pore (mPTP) is opened due to mitochondrial dysfunction, Ca2+ overload, and accumulation of reactive oxygen species (ROS). The transition pore opening initiates the intrinsic apoptotic pathway, which is connected with the mitochondrial outer membrane permeabilization, awakening cytochrome c release, and activation of caspase-3 [66, 67]. Bcl-2 inhibits the activation of proapoptotic factors such as Bcl-2-associated X protein (Bax) and Bcl-2-associated K protein (Bak), thus suppressing the release of cytochrome c from mitochondria. The Bax/Bcl-2 ratio imbalance often occurs during the process of apoptosis [68]. MSC mitochondrial transfer through regulation of the balance of Bax/ Bcl-2 and reduction of the expression of caspase-3 can reduce apoptosis levels and promote cell viability in recipient cells [69, 70].

Recent studies have demonstrated that MSCs have the potential to transfer the defective mitochondria between MSCs and aging cells [71]. For the first time, the MSC mitochondria transfer was shown in A549 cells with mtDNA deletions after

their co-culture with human MSCs. This work demonstrated the recovery of function by mitochondrial activities such as increased oxygen consumption, membrane potential, and intracellular ATP levels [72].

It is worth mentioning that the transfer of dysfunctional mitochondria from damaged cells to MSC also can occur. Gozzelino et al. showed that mitochondria released from damaged cardiomyocytes or endothelial cells could be "swallowed" by MSCs. This event rigger increases the expression of Heme oxygenase-1 (HO-1), a protein that protects against programmed cell death, and increases mitochondria in MSCs, which in turn induces an adaptive reparative response [73, 74].

Fluorescence microscopy studies revealed MSC mitochondria transfer in astrocytes and neuron-like pheochromocytoma cells. MSC mitochondria transfer to astrocytes was more efficient when the astrocytes were subjected to ischemic damage associated with elevated ROS levels. The ROS accumulation in normal aging or disease leads to increasing the rate of mitophagy and decreasing the level of mitochondrial biogenesis, which reduces mitochondrial mass [75]. Such mitochondria transport re-established the bioenergetics of the recipient cells and stimulated their proliferation. Furthermore, the authors showed that MSCs mitochondria transferability may be enhanced by upregulation of Miro1 (adaptor protein participating in mitochondria moving along microtubules [76] therefore, this study showed that mitochondrial impairment in differentiated cells can be restored after MSC healthy mitochondria transfer and this approach may serve as a promising treatment for neurological diseases [77].

#### *4.2.2 MSC mitochondria transfer and inflammation*

Tissue injury or degeneration is usually followed by inflammation, which is a driving force for mitochondrial transfer. In HD, massive neuroinflammation in the striatum and caudate nucleus are already present before patients develop any symptoms [21, 78, 79]. The therapeutic effects of MSC are mediated mainly by its secretome/exosomes since in response to a combination of molecules present in the inflamed microenvironment, these cells undergo a process activation or "licensing," acquiring an anti-inflammatory phenotype and producing large amounts of immunomodulation factors, growth factors and specific chemoattractants, being able to modulate significantly innate and adaptive immune cells [38, 80].

The MSCs secreted cytokines that immunomodulate various immune cells, such as T cells, B cells, natural killer cells, and macrophages [81]. Recent studies demonstrated that between MSCs and immune cells MSC mitochondrial transfer can occur, such influencing the functions of the immune cells. Jackson et al. showed MSC mitochondria transfer occurs in an acute respiratory distress syndrome (ARDS) model. MSC provides mitochondria to host macrophages, thus enhancing the phagocytic capacity and bioenergetics of macrophages. This MSC mitochondria transfer leads to improved clearance of pathogenic bacteria [82]. Using the same model Morrison et al. showed that MSC exosomes mediated transfer of mitochondria, which can induce monocyte-derived macrophages to differentiate to an M2 phenotype with a high phagocytic capacity [58]. In addition, MSC mitochondria transfer regulates T cell differentiation. Some authors reported that when healthy donor-derived bone marrow-derived MSC (BMMSC) is cocultured with primary Th17 effector cells, the mitochondrial transfer occurs, increasing respiration in recipient Th17 cells [59].

HD demonstrates typical cellular and molecular features of inflammation, such as cytokine expression and microglia activation. However, no immune cell infiltration from the bloodstream was observed [83, 84]. Nevertheless, HD is characterized by a

#### *Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease… DOI: http://dx.doi.org/10.5772/intechopen.102539*

chronic increase of systemic pro-inflammatory cytokine production. Microglia and astrocytes are non-neuronal cells in the brain that participate in tissue homeostasis and support neuronal function. Under pathologic conditions, these cells become 'activated.' They start to produce numerous mediators promoting inflammation. These cells change their morphology and, can divide, thus increasing cell numbers, an event named 'gliosis.' Recent studies suggest that cell-autonomous pro-inflammatory activation of microglia occurs due to the expression of mutant HTT, thus contributing to the progression of HD pathogenesis [21].

MSC's metabolic state is characterized by the balance between the intrinsic necessities of the cell and limitations imposed by extrinsic conditions. Under pathogenic conditions or inflammation, MSCs respond to reactive oxygen species (ROS), damage-associated molecular patterns (DAMPs), damaged mitochondria, and mitochondrial products, thus transferring their mitochondria to damaged cells. MSC therapies can protect the potentially damaged cells by regulating cellular metabolism in injured tissues, modulating ROS and endogenous MSCs.

Furthermore, to treat such complex diseases like Huntington's, we should develop new complex therapies acting on multiple targets. MSC, due to the wide range de therapeutic molecules they produced and the different mechanisms they used to fight the disease, these cells are a good candidate for the new class of such therapeutics.

#### **5. Cell-free therapy: novel perspectives for the treatment of HD**

For a long, it was considered that the therapeutic effects of the stem cells were associated with the replacement of dead cells [73, 74]. However, in a model of kidney injury caused by the injection of toxic doses of glycerol, it was verified that transplanted stem cells remain in the injury site for up few days and, subsequently, are not found in the tissue [73, 75, 76]. These data provide evidence that the therapeutic potential of MSCs is mediated by trophic factors naturally produced and secreted by these cells in an accessible form or into EVs [1]. However, whereas the bioactive molecules present in the extracellular medium are subjected to rapid hydrolysis and oxidative effects, the biomolecules present in EVs are more stable [73]. For this reason, the EVs (particularly exosomes) have been biotechnology explored in a novel therapeutic approach known as cell-free therapy [26, 77, 78].

Cell-free therapy possesses different advantages when compared with cell-based treatment. Among these advantages are: [1] EVs can be easily prepared, stored for a relatively long period without any toxic cry preservative such as dimethylsulphoxide (DMSO) and transported; [2] therapeutic application of exosomes have been demonstrated to be well tolerated; [3] the use of EVs instead of whole cells avoids possible complications associated with pulmonary embolism after intravenous infusion of MSCs; [4] avoids the risk of unlimited cell growth and tumor formation since EVs are not dividing; [5] exosomes from MSCs, and epithelial cells do not induce toxicity when repeatedly injected; [6] EV may be isolated from unmodified or genetically modified human stem cells; [7] evaluation of culture medium for safety and efficacy is much simpler and analogous to conventional pharmaceutical agents [1, 73, 79–82]. Further, the cell-free therapy allows biotechnologically exploring the use of the culture medium, which is generally discarded as a byproduct of the in vitro expansion of MSCs. This is because this culture medium—also termed conditioned medium (CM) [79]—is an essential source of bioactive molecules, which can find in an accessible form or an extracellular vesicle (EVs) [1].

#### **5.1 Perspectives and challenges in cell-free therapy**

Although, different strategies have been successfully used to isolate exosomes, they represent the main obstacle to the therapeutic application of EV since these procedures are time-consuming and generally provide few quantities of EVs [1, 73]. However, novel methodologies have been proposed to solve these problems. Based on this, we aimed to summarize the pros and cons of each available method for isolating exosomes.

Ultracentrifugation (UC) and commercial kit rooted in polymer-based precipitation are the most well-established and standard methods used for isolating exosomes [74], being adopted as a strategy in about 81% of researches [78]. Ultracentrifugationbased methods can be divided into two major types of techniques according to the separation mechanism: (i) differential ultracentrifugation and (ii) density gradient ultracentrifugation [78]. For both methods, death cells, cellular debris, and large EVs (>200 nm) are separated using low centrifugal forces (300–2000 × *g*) for 10–30 min at room temperature, as verified in the most protocol, as already revised by us [1]. An additional filtration step using a 0.22–0.45 μm-membrane filter can increase the exosome purity. In differential ultracentrifugation, the particles are separated using a serial of differential centrifugal forces (100,000–120,000 × *g*) and time (70 min to 12 h). At the end of the process, the pellet of exosomes is washed with phosphate saline buffer (PBS) or 0.9% NaCl solution to remove remaining proteins co-isolated with the EVs. Differential ultracentrifugation provides pure EVs for both scientific and clinical purposes. However, the majorities of UC-based proposed methods are laborious, time-consuming, and unsuitable for mass-scale EV production, making it difficult for therapeutics [1]. In density gradient, ultracentrifugation (DGUC) is employed as a sucrose density gradient, which reduces the destructive effects of centrifugal force on exosomes [58, 78]. According to the exosome buoyant density in aqueous sucrose (1.10–1.20 g/mL), the exosomes can be easily isolated [59, 78]. Although, this method provides the highest efficiency for exosome purification, its suitability for clinical purposes is questionable due to the difficulty in upscaling and automating the process [83, 84]. Moreover, the wash step is mandatory for this method to remove eventual residues of CsCl or sucrose used to obtain the gradient density.

Another strategy commonly employed to isolate exosomes is coprecipitation. This method uses polymers, such as polyethylene glycol (PEG) 6000 or 8000, which can coprecipitate with hydrophobic proteins and lipid molecules present in exosome membranes [78]. Although, most simple and less expensive than the methods based on ultracentrifugation, the isolation using coprecipitation is not scalable, limiting its use for therapeutic purposes. Moreover, this technique requires sample incubation with the polymers for a long time (generally 12–16 h) [1].

The differential expression of specific surface biomarkers, such as CD9, CD63, and CD83, also provides an excellent opportunity to develop immunoaffinity-capturebased techniques for exosomes isolation using modified magnetic beads or microchannels surfaces with specific antibodies [1, 78]. Although, this technique allows isolating only exosomes, it works with few volumes, limiting its use for therapeutic purposes, which require scalable methods. Moreover, this method generally requires a pre-enrichment step, which is commonly performed using commercial kits based on coprecipitation, resulting in PEG contamination [1].

Another strategy used to isolate exosomes is the size-based isolation technique. This technique can be based on sequential filtration, size-exclusion chromatography (SEC), and size-dependent microfluids. These methods allow isolating the EVs

#### *Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease… DOI: http://dx.doi.org/10.5772/intechopen.102539*

according to their size [78]. EVs are separated using membrane filters with different size or molecular weight exclusion limits in sequential filtration. First, the CM is filtered using a 0.22 μm membrane filter. Then, proteins with a 500 kDa molecular weight are purified using a dialysis bag. Finally, the samples are filtered with a 100 nm membrane filter [78]. The SEC is based on particle size filtration through a porous stationary phase composed of spherical gel beads with pores of specific size [78]. Large particles are eluted when the sample passes through the stationary phase, whereas small particles pass through the pores [78]. The size-dependent microfluidics uses a viscoelastic microfluidics device, composed of a microchannel, two inlets, and three inlets, to fractionate exosomes from other types of EVs [78]. These techniques are faster than those based on ultracentrifugation and do not require special equipment. Moreover, they avoid PEG contamination, frequently observed in coprecipitation-based methods. However, the size-based isolation techniques are relatively expensive and cannot separate exosomes from other EVs, requiring additional steps for exosome purification [1].

#### **5.2 Cell-free therapy for the treatment of HD**

Due to their ability to cross the blood-brain barrier and biocompatibility, exosomes are promising therapeutic drug carriers into the CNS. In HD, exosomes are exceptionally efficient in delivering specific microRNAs (miRs), short non-coding RNAs of about 22 nucleotides that regulate gene expression by suppressing the translation of mRNA, which are found deregulated in HD patients.

In this sense, several miRs had already been identified as deregulated in HD, including the miR-124, which was found downregulated in HD patients [85]. The decreased expression of miR-124 increases the levels of its target gene (REST), which acts as a repressor of BDNF [85]. By contrast, the expression of miR-124 induces adult neurogenesis in the subventricular zone (SVZ) and regulates the cell cycle in striatal neurons. Considering that the HD striatum exhibits decreased neurogenesis, which leads to brain atrophy, it was hypothesized that the delivery of miR-124 may be a feasible way to induce neural regeneration. However, naked miRs are vulnerable to degradation.

In this regard, exosomes emerge as candidates for the miR-124 delivery to recipient cells. Based on this, Lee et al. [85] injected exosomes derived from HEK 293 cells overexpressing miR-124 within (Exo-124) the striatum of 6-week-old R6/2 transgenic mice. Using ex vivo imaging, the authors demonstrated the presence and maintenance of the exosomes within the striatum even after one week later the Exo-124 administration. Furthermore, it was verified that Exo-124-treated R6/2 mice exhibited slightly higher levels of miR-124 when compared to the non-treated mice (control). However, no statistically significant differences between the treated and control mice were reported. By contrast, the Exo-124-treated R6/2 mice exhibited lower levels of REST protein concerning the control. Although, the study had provided a proof of concept for exosome-based delivery of miRNAs to the brain, the therapeutic efficacy of Exo-124 was modest, suggesting the need to increase the dose of miRNAs packed in the exosomes or to combine this miRNA with other candidate miRNAs such as miR-9, miR-22, miR-125b, miR-146a, miR-150, and miR-214.

In this sense, the exosomes derived from MSCs can be considered an important source of these miRs and other mRNAs and proteins deregulated in HD physiopathology. Supporting this, Lee et al. [86] showed that exosomes derived from an adipose-derived stem cell (ASC-exo) decreased mHtt aggregates in R6/2 mice-derived neuronal cells through the upregulation of PGC-1, phospho-CREB.

#### **6. Conclusion**

In this review, we demonstrated that Huntington's disease is devastating and affects brain cells and the organism as a whole. Although, the main cause of HD patients' death is medium spiny neurons, many specific events occur at presymptomatic and symptomatic HD phases. Currently, Huntington's chorea is in the focus of pharmaceutical companies, producing drugs able to combat this HD symptom. However, these drugs are always not possible to delay the disease and present moderate to severe side effects.

In contrast to conventional drugs, MSC is safe, and they did not present any side effects as shown in multiple clinical trials. MSC showed therapeutic potential distinct from, for example, small molecules and biologics. Cells are deposited multiple drugs, they can sense diverse signals, migrate to specific sites in the body, make decisions, and carry out complex responses inside one specific tissue environment.

Our knowledge about the biology and therapeutic potential of these cells is still minimal; however, as demonstrated by scientific literature, these cells and their derivatives as exosomes and mitochondria have tremendous therapeutic potential. Pre-clinical studies provided evidence about the paracrine effect of these cells' such as regenerative, anti-apoptotic, anti-fibrotic anti-inflammatory, immunosuppressive, immunomodulatory, and angiogenic.

More recently, the potential effect of MSC against different pathophysiological aspects of HD, such as mitochondrial dysfunction; transcriptional dysregulation; altered axonal transport of critical factors; disrupted calcium signaling; abnormal protein interactions, and impaired autophagy, has been demonstrated.

This review tries to provide insight into cellular and cell-free technologies from the exact cellular origin. These cell and cell-free products may share similar features and present specific characteristics, as demonstrated for MSC, exosomes, and mitochondria. We tried to clarify that these products aim at different cellular targets or molecular pathways involved in Huntington's disease. Therefore, we should study how to use these new therapeutics, which can delay or even stop neurodegenerative devasting diseases.

*Advances in Cellular and Cell-Free Therapy Medicinal Products for Huntington Disease… DOI: http://dx.doi.org/10.5772/intechopen.102539*

#### **Author details**

Irina Kerkis1 \*, Rodrigo Pinheiro Araldi2 , Cristiane Valverde Wenceslau3 and Thais Biude Mendes1

1 Genetics Laboratory, Butantan Institute, São Paulo, Brazil

2 Federal University of São Paulo, Cellavita Pesquisas Científicas, São Paulo, Brazil

3 Cellavita Pesquisas Científicas, Valinhos, SP, Brazil

\*Address all correspondence to: irina.kerkis@butantan.gov.br

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 7**

## Nrf2 as a Potential Therapeutic Target for Treatment of Huntington's Disease

*Saravanan Jayaram, Praveen Thaggikuppe Krishnamurthy, Meghana Joshi and Vishnu Kumar*

#### **Abstract**

Oxidative stress-induced neuronal damage plays a significant role in pathogenesis of several neuro-degenerative disorders including Huntington's disease. In Huntington's disease, oxidative stress-induced neuronal damage is reported to be mediated by PGC-1α and microglial cells. This development led to various clinical trials that tested the efficacy of several exogenous antioxidants such as vitamin E, vitamin C, etc. to prevent the oxidative stress-induced cell damage in several neuro-degenerative disorders. But these randomized clinical trials did not find any significant beneficial effects of exogenous antioxidants in neuro-degenerative disorders. This forced scientists to search endogenous targets that would enhance the production of antioxidants. Nrf2 is one such ideal target that increases the transcription of genes involved in production of antioxidants. Nrf2 is a transcription factor that controls the expression of antioxidant genes that defend cells against oxidative stress. This chapter focuses on the role of oxidative stress in Huntington's disease and explores the therapeutic benefits of Nrf2 activators.

**Keywords:** Nrf2, oxidative stress, Keap 1, Huntington's disease

#### **1. Introduction**

Huntington's disease is an inherited autosomal dominant neurodegenerative disorder characterized by a triad of psychiatric, cognitive and motor symptoms. Every human has two copies of the huntingtin gene (HTT) that codes for huntingtin protein (htt) [1]. The exact functions of huntingtin protein still remain unclear, but it is believed to be involved in the development and formation of cortical and striatal excitatory synapses, surveillance and biogenesis of mitochondrial function, activation of glial cells, upregulation of the expression of brain derived neurotrophic factor, balance of histone acetylation and deacetylation, axonal transport, regulation of signaling pathways and autophagy [2–5]. The HTT gene, also called as *IT15* ('interesting transcript 15') gene, is located on the short arm (p) of chromosome number 4 at 4p16.3 [6]. The 5′ end of the HTT gene comprises a three-nucleotide base sequence, cytosine-adenine-guanine (CAG), which is repeated multiple times

and codes for the amino acid glutamine. The number of CAG repetitions in a healthy individual varies between 7 and 35. This region of CAG repeats called the trinucleotide repeats varies in length from person to person and may vary in length from generation to generation. The length of the CAG region in the HTT gene is increased due to a hereditary mutation in the HTT gene. The length of the CAG repeats ranges between 36 and 120 in people with HD. Individuals with CAG repeats between 36 and 39 may or may not develop signs of Huntington's disease, whereas individuals with 40 or more repeats always display the characteristic signs and symptoms of Huntington's disease. This expansion of CAG repeats due to the inherited mutation in the HTT gene leads to the production of an unusually long version of huntingtin protein (mHtt) [7]. The mutant huntingtin protein is highly susceptible to cleavage, and this results in the creation of shorter fragments containing polyglutamine expansion. These protein fragments are susceptible to misfolding and aggregation, producing fibrillar aggregates in which non-native polyglutamine strands from different proteins are bonded together by hydrogen bonds. These aggregates share the same basic β-amyloid structure seen in other protein deposition diseases [8]. One of the pathways through which mHtt causes cell death is mitochondrial dysfunction [9]. The impairment of mitochondrial electron transport chain by mHtt increases the level of free radicals and oxidative stress [10]. Following the irrefutable role of oxidative stress and associated neuroinflammation in the pathogenesis of neurodegenerative disorders including Huntington's disease, several exogenous antioxidants were expected to have protective and therapeutic benefits in these degenerative diseases of the brain. But largescale randomized clinical trials failed to establish any conclusive data to support the hypothesis that exogenous antioxidants could possess neuroprotective or therapeutic benefit in neurodegenerative diseases. Nevertheless, these clinical trials do not refute the fact that oxidative stress and associated neuroinflammation play a key role in the pathogenesis of neurodegenerative diseases. So, it appears logical to stimulate endogenous targets that would reduce oxidative stress and associated neuroinflammation in diseases associated with oxidative stress. Nuclear factor-erythroid-2-related factor 2 (Nrf2) is one such target. Nrf2 is a transcription factor present in the cytoplasm of cells [11]. By upregulating the expression of almost 200 cytoprotective genes, Nrf2 assists cells adapt to inflammation and oxidative stress. Keap1, a repressor protein, controls the level of Nrf2 in the cytoplasm. Keap1 is a cysteine-rich protein that binds to Nrf2 and activates the ubiquitin-proteasome pathway to degrade Nrf2 in the cytoplasm. During oxidative stress, the degradation of Nrf2 by Keap1 is blocked. This results in an increased level of Nrf2 in the cytoplasm of cells. The free Nrf2 moves into the nucleus of cell and increases the transcription of many genes that code for detoxification enzymes and cytoprotective proteins [12]. The potential of Nrf2 to negate oxidative stress and associated neuroinflammation makes it an effective target in the prevention and treatment of Huntington's disease. The focus of this chapter is to review the role of oxidative stress and associated neuroinflammation in Huntington's disease and the potential beneficial effects of Nrf2 activators in Huntington's disease.

#### **2. Oxidative stress in Huntington's disease**

Reactive oxygen species (ROS) are highly reactive molecules or molecular fragments formed from oxygen through biochemical reactions that occur during cellular respiration. Reactive oxygen species and reactive nitrogen species exert both beneficial and harmful effects on the living systems [13]. At low to moderate cellular levels, free radicals play a

*Nrf2 as a Potential Therapeutic Target for Treatment of Huntington's Disease DOI: http://dx.doi.org/10.5772/intechopen.103177*

physiological role in destroying the invading pathogenic microorganisms, regulation of signaling pathways and induction of mitogenic response. At high cellular concentrations, free radicals exert a deleterious effect on lipids, proteins, nucleic acids and other cellular structures [14]. In many pathological conditions including Huntington's disease, an increase in the level of free radicals and cellular damage due to free radicals is observed. But it still remains unclear whether free radical induced damage in pathological conditions is a cause or downstream consequence of the underlying pathological process.

#### **3. Antioxidants in Huntington's disease**

Antioxidants are substances that are capable of scavenging the free radicals and thereby counteracting the free radical induced oxidative damage and inflammation. There are two classes of antioxidants—enzymatic and non-enzymatic antioxidants. Enzymatic antioxidants include superoxide dismutase (SOD), glutathione peroxidase (Gpx) and catalase (CAT). Ascorbic acid (Vitamin C), carotenoids, α-tocopherol, glutathione (GSH), retinoic acid and flavonoids are examples of non-enzymatic antioxidants. Many of these antioxidants have proven their efficacy in several in vitro and animal models but not in randomized clinical trials. The following table summarizes the findings of the studies that evaluated the efficacy of antioxidants in Huntington's disease (**Table 1**).



#### **Table 1.**

*List of antioxidants studied in Huntington's disease.*

#### **4. PGC1α-mediated oxidative stress in Huntington's disease**

The peroxisome proliferator-activated receptor co-activator-1α (PGC1α) is a transcriptional regulator present in tissues that have a high energy demand such as the brain, liver, cardiomyocytes, adipocytes, skeletal muscles and the kidneys [27, 28]. PGC1α plays a key role in mitochondrial biogenesis, metabolism, peroxisomal remodeling and detoxification of reactive oxygen species [29]. An important and effective mechanism through which PGC1α confers neuroprotection is by its antioxidant activity. Oxidative stress is suppressed by PGC1α by inducing the formation of antioxidant enzymes such as SOD1, SOD2, Gpx-1 and mitochondrial uncoupling proteins [30]. PGC1α also regulates the expression of SIRT3 in mitochondria and SIRT3 in turn activates SOD2 via deacetylation and reduces the level of reactive oxygen species [31, 32]. In short, PGC1α plays a key role in improving mitochondrial function, biogenesis, expression of antioxidant enzymes and amelioration of oxidative stress induced neuronal damage. A deficiency of PGC1α in the brain affects the integrity of mitochondrial structure and increases the level of reactive oxygen species leading to cellular senescence and disorders related to aging [33]. PGC1α expression has been found to be disturbed in neurodegenerative diseases such as Huntington's disease, Parkinson's disease and multiple sclerosis, resulting in mitochondrial abnormalities and elevated ROS levels [34–36]. Therapeutic agents that can activate endogenous antioxidant systems such as Nrf2/ARE pathway leading to increased expression of antioxidant enzymes hold great promise as neuroprotective agents in Huntington's disease. Transcriptional modification of Nrf2 pathway, therefore, is considered an excellent approach to counteract the oxidative stress-mediated neuronal damage in Huntington's disease.

In Huntington's disease, mHtt causes an increase in oxidative stress mediated by PGC1α. mHtt binds to the promoter sequence of PGC1α and reduces the transcriptional level of PGC1α [37]. mHtt also supresses the expression of mitochondrial uncoupling proteins and antioxidant enzymes by direct binding and inactivation of PGC1α [30].

*Nrf2 as a Potential Therapeutic Target for Treatment of Huntington's Disease DOI: http://dx.doi.org/10.5772/intechopen.103177*

**Figure 1.** *PGC1α-mediated oxidative stress in Huntington's disease.*

mHtt disrupts the balance between mitochondrial fission-fusion process by interfering with the function of Drp1 [38]. mHtt induces leakage of calcium ions through the calcium channel ryanodine receptors, further resulting in opening of the mitochondrial permeability transition pore (mPTP), which contributes to mitochondrial oxidative stress [39]. PGC-1α transcription and activity impact the enzyme system that combats reactive oxygen species (ROS). As a result, ROS defense genes such as SOD1, SOD2 and glutathione peroxidase (GPx1) are downregulated, resulting in increased oxidative damage and neuronal death in Huntington's disease (**Figure 1**).

#### **5. Microgliosis, oxidative stress and associated neuroinflammation in Huntington's disease**

Microglial cells are the resident immune cells of the central nervous system (CNS) and make up between 10 and 15% of all glial cells in the brain. Microglial cells develop from pro-erythromyeloid progenitor cells in the yolk sac during embryogenesis and go through three stages of development: early, pre and adult microglia. They then migrate into the CNS, using white matter tracts as guiding structures, until the blood-brain barrier is formed. Microglial cells, once inside the CNS, multiply and disseminate evenly to various regions of the brain and maintain a constant population through self-renewal [40]. Microglial cells have numerous slender and elongated processes branching from the small oval-shaped body, which makes them appear ramified. However, when the brain is exposed to potential dangers such as infection, trauma or other factors, these cells lose their branches and take on an amoeboid shape. Microglial cells in the CNS are involved in the establishment and remodeling of neural circuits, protection and repair of the brain, phagocytosis of apoptotic cells in the developing brain, organization of synapses, neurogenesis, control of myelin turnover, control of neuronal excitability and programmed cell death [41, 42]. Homeostatic microglial cells interact with practically every component of the CNS to maintain homeostasis, development and repair by continuously monitoring ongoing actions in the brain. When microglial cells detect a threat to the CNS's homeostasis, they become activated and produce a variety of cytokines and pro-inflammatory mediators to neutralize the threat. Although this acute response of microglial cells is protective and necessary for maintaining CNS homeostasis, over-activation of microglial cells has been linked to a variety of neurodegenerative diseases [43]. Microglial cells, after activation, release pro-inflammatory mediators and several

cytokines that lead to severe oxidative stress and neuroinflammation. According to recent research, activated microglial cells release cytokines and pro-inflammatory mediators, which are the main contributors to neuroinflammation in neurodegenerative diseases [44–46].

A significant increase in microgliosis has been observed in the autopsied brains of the patients with Huntington's disease compared with the controls. Accumulation of glial cells has been observed in all grades of Huntington's disease, and the density of microglial cells finely correlates with the degree of neuronal loss [47, 48]. A significant activation of microglial cells in the regions of the brain affected by Huntington's disease has been reported in an *in vivo* positron emission tomography [49, 50]. In Huntington's disease, microglial cells are activated by mHtt protein, and activated glial cells cause degeneration of neurons in the striatal region of the brain by releasing a variety of proinflammatory cytokines and free radicals [51, 52].

#### **6. Structure of Nrf2**

The Nrf2 protein contains 6 highly conserved regions called Nrf2-ECH (Neh) homology domains. The first domain (Neh1) carries the CNC-bZIP domain that mediates heterodimerization with Maf (musculoaponeurotic fibrosarcoma oncogene homolog) proteins. Two degrons called DLG and ETGF, present in the second domain (Neh2) specifically bind to Keap1 protein that leads to degradation of Nrf2 [53]. The third domain (Neh3) is considered to improve the stability of Nrf2 and also acts as the transactivation domain. The fourth (Neh4) and fifth (Neh5) domains of Nrf2 also act as transactivation domains by binding to cAMP response Element Binding Protein (CREB). The sixth domain (Neh6) plays a role in the degradation of Nrf2 by E3 ubiquitin ligase [54].

#### **7. Structure of Keap1**

'Kelch-like ECH-associated protein 1(Keap 1) is a protein that interacts with Nrf2 leading to degradation of Nrf2. Keap1 is a protein of BTB-Kelch family, composed of four domains. The N-terminal domain—Broad complex, Tramtrack and Bric a Bric (BTB) control homodimerization of Keap 1 and its interaction with cul3. This domain also contains Cys-151 residue that plays an important role in sensing oxidative stress. The second domain called the intervening region (IVR) domain contains Cys-273 and Cys-288. These two cysteine residues play a secondary role in sensing oxidative stress. The third domain, double glycine repeat (DGR) and the fourth domain, C-terminal region (CTR) binds to ETGE and DLG motifs of Nrf2 and causing its degradation (**Figure 2**) [54].

#### **8. Mechanism of action of Nrf2 activators**

Nrf2 is a transcription factor that regulates the expression of many antioxidant enzymes, phase I and phase II enzymes and several anti-inflammatory mediators. Nrf2 acts as an important defense mechanism in the neurons and glial cells against oxidative stress, neuroinflammation and other pathological insults. Nrf2 dysregulation has been reported in many oxidative-stress-related diseases such as Huntington's

*Nrf2 as a Potential Therapeutic Target for Treatment of Huntington's Disease DOI: http://dx.doi.org/10.5772/intechopen.103177*

*Structure of Nrf2 and Keap1.*

**Figure 3.** *Mechanism of action of Nrf2 activators.*

disease [54]. This makes Nrf2 activators excellent agents to increase antioxidant capacity, decrease neuroinflammation and alleviate pathology in Huntington's disease (**Figure 3**).

Nuclear factor E2-related factor (Nrf2) is a transcription factor composed of 605 amino acids that controls the expression of as many as 200 genes [55–57]. The proteins encoded by Nrf2 genes are control several functions such as anti-inflammation, antioxidant defense, apoptosis, detoxification, removal of oxidized protein by proteasome and DNA repair [58–60]. In physiological conditions, the half-life of Nrf2 is very short (<20 minutes) as it is continuously degraded by Kelch-like ECH-associated protein 1 (Keap 1) [61]. Keap 1 is a regulatory protein that regulates the levels of Nrf2 in the cytoplasm of cell. In basal conditions, the Neh2 domain of Nrf2 binds to the β-barrel structure of Keap-1. This is followed by binding of Cullin-3 to Keap-1-Nrf2

complex, and this results in the formation of ubiquitin 3-ligase complex. The ubiquitin 3-ligase complex bins to many ubiquitin molecules resulting in polyubiquitination of Nrf2, which serves as a signal for proteasomal degradation [62]. Keap-1 contain a lot of cysteines in their structure and the free sulfhydryl (∙SH) of cysteine help keap-1 to act as sensors of oxidative stress. During oxidative stress, electrophiles alkylate keap-1 and prevent keap-1 from degrading Nrf2. This leads to accumulation of recently synthesized Nrf2 that increases the antioxidant potential by promoting the transcription of antioxidant and detoxifying genes. In an alternative pathway, Nrf2 is degraded by phosphorylation by glycogen synthase kinase 3β (GSK3β). This degradation of Nrf2 by GSK3β is also blocked by elevated levels of oxidants that leads to accumulation of freshly synthesized Nrf2 [63]. In another pathway, Keap-1 itself is degraded by p62. In this pathway, p62 is phosphorylated by TANK-binding kinase 1 (TBK1) and mechanistic target of rapamycin complex 1 (mTORC1). The phosphorylated p62 makes a complex with keap-1, and this complex is degraded by autophagy in cells [63]. Activation of all these pathways by oxidants leads to accumulation of newly synthesized Nrf2. Nrf2 escapes breakdown into the nucleus and forms heterodimers with sMaf (Nrf2/sMaf). In the nucleus, the activity of Nrf2 is negatively regulated by BACH-1, which competes with Nrf2 to form heterodimers with sMaf [63]. The binding of Nrf2/sMaf to antioxidant response elements promotes the expression of as many as 200 cytoprotective genes.

#### **9. Nrf2 activators in Huntington's disease**

Minhee Jang et al. have reported that gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, alleviated the severity of neurological impairment and lethality following 3-nitropropionic acid treatment in laboratory animals through activation of Nrf2. The authors of this study conclude that gintonin might be an innovative therapeutic candidate to treat HD-like syndromes because of its potential to activate Nrf2 and decrease oxidative stress and neuroinflammation [64]. A similar study evaluated the effect of Sulforaphane in animal model of 3-NP acid-induced Huntington's disease. The study revealed that pre-treatment with sulforaphane activated Nrf2 in animals and decreased the formation of a lesion area, neuronal death, succinate dehydrogenase activity, apoptosis, microglial activation and expression of inflammatory mediators, including tumor necrosis factor-alpha, interleukin (IL)-1β, IL-6, inducible nitric oxide synthase and cyclooxygenase-2 in the striatum after 3-NP treatment [65]. Similarly, curcumin is also reported to have beneficial effects in HD via activation of Nrf2 [66]. D. Moretti et al. have reported that compound 2, a covalent KEAP1 binder, demonstrated an ability to stimulate the expression of genes known to be regulated by Nrf2 in neurons and astrocytes separated from wild-type rat, wild-type mouse and zQ175 (an HD mouse model) embryo [67].

#### **10. Challenges facing Nrf2 activators**

One of the main challenges associated with Nrf2 activators is achieving effective therapeutic concentrations as these agents are metabolized faster leading to a low bioavailability in distal organs [68–70]. The second concern with Nrf2 activators is lack of selectivity as these agents have been reported to act on other signaling pathways

*Nrf2 as a Potential Therapeutic Target for Treatment of Huntington's Disease DOI: http://dx.doi.org/10.5772/intechopen.103177*

and affect associated physiological processes. For instance, sulforophane, a widely reported Nrf2 activator, suppresses the activation of inflammosome [71, 72] and causes cell arrest [73]. Nrf2 activators have been reported to promote the development of cancer [74–76] and development of resistance to anti-cancer drugs [77–80].

#### **11. Current status of Nrf2 activators**

Oxidative stress plays a significant role in pathophysiology of numerous diseases. Initially, exogenous antioxidants were expected to have a protective and therapeutic role in the management of diseases associated with oxidative stress. But randomized clinical trials failed to find any significant therapeutic benefits of exogenous antioxidants. This unexpected outcome led to a search for endogenous targets that would enhance the antioxidant potential of the cells and tissues to prevent oxidative stressinduced damage. This quest for an endogenous antioxidant target led to the discovery of Nrf2 in the year 1994 [81]. Five years later, in 1999, it was discovered that the levels of Nrf2 in the cytoplasm are controlled by a negative regulator, Keap-1 [61]. In recent years, many potential Nrf2 activators are in pre-clinical and different stages of clinical trials for various diseases associated with oxidative stress. **Table 2** provides a list of potential Nrf2 activators in clinical trials and possible indications.



#### **Table 2.** *List of current Nrf2 activators.*

#### **12. Conclusion**

As free radicals-induced oxidative stress has been proven to play a major role in the pathogenesis of several diseases, it is quintessential to develop antioxidant therapies to negate oxidative stress-induced damage. The initial expectation that exogenous antioxidants such as vitamin E, vitamin C might have a therapeutic benefit in diseases associated with oxidative stress has failed to find any significant beneficial proof in randomized clinical trials. So, it is time to find agents that activate endogenous antioxidant mechanisms such as Nrf2. Nrf2 activators might offer a beneficial action in diseases associated with oxidative stress such as Huntington's disease.

#### **Acknowledgements**

The authors would like to thank the Department of Science and Technology— Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions (DST-FIST), New Delhi, for their infrastructure support to our department. We acknowledge the generous research infrastructure and support from JSS College of Pharmacy, JSS Academy of Higher Education & Research, Rocklands, Ooty, The Nilgiris, Tamil Nadu, India.

### **Author details**

Saravanan Jayaram\*, Praveen Thaggikuppe Krishnamurthy, Meghana Joshi and Vishnu Kumar Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Ooty, Tamil Nadu, India

\*Address all correspondence to: getsarwan@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Natalia Szejko*

Huntington's Disease (HD) is a progressive neurodegenerative disease leading to a variety of neurological and psychiatric symptoms such as chorea, parkinsonism, oculomotor symptoms, cognitive decline, depression, suicidal ideation, and psychosis. Currently, only symptomatic treatment is available. In recent years, there has been a growing number of publications regarding pathophysiology, disease biomarkers, and possible avenues for causal therapy of HD. This book presents an overview of the most important research updates in the pathophysiology and treatment of HD, with particular reference to advances in HD neuropathology, neuroimaging, and biomarkers of HD. We also summarize possible new therapeutic targets, including cannabis-based medicine, cellular, and cell-free therapeutics. Importantly, researchers from different regions of the world have contributed to this volume as we wanted to create a balanced, inclusive, and interdisciplinary review of the topics. We hope that with this book readers will be offered a compact summary of up-to-date trends in HD research which, ultimately, will enable better diagnosis and treatment for HD patients.

Published in London, UK © 2022 IntechOpen © dani3315 / iStock

From Pathophysiology to Treatment of Huntington's Disease

From Pathophysiology to

Treatment of Huntington's

Disease

*Edited by Natalia Szejko*