Systemic manifestation and contribution of peripheral tissues to Huntington’s disease pathogenesis

Abstract

Huntington disease (HD) is an autosomal dominant neurodegenerative disease that is caused by expansion of cytosine/adenosine/guanine repeats in the huntingtin (HTT) gene, which leads to a toxic, aggregation-prone, mutant HTT-polyQ protein. Beyond the well-established mechanisms of HD progression in the central nervous system, growing evidence indicates that also peripheral tissues are affected in HD and that systemic signaling originating from peripheral tissues can influence the progression of HD in the brain. Herein, we review the systemic manifestation of HD in peripheral tissues, and the impact of systemic signaling on HD pathogenesis. Mutant HTT induces a body wasting syndrome (cachexia) primarily via its activity in skeletal muscle, bone, adipose tissue, and heart. Additional whole-organism effects induced by mutant HTT include decline in systemic metabolic homeostasis, which stems from derangement of pancreas, liver, gut, hypothalamic–pituitary–adrenal axis, and circadian functions. In addition to spreading via the bloodstream and a leaky blood brain barrier, HTT-polyQ may travel long distance via its uptake by neurons and its axonal transport from the peripheral to the central nervous system. Lastly, signaling factors that are produced and/or secreted in response to therapeutic interventions such as exercise or in response to mutant HTT activity in peripheral tissues may impact HD. In summary, these studies indicate that HD is a systemic disease that is influenced by intertissue signaling and by the action of pathogenic HTT in peripheral tissues. We propose that treatment strategies for HD should include the amelioration of HD symptoms in peripheral tissues. Moreover, harnessing signaling between peripheral tissues and the brain may provide a means for reducing HD progression in the central nervous system.

1. INTRODUCTION

Historically, research on neurodegenerative diseases has mainly focused on cell-autonomous mechanisms that occur in neurons and nearby support cells in the brain. However, increasing evidence indicates that also peripheral tissues play important roles in the progression and prognosis of neurodegenerative diseases, and that there is active crosstalk between peripheral tissues and the brain (Cao and Zheng, 2018; Trushina, 2019). For example, pathogenic alpha-synuclein proteins associated with Parkinson’s disease are generated also at non- neuronal sites and can spread to other tissues (Adler et al., 2016; Zange et al., 2015). Moreover, the gut microbiota was found to influence Parkinson’s disease pathogenesis in the brain (Sampson et al., 2016).

Huntington’s disease (HD) is an autosomal dominant inherited neurodegenerative disorder with age-dependent progression that leads to behavioral, cognitive, and motor symptoms (Bates et al., 2015; Labbadia and Morimoto, 2013). It is caused by expansion of trinucleotides (cytosine/adenosine/guanine [CAG]) encoding for glutamine (Q) residues in the huntingtin (HTT) gene, which leads to a toxic, aggregation-prone, mutant HTT-polyQ protein. Whereas normal non-pathogenic HTT has between 6 and 35 glutamine residues, mutant HTT has >36–250. Similar to wild-type HTT, mutant HTT is expressed in most, if not all, tissues (Bates et al., 2015; Labbadia and Morimoto, 2013).

Toxicity of pathogenic HTT is currently thought to arise from the capacity of HTT monomers/oligomers to interact with and promote misfolding and/or dysfunction of endogenous proteins and protein complexes, whereas sequestration of pathogenic HTT into larger protein aggregates may limit such interactions and hence may represent a means of reducing toxicity (Hoffner and Djian, 2014; Truant et al., 2008), as observed for other aggregation-prone proteins (Cohen et al., 2006; Cohen et al., 2009; Douglas and Dillin, 2010).

In this review (and in most of the studies here cited), there is no distinction made between these different forms of pathogenic HTT. Therefore, in the absence of more detailed analyses, studies reporting changes in HTT protein aggregates should be interpreted as indicating overall changes in HTT protein levels, rather than specific changes in the status of mutant HTT aggregation.

Here, we examine studies from different disease models and organisms that have investigated the systemic consequences of mutant HTT expression in peripheral tissues (Figure 1), and the role of systemic intertissue signaling in Huntington’s disease pathogenesis (Figure 2). In this review, symptomatic HD patients were identified based on the Unified Huntington’s Disease Rating Scale (Kieburtz et al., 2001) or Total Functional Capacity scale (Paulsen et al., 2010; Shoulson and Fahn, 1979) whereas postmortem brain tissues were defined based on the neuropathological classification (Vonsattel et al., 1985). Studies from several HD animal models, including mouse, rat, minipig, Drosophila, and C. elegans, are discussed below. The background information for different HD mouse models is summarized in Table 1.

Figure 1. Summary of the systemic manifestation and influence of peripheral tissues in the progression of Huntington’s disease.

Huntington’s disease is a systemic disorder. In this review, we highlight three major axes that characterize the systemic effects found in Huntington’s disease: body wasting syndrome, systemic effects via the bloodstream, and systemic metabolic dysfunction. The main phenotypes due to mutant HTT and manifested in each peripheral organ/tissue are listed. The arrows depict possible inter-tissue crosstalk between peripheral tissues and the brain. We propose that the systemic environment influences local tissueautonomous events in HD pathogenesis in the brain, as well as in other tissues.

Figure 2. Inter-tissue signaling initiated by mutant HTT and its transmission across tissues.

HTT-polyQ transmission may occur via the bloodstream and may enter the brain via a leaky blood brain barrier. Moreover, HTT-polyQ could spread across nearby cells and travel long distance via its uptake by neurons and its axonal transport from the peripheral to the central nervous system. However, the significance of such HTT-polyQ spreading is unclear as HTT is widely expressed. Lastly, there could be signaling factors that are produced and/or secreted in response to therapeutic interventions such as exercise or by HTT activity in peripheral tissues. Such signaling factors may in turn impact whole-body processes and perhaps even the activity of pathogenic HTT in the brain. In summary, we propose that HD is a systemic disease that is influenced by inter-tissue signaling and by the action of HTT in peripheral tissues.

Table 1.

Mouse and rat HD animal models, which are used to investigate the impact of HTT-polyQ in different tissues. The expression of HTT-polyQ is ubiquitous in these animal models except for N171-82Q mice, which express HTT-polyQ mainly in brain neurons and heart.

Mouse or Rat HD Model	Strain	CAG number	Expression	Reference
R6/1 mouse	CBA × C57BL/6	115 CAG	Whole body	Mangiarini et al., 1996
R6/2 mouse	CBA × C57BL/6	150 CAG	Whole body	Mangiarini et al., 1996
BACHD mouse	FBV/NJ	97 CAA-CAG	Whole body	Gray et al., 2008
BACHD rat	Sprague Dawley	97 CAA-CAG	Whole body	Yu-Taeger et al., 2012
CAG140 knock-in mouse	C57BL/6	140 CAG	Whole body	Menalled et al., 2003
zQ175 mouse	C57BL/6J	175 CAG	Whole body	Menalled et al. 2012
N171-82Q mouse	C3H/HEJ × C57BL/6JFl	82 CAG	Brain neurons and heart	Schilling et al. 1999
Hdh150 knock-in mouse	C57BL/6J	150 CAG	Whole body	Lin et al. 2001

2. BODY WASTING SYNDROME (CACHEXIA) IN HUNTINGTON’s DISEASE

2.1. Bone.

Because body weight loss is one of the clinical symptoms of HD, many studies have analyzed the body composition of pre-HD carriers and early HD patients with the intent of identifying biomarkers for early diagnosis of HD. These studies have found that bone mineral density is significantly lower in pre-HD carriers compared to healthy controls (Goodman and Barker, 2011). Besides bone mineral density, lean body mass (e.g. skeletal muscle) and fat mass are also significantly lower in early HD patients compared to the control group, especially in male patients (de Miranda et al., 2019; Mielcarek, 2015). Similar results were observed in the R6/2 mouse model (Bjorkqvist et al., 2006). Altogether, these studies indicate that loss of bone mineral density contributes to the body wasting see in HD patients. However, the mechanisms by which pathogenic huntingtin affects bone density are still largely unexplored.

2.2. Skeletal muscle.

Because skeletal muscle constitutes around 40–50% of the human body (Piccirillo et al., 2014), skeletal muscle wasting is considered the major contributor to weight loss that occurs in HD (de Miranda et al., 2019). In this respect, many studies have determined the tissue and cellular defects induced by pathogenic huntingtin and responsible for the reduction in muscle mass and strength that is found in HD (Busse et al., 2008; Mielcarek, 2015).

At the cellular level, in addition to protein aggregates of pathogenic huntingtin (Orth et al., 2003; Sathasivam et al., 1999), inclusions of poly-ubiquitinated proteins were also found to increase within muscle cells (myofibers) and myonuclei in R6/2 mice (Ribchester et al., 2004), and in muscle cell cultures (Orth et al., 2003). In muscle that express pathogenic huntingtin, dysfunction of chloride and potassium ion channels results in decreased resting conductance, which leads to hyperexcitability of myofibers, and has been suggested as a potential pathway to damage mitochondria in R6/2 mouse muscle (Waters et al., 2013). It has also been proposed that muscle mitochondria are more vulnerable in mice with HD than in wild-type mice due to Ca²⁺-induced stress (Gizatullina et al., 2006). Additionally, in transgenic HD minipig animal models, expression of HTT-polyQ results in decreased levels of the mitochondrial fusion protein optic atrophy 1 (OPA1), which is followed by increasing levels of the mitochondrial fission protein dynamin-related protein 1 (DRP1) (Rodinova et al., 2019). These results suggest that imbalance in the function of the fission-fusion machinery may contribute to derangement of the mitochondrial ultrastructure and consequent mitochondrial dysfunction of muscle (Squitieri et al., 2010), which may lead to cytochrome c release and increase in apoptosis (Ciammola et al., 2006). In summary, via direct and indirect mechanisms, pathogenic HTT-polyQ impairs protein quality control and ion channel function, and induces mitochondrial abnormalities and apoptosis (Table 2).

Table 2.

HTT-polyQ-induced phenotypes in skeletal muscle include myofiber atrophy, mitochondrial abnormalities, ion channel dysfunction, and impairment of protein quality control.

Skeletal muscle	Phenotype	Model	Stage of HD	Reference
	Transition from fast-twitch to slow-twitch myofibers	R6/2 mice	Late stage	Strand et al., 2005
	Altered morphology of mitochondria	Primary cell cultures from HD patients	Stage II to IV	Squitieri et al., 2010
	Slowed phosphocreatine recovery after exercise	Pre-HD carriers	Asymptomatic	Saft et al., 2005
	Deficit in mitochondrial ATP production rate after exercise	Pre-HD carriers	Asymptomatic	Lodi et al., 2000
	Reduced muscle strength	HD patients	Middle to late stage	M. E. Busse et al., 2008
	Release of cytochrome C, increase in apoptosis, and formation of inclusion bodies	Primary cell cultures from HD patients	Stage I to V	Ciammola et al., 2006
	Skeletal muscle atrophy and formation of HTT-polyQ inclusion bodies	R6/2 mice	Late stage	Orth et al., 2003
	Formation of HTT-polyQ inclusion bodies	R6/2 mouse primary cell culture	Late stage	Sathasivam et al., 1999
	Reduced number and size of limb motor neurons, muscle atrophy, decreased number of glycolytic fast type IIB fibers, fragmentation of neuromuscular junctions	BACHD mice	Late stage	Valadao, de Aragao, et al., 2019
	Fragmentation of neuromuscular junctions in sternomastoid muscles, axon degeneration, motoneuron death	BACHD mice	Late stage	Valadão et al., 2017
	Myofiber atrophy, lower resting potential, abnormality in electrophysiological tests, ubiquitin inclusions in myonuclei and myofibers of R6/2 mice	R6/2 mice	Middle to late stage	Ribchester et al., 2004
	Reduced life span	Drosophila	Specific expression of HTT-polyQ in skeletal muscle	Demontis & Perrimon, 2010
	Ion channel dysfunction, instable calcium homeostasis	R6/2 mice	Late stage	Gizatullina et al., 2006
	Dysfunction of chloride and potassium ion channels	R6/2 mice	Late stage	Waters et al., 2013
	Lower levels of mitochondrial fusion protein optic atropy 1 (OPA1), higher levels of the mitochondrial fission protein dynamin-related protein 1 (DRP1)	Transgenic minipig model	Early stage (about three year)	Rodinova et al., 2019
	Increased detergent-soluble levels of HTT-polyQ, accelerated age-dependent onset of tremors, and reduced lifespan, body weight, and fat mass in Scn4a mutant mice	N171-82Q mice	Early stage	Corrochano et al., 2018

In addition to cellular abnormalities, pathogenic huntingtin was found to lead to several tissue-scale defects, such as myofiber atrophy, myofiber type switching, and denervation. Specifically, in BACHD mice, the cross-sectional area and number of myofibers in limb muscles, especially type IIB myofibers, were reduced compared to wild-type mice (Valadao et al., 2019). Moreover, in the sternomastoid muscle, there were more type IIA and fewer type IIX myofibers (Valadão et al., 2017). Besides myofiber atrophy and changes in the proportion and number of myofiber types, denervation and fragmentation of neuromuscular junctions were found to occur in BACHD mice (Valadao et al., 2019; Valadão et al., 2017). Similarly, in R6/2 mouse models, transition from fast-twitch glycolytic myofibers (type IIB) to slow-twitch myofibers (type I) was observed (Strand et al., 2005) as well as atrophy of all myofiber types, lower resting potential, and abnormalities in electrophysiological functions (Ribchester et al., 2004).

Besides muscle functional defects at steady state, recovery from exercise is also affected by pathogenic huntingtin, as indicated by slow phosphocreatine recovery (Saft et al., 2005) and deficits in mitochondrial adenosine triphosphate (ATP) production after exercise (Lodi et al., 2000).

In addition to contributing to the whole-body manifestation of HD, the action of pathogenic huntingtin in skeletal muscle has allowed the use of this tissue as an experimental system for discovering HD modifiers. For example, the capacity of puromycin-sensitive aminopeptidase (Psa) to degrade pathogenic huntingtin was established in mouse skeletal muscles (Menzies et al., 2010). Similarly, RNAi screening in C. elegans worms that express HTT-polyQ in their skeletal muscle was used to identify genetic modifiers of HD (Silva et al., 2011). Moreover, in N171-82Q mice, mutations in the skeletal-muscle specific sodium channel gene Scn4a were found to increase the detergent-soluble levels of HTT-polyQ, to accelerate the age-dependent onset of tremors, and to reduce lifespan, body weight, and fat mass (Corrochano et al., 2018).

Altogether, clinical observations in HD patients and studies in mouse disease models indicate that HTT-polyQ reduces skeletal muscle function by inducing myofiber atrophy, by reducing the number of myofibers, and by altering many cellular processes such as protein quality control and mitochondrial function. Moreover, muscle-specific overexpression of HTT-polyQ was found to reduce lifespan in Drosophila, indicating that the function of pathogenic huntingtin in skeletal muscle is per se an important determinant of organismal survival in HD (Demontis and Perrimon, 2010). Moreover, high levels of the stress hormone glucocorticoids, which are known inducers of myofiber atrophy (Braun and Marks, 2015), are found in HD patients (discussed below in section 3.4) and contribute to muscle wasting.

2.3. Adipose tissue.

In addition to loss of bone density and skeletal muscle mass, HD is also characterized by a decline in fat mass, which occurs in a manner dependent on its anatomical location and HD stage. Indication that fat mass could be altered in HD came from several studies, including the observation that HD patients have lower plasma levels of leptin (Popovic et al., 2004). Because circulating leptin positively reflects adipose tissue size (Munzberg and Morrison, 2015), it was proposed that there might be a decrease in fat mass in HD. Indeed, it was found that truncal fat mass was significantly lower in HD patients compared to controls (de Miranda et al., 2019). Similarly, R6/2 mice display reduced fat mass, a component of severe body wasting that leads to their premature death between 12 and 15 weeks of age. Interestingly, such low fat mass of R6/2 HD mice can be resolved with an appropriate diet. Specifically, a high fat and high sugar diet elevated the amount of fat mass and the concentration of serum leptin, and caused an increase in adipocyte cell size largely because of defective fat breakdown (Fain et al., 2001). Moreover, this diet-induced obesity was not accompanied by symptoms of diabetes, i.e. there were no defects in circulating glucose and insulin levels, and in insulin sensitivity (Fain et al., 2001).

Besides, in R6/2 HD and CAG140 knock-in mouse models, it was found that there is fat mass reduction in early stages and that fat mass reduction becomes more pronounced at later stages (Phan et al., 2009). In parallel, there was a reduction in the release of the adipokines (adipose tissue-derived hormones) leptin and adiponectin, which respectively repress food intake and improve glucose metabolism (Phan et al., 2009). Importantly, experimental ablation of leptin decreased energy consumption and increased white adipose tissue content and body weight in R6/2 mice although leptin deficiency did not improve motor behavior and HTT-polyQ-induced degeneration in the brain (Sjögren et al., 2019).

At the cellular level, adipocytes acquire an immature de-differentiated phenotype in response to expression of pathogenic huntingtin, which is due to interference of mutant huntingtin with PGC-1α activity and consequent decreased expression of fat storage genes (Phan et al., 2009). Similarly, there was lower PGC-1α activity, reduced UCP-1 expression, and dysfunctional mitochondria in brown adipose tissue of N17-182Q transgenic mice, which led to hypothermia (Weydt et al., 2006). These mice also displayed hypophagia and increased energy consumption during fasting (Weydt et al., 2006).

In addition to mice, overexpression of HTT-polyQ in the Drosophila fat body alone was sufficient to cause chronic weight loss in spite of higher food intake (Lakra et al., 2019). Specifically, there was an age-dependent decrease in lipid content, lipid droplets, and carbohydrates and a higher rate of apoptosis associated with mutant huntingtin expression in adipocytes.

Taken together, these studies indicate that pathogenic huntingtin impairs the normal function of adipocytes, and that this contributes to the systemic wasting syndrome associated with HD (Table 3).

Table 3.

HTT-polyQ reduces release of leptin and adiponectin, causes hypothermia, and affects feeding behavior.

Adipose tissue	Phenotype	Model	Stage of HD	Reference
	Decreased levels of leptin	HD patients	Middle stage	Popovic et al., 2004
	Decreased amount of truncal fat mass	HD patients	Early stage	de Miranda et al.,2019
	Reduced fat mass in early stage but increased fat mass in late stage in R6/2 mice, reduced fat mass in late stage in CAG140 mice, reduction in the release of leptin and adiponectin, downregulation of PGC-lα	R6/2 mice and CAG140 knock-in mice	Early to late stage	Phan et al., 2009
	Hypothermia, increased energy consumption, lower food intake, mitochondrial abnormalities, reduced level of PGC-lα in HD patients	N171-82Q mice and HD patients	Early stage both in N171-82Q mice and HD patients	Weydt et al., 2006
	In R6/2 mice with leptin-deficiency: increased body weight, decreased energy consumption, and elevation of white adipose tissue but no improvement in motor behavior and HTT-polyQ-induced degeneration in the brain	R6/2 mice	Middle to late stage	Sjögren et al., 2019
	Weight loss, impact on feeding behavior, decreased lipid content, decreased lipid droplets, decreased carbohydrates, HTT-polyQ aggregates in adipocytes	Drosophilo	Specific expression of HTT-polyQ in fat body	Lakra et al., 2019

2.4. Heart.

Heart disease is the second leading cause of death in HD patients (Sorensen and Fenger, 1992) and may be a component of HD-associated cachexia as found in other disease contexts (Anker and Sharma, 2002; Florea et al., 2002).

Clinical studies indicate that HD-associated cardiac dysfunction derives from dysfunction of the autonomic nervous system (ANS), i.e. the control system that acts largely unconsciously to regulate heart rate. Specifically, analysis of the ANS by using heart rate variability analyses in HD patients showed reduced heart rate variability, which suggested an imbalance in the function of the sympathetic and parasympathetic nervous systems (Andrich et al., 2002; Bär et al., 2008; Kobal et al., 2004; Sharma et al., 1999). Notably, there is a significant reduction in vagal modulation, i.e. the control of the heart rate by the vagus nerve, in HD patients compared to healthy controls (Andrich et al., 2002). Moreover, sympathetic hyperfunction was present in asymptomatic mutant HTT gene carriers, which indicates that derangement of the ANS is an early event in HD (Kobal et al., 2004).

Clinical studies indicate that the precerebral arteries are also impacted in HD. Specifically, there is higher arterial stiffness, which in turn can contribute to cardiac defects through ANS dysfunction (Kobal et al., 2017; Kobal et al., 2010). Other defects observed in HD patients (based on electrocardiograms) consist of an abnormal slow heart action and conduction abnormalities (Stephen et al., 2018), which might also be explained on the basis of ANS impairment (Melik et al., 2012). Altogether, these studies indicate that cardiac defects arise primarily from ANS dysfunction in HD.

Similar evidence has also been found in mouse models of HD. Specifically, by analyzing electrocardiograms, unstable heart beats and arrhythmia were found in R6/1 (Kiriazis et al., 2012) and BACHD mice (Schroeder et al., 2016), and these were ascribed to dysregulation of the ANS. Additionally, reduced heart size was observed in early stages of disease progression in R6/1 mice. Additionally, by measuring the baroreceptor reflex, a homeostatic mechanism which regulates blood pressure and that is regulated by the ANS, a significantly higher blood pressure was found in BACHD mice compared to controls (Schroeder et al., 2011).

Besides ANS dysfunction, a number of changes have been found in the heart at the cellular and tissue level in HD. Specifically, heart fibrosis and gap junction remodeling were found in HD, and presumably resulted from progressive cardiomyocyte loss via apoptosis in R6/2, HdhQ150 (Mielcarek et al., 2014), and BACHD mice (Schroeder et al., 2016). Mechanistically, pathogenic huntingtin was found to be responsible for these cellular defects via inhibition of the proteasome in the cytosol and in the nucleus of cardiomyocytes of R6/2 mice (Mihm et al., 2007).

Mitochondria were also profoundly affected in cardiomyocytes of HD models. Specifically, studies in cardiomyocytes from BACHD and R6/2 mice revealed alterations in mitochondrial ultrastructure and dynamics, which also contribute to cardiomyocyte apoptosis (Mihm et al., 2007; Schroeder et al., 2016). Similar results are also observed in Drosophila: expression of HTT-polyQ specifically in the heart leads to cardiac dysfunction because of impaired mitochondrial function, increased production of reactive oxygen species, autophagic defects, and cell death (Melkani et al., 2013).

Concerning the modulation of signal transduction pathways in HD, cardiac mTORC1 activity (which is known to regulate stress responses and heart size) was decreased in N171-82Q mice, as assessed based on the phosphorylation status of S6 and 4E-BP, and this was rescued by knockdown of HTT-polyQ (Child et al., 2018). Conversely, the activity of Ca²⁺/calmodulin protein kinase II (CaMKII) was unusually high and contributed to oxidative damage and cellular arrhythmia in BACHD mice (Joviano-Santos et al., 2019).

Taken together, these studies show that cardiac dysfunction is a prominent feature in HD that derives from dysfunction of the ANS and a number of cellular and tissue defects (mitochondrial defects, proteasome inhibition, deregulated signal transduction, and apoptosis) which arise from pathogenic huntingtin expression (Critchley et al., 2018; Melkani, 2016) (Table 4).

Table 4.

HTT-polyQ-related phenotypes are observed in cardiomyocytes. Cardiac dysfunction is mainly due to HTT-polyQ aggregates in cardiomyocytes and autonomous nerve system dysfunction.

Cardiomyocytes	Phenotype	Model	Stage of HD	Reference
	Lower heart rate variability, reduced vagal modulation only in middle stage HD patients	HD patients	Early to middle stage	Andrich et al., 2002; Bär et al., 2008
	Sympathetic hyperfunction in pre-HD carriers and early HD patients, sympathetic hypofunction in middle and late HD patients	Pre-HD carriers and HD patients	Asymptomatic and early to late stage	Kobal et al., 2004
	Prolonged skin sympathetic response latency and smaller amplitude	HD patients	NA	Sharma et al., 1999
	Lower relative heat rate and diastolic pressure	Pre-HD carriers and HD patients	Asymptomatic and early stage	Kobal et al., 2010
	Higher parameters for arterial stiffness and lower for carotid artery	Pre-HD carriers and HD patients	Asymptomatic and early stage	Kobal et al., 2017
	Slow heart action and conduction abnormalities	HD patients	Stage I	Stephen et al., 2018
	Reduction of cutaneous Laser-Dopplerflux, abnormal heart rate variability	Pre-HD carriers	Asymptomatic	Melik et al., 2012
	Unstable heart beats, arrhythmia, reduced heart size	R6/1 mice	Early stage	Kiriazis et al., 2012
	Cardiac fibrosis and increased the level of caspase 3 in late stage, unstable heart beats in early stage, alterations in mitochondrial ultrastructure, abnormal mitochondrial dynamics	BACHD mice	Early and late stage	Schroeder et al., 2016
	Higher blood pressure, increased heart weight	BACHD mice	Middle stage	Schroeder et al., 2011
	Gap junction abnormality, progressive apoptotic cardiomyocyte loss, cardiac fibrosis	R6/2 mice and Hdh150 mice	Middle to late stage	Mielcarek et al., 2014
	Cardiac hypertrophy and fibrosis, HTT-polyQ aggregates both in the cytosol and nucleus, proteasome impairment, increased protein oxidation, alterations in mitochondrial ultrastructure, abnormal mitochondrial dynamics	R6/2 mice	Middle to late stage	Mihm et al., 2007
	Dysregulation of cardiac mTORC1 activity due to HTT-polyQ aggregates	N171-82Q mice	Middle to late stage	Child et al., 2018
	Increased the activity of Ca2+/calmodulin protein kinase II	BACHD mice	Late stage	Joviano-Santos et al., 2019
	Modulation of protein folding pathway and oxidative stress suppress HTT-polyQ-induced mitochondrial abnormalities and cardiac amyloidosis	Drosophila	Specific expression of HTT-polyQ in cardiomyocytes	Melkani et al., 2013

3. SYSTEMIC METABOLIC DYSFUNCTION IN HUNTINGTON’s DISEASE

3.1. Gut.

Gut microbiota dysbiosis has been linked to neurodegenerative diseases in humans. Although there are several studies that have examined the impact of the microbiota on Alzheimer’s and Parkinson’s disease (Sampson et al., 2016; Seo et al., 2019), less is known on the impact of the microbiota on HD and vice versa. By analyzing gut bacteria-derived metabolites found in the blood (Wikoff et al., 2009), a study investigated the changes in such metabolites associated with early symptomatic HD patients, pre-manifest HD carriers, and healthy controls. Alterations in purine, tryptophan, tyrosine, and anti-oxidant pathways that occur in the gut microbiome were found to be different in pre-manifest HD carriers compared to early symptomatic HD patients (Rosas et al., 2015). In addition, by comparing the composition of the gut microbiota, significant differences were found between HD patients and pre-manifest HD carriers, especially in males (Wasser et al., 2020). Three phyla, Euryarchaeota, Verrucomicrobia, and Firmicutes, are significantly different but also other families are modulated in males including Acidaminococcaceae, Akkermansiaceae, Bacteroidaceae, Bifidobacteriaceae, Christensenellaceae, Clostridiaceae, Coriobacteriaceae, Eggerthellaceae, Enterobacteriaceae, Erysipelotrichaceae, Flavobacteriaceae, Lachnospiraceae, Methanobacteriaceae, Peptococcaceae, Peptostreptococcaceae and Rikenellaceae.

Interestingly, there seems to be a connection between changes in the gut microbiota and related metabolic pathways and neuroinflammation. Specifically, there is a positive relationship between the abundance of Eubacterium hallii and the preservation of motor function in HD patients, but not in pre-manifest HD carriers. Mechanistically, E. hallii produces short-chain fatty acids which may help maintain gut homeostasis and improve brain health (Bourassa et al., 2016). Similarly, there is a significant difference in the composition of the gut microbiome between R6/1 HD mice and wild-type controls. In this study, researchers extracted bacterial 16S rRNA DNA from fecal samples of R6/1 mice to analyze the composition of the gut microbiome. There were two dominant phyla (about 98% of total abundance), Bacteroidetes and Firmicutes, whereas the rest of the phyla consisted of Actinobacteria, Proteobacteria, Cyanobacteria, Deferribacteres, and Tenericutes (Kong et al., 2018). Importantly, a significant increase in Bacteroidetes and a significant decrease in Firmicutes were found in R6/1 mice compared to controls. Additionally, significantly increases in Actinobacteria and Proteobacteria and a significant decrease in Deferribacteres were found in male but not in female R6/1 mice compared to wild-type mice. These abnormalities in microbial diversity were associated with decreased gut mucosal thickness and villus length, diarrhea, and malabsorption of food, which led to body weight loss in R6/1 and R6/2 mice (Kong et al., 2018; van der Burg et al., 2011). In addition, gut dysbiosis was exacerbated by dysfunction of the enteric nervous system, resulting from loss of cholinergic neuronal activity due to mutant huntingtin (Moffitt et al., 2009; van der Burg et al., 2011).

In addition to the microbiota, the digestive tract may provide a route for modulating HD pathogenesis via dietary interventions. For example, dietary supplementation of arginine rescued body weight loss and improved motor ability due to higher cerebral blood flow (CBF) in R6/1 mice (Deckel et al., 2000a). Such effects of dietary arginine may derive from increased synthesis of nitric oxide (which improves CBF) by nitric oxide synthase, which utilizes arginine as substrate. Because clinical research indicates higher resting CBF in HD patients (Deckel et al., 2000b), dietary supplementation with arginine may prove useful in the clinic.

Dietary/calorie restriction is a known intervention to delay aging and age-related diseases in a number of organisms (Kapahi et al., 2017). Consistently, it was found that dietary restriction extends life span, prevents weight loss, and delays the onset of motor deficits in N171-82Q mice (Duan et al., 2003). These effects stem from reduction in Htt-polyQ levels and brain atrophy, presumably because of upregulation of BDNF (brain-derived neurotrophic factor) and Hsp70 (heat shock protein 70), which have neuroprotective functions (Duan et al., 2003).

Interestingly, other dietary treatments, some of which have been proposed to work via hormesis, have been found to offer protection from HD onset and progression (Calabrese et al., 2019). For example, olive oil polyphenols (hydroxytyrosol and oleuropein aglycone) have been reported to prevent or delay the onset of neurodegeneration in neuroblastoma cells (Sirangelo et al., 2020), C. elegans and mice (Brunetti et al., 2020; Di Rosa et al., 2020; Siracusa et al., 2020). Similarly, Ginkgo biloba (Calabrese et al., 2020) and Mediterranean and Asian diets (Leri et al., 2020) have been reported to delay aging and neurodegeneration.

Altogether, gastrointestinal dysfunction due to expression of pathogenic huntingtin in the gastrointestinal tract and changes in the composition of the microbiome contribute to systemic degeneration in HD. Moreover, dietary interventions are a promising avenue to delay the onset and progression of HD (Table 5).

Table 5.

HTT-polyQ-related phenotypes are observed in the gut. Alterations of gut microbiome-derived metabolites are found in the blood of pre-HD carriers and early stage HD patients, and can be potential indicators of HD diagnosis. Furthermore, bacteria floras are also different in HD models compared to wild-type mice, which may lead to weight loss and impairment of the enteric nervous system.

Gut	Phenotype	Model	Stage of HD	Reference
	Alteration in gut microbiome-derived metabolites	Pre-HD carriers and HD patients	Asymptomatic and early stage	Rosas et al., 2015
	Increased Bacteroidetes and decreased Firmicutes both in male and female R6/1 mice, increased Actinobacteria and Proteobacteria and decreased Deferribacteres in male R6/1 mice	R6/1 mice	Early stage	Kong et al., 2018
	Gastrointestinal dysfunction contributing to weight loss, decreased mucosal thickness and villus length, loss of neuropeptides in the enteric nervous system	R6/2 mice	Middle to late stage	van der Burg et al.,2011
	Less gut richness and alteration in gut microbiome	HD patients	Stage I	Wasser et al., 2020
	Feeding with arginine rescued body weight loss and improved motor ability due to increased cerebral blood flow	R6/1 mice	Early stage	Deckel et al., 2000a
	Dietary restriction extended life span, reduced weight loss, delayed the onset of motor deficits, reduced brain atrophy, decreased Htt-polyQ levels, and upregulated the level of BDNF and Hsp70	N171-82Q mice	Middle to late stage	Duan et al., 2003

3.2. Pancreas.

HD patients suffer from abnormalities in energy homeostasis and some of them develop type 2 diabetes mellitus (Farrer, 1985), which suggests that the function of the pancreas is impacted by mutant huntingtin. Indeed, by using the oral glucose tolerance test, it was found that HD patients have impaired glucose tolerance (Kremer et al., 1989). Similar results were also observed in R6/2 mice, which develop type 2 diabetes (Hurlbert et al., 1999). Immunohistochemical analyses found reduced levels of glucagon in α-cells and of insulin in β-cells, which was the cause of type 2 diabetes (Hurlbert et al., 1999). Mechanistically, the lack of secretory granules in β-cell and β-cell dysfunction are the main reasons for the development of type 2 diabetes in R6/2 mice and these defects arise from the action of pathogenic huntingtin in the pancreas (Björkqvist et al., 2005). In addition, HTT-polyQ aggregates associate with reduced expression of other islet hormones such as glucagon and somatostatin (Andreassen et al., 2002).

Intriguingly, treatment with exendin-4 ameliorates plasma glucose concentrations, improves α-cell and β-cell function, reduces mutant huntingtin aggregates in both the pancreas and the brain, rescues motor deficits, and extends survival in N171-82Q mice (Martin et al., 2009). Exendin-4 is an FDA-approved agonist of glucagon-like peptide 1 receptor, which increases the production and release of insulin and also elevates insulin sensitivity in the brain and pancreas. On this basis, this study highlights the systemic influence of the pancreas in modulating HD pathogenesis in the brain. In summary, mutant huntingtin impairs the function of α-cells and β-cells in the pancreas and leads to type 2 diabetes mellitus. Importantly, rescuing the function of the pancreas reduces HD pathogenesis in the brain (Table 6).

Table 6.

HTT-polyQ-related phenotypes are observed in the pancreas. HTT-polyQ aggregates lead to diabetes mellitus and impair the normal function of pancreatic α-cells and β-cells.

Pancreas	Phenotype	Model	Stage of HD	Reference
	Type 2 diabetes mellitus	HD patients	NA	Farrer, 1985
	Impaired glucose tolerance	HD patients	NA	Kremer et al., 1989
	Type 2 diabetes mellitus, reduction in the level of glucagon in α-cells and insulin in β-cells	R6/2 mice	Late stage	Hurlbert et al., 1999
	Impaired insulin gene expression, progressive defects in glucose tolerance, HTT-polyQ aggregates in pancreatic islets, reduced glucagon and somatostatin hormone expression	R6/2 mice	Middle to late stage	Andreassen et al., 2002
	β-cell dysfunction	R6/2 mice	Late stage	Björkqvist et al., 2005
	Higher production and release of insulin and insulin sensitivity in brain and pancreas improve α-cell and β-cell abnormalities, rescue motor defects, and reduce HTT-polyQ aggregates	N171-82Q mice	Middle to late stage	Martin et al., 2009

3.3. Liver.

Clinical studies have found increased lactate concentration in the blood of HD patients (Harms et al., 1997; Jenkins et al., 1993; Nielsen, 1999), suggesting that there might be defects in lactate utilization by the liver in HD. In R6/2 mice, besides changes in blood glucose and lactate, the enzyme activity of pyruvate kinase in the liver is increased whereas the activity and the mRNA level of phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, are decreased in the liver of R6/2 mice compared to wild-type controls (Josefsen et al., 2010). Additionally, intraperitoneal injection of lactate into R6/2 mice revealed that lactate-based gluconeogenesis in the liver is progressively impaired as evinced from the higher blood levels of lactate and lower glucose levels.

At the cellular level, as found in other tissues (see paragraphs above), mutant huntingtin negatively impacts mitochondrial function also in the liver. Specifically, there is progressive hepatic mitochondrial dysfunction in HD patients and pre-HD carriers, as assessed based on the analysis of methionine metabolization by hepatic mitochondrial decarboxylation (Hoffmann et al., 2014; Stuwe et al., 2013). Mechanistically, there are lower mRNA levels of the transcription factors PGC-1a and Tfam (mitochondrial transcription factor A) in the liver of N171-82Q mice. Because PGC-1a and Tfam promote mitochondrial gene expression and biogenesis (Scarpulla, 2011), their reduced levels likely leads to defects in glucose metabolism by the liver at least in part due to defects in mitochondrial function (Chaturvedi et al., 2010).

Additionally, the urea cycle is also affected in HD. Specifically, R6/2 mice display a higher blood concentration of ammonia and poor motor function, in parallel with HTT-polyQ aggregates and with a reduction in chaperone (Hsp70 and Hsp27) levels in the liver (Chiang et al., 2007). Mechanistically, studies in R6/2 mice have demonstrated that defects in urea metabolism arise from a decline in the expression in the liver of argininosuccinate lyase, which is a key enzyme for the urea cycle (Chiang et al., 2007). The activity of mutant huntingtin in the liver decreases argininosuccinate lyase expression by repressing the function of the transcription factor C/EBPa. Interestingly, defects in the urea cycle are improved by low-protein diet (Chiang et al., 2007). Besides C/EBPa, another transcription factor, PPARg, is also affected in R6/2 mice. Importantly, treatment with PPARg agonists not only elevated the mRNA level of PPARg and its downstream genes but also reduced HTT-polyQ levels (Chiang et al., 2011).

Altogether, these studies indicate that mutant huntingtin leads to hepatic dysfunction and in turn to defects in systemic metabolic homeostasis (Table 7).

Table 7.

HTT-polyQ-related phenotypes observed in the liver include impairment of gluconeogenesis and hepatic mitochondrial dysfunction. HTT-polyQ aggregates in the liver decrease the levels of PGC-1α, PPARγ, and further cause C/EBPα dysfunction and higher blood concentration of ammonia in HD mouse models.

Liver	Phenotype	Model	Stage of HD	Reference
	Reduced gluconeogenesis and lactate, decreased activity and mRNA levels of phosphoenolpyruvate carboxykinase, increased enzyme activity of pyruvate kinase in liver	R6/2 mice	Middle stage	Josefsen et al., 2010
	Hepatic mitochondrial dysfunction	Pre-HD carriers	Asymptomatic	Hoffmann et al., 2014
	Hepatic mitochondrial dysfunction	HD patients	Early stage	Stuwe et al., 2013
	Decreased mRNA levels of PGC-lα and mitochondrial transcription factor A (Tfam)	N171-82Q mice	Middle stage	Chaturvedi et al., 2010
	Higher blood concentration of ammonia, reduction of protein chaperones, reduced mRNA levels of argininosuccinase acid lyase, loss of function of C/EBPα	R6/2 mice	Late stage	Chiang et al., 2007
	Elevation of the mRNA levels of PPARγ and its downstream genes reduced HTT-polyQ aggregates	R6/2 mice	Late stage	Chiang et al., 2011

3.4. Adrenal glands and the endocrine stress response.

Adreno-corticotropic hormone (ACTH; also called corticotropin) is produced and secreted by the pituitary gland. ACTH is a key component of the hypothalamic–pituitary–adrenal (HPA) axis. HPA hyperactivity is an early symptom in patients with HD due to hypothalamic dysfunction (Aziz et al., 2009b). ACTH controls the release of the glucocorticoid hormone cortisol; in turn, excessive levels of cortisol contribute to muscle wasting and cognitive deficits in HD patients (Heuser et al., 1991), as observed in R6/2 HD mice, which have enlarged adrenal glands (Bjorkqvist et al., 2006). Interestingly, a dysfunctional HPA axis and abnormally high ACTH levels can be corrected by environmental enrichment (Du et al., 2012), i.e. by housing conditions that enhance sensory functions, motor performance, and cognitive stimulation, and that can be used to delay HD (Nithianantharajah and Hannan, 2006; Van Dellen et al., 2000).

Consistent with a role for stress hormones in HD, oral administration of the stress hormone corticosterone led to short-term memory deficits specifically in male but not in female R6/1 mice, whereas motor function was not affected (Mo et al., 2014a). Additionally, in a model of stress induced by restraint, olfactory sensitivity was reduced in males, and to a lesser extent in females (Mo et al., 2014b). Motor coordination and locomotor activity were enhanced by chronic restraint in both R6/1 and wild-type male mice whereas there was no effect in females (Mo et al., 2014b). Taken together, these results demonstrate that HPA axis dysfunction is an early symptom in HD pathology and that excess cortisol resulting from HPA hyperactivity leads to cognitive deficits and muscle wasting (Table 8).

Table 8.

Hypothalamic–pituitary–adrenal (HPA) axis dysfunction is an early symptom in HD pathology and contributes to cognitive deficits and stress response abnormality.

Adrenal gland & stress response	Phenotype	Model	Stage of HD	Reference
	HPA hyperactivity is an early symptom in patients with HD due to hypothalamic dysfunction	HD patients	Early stage	Aziz et al., 2009b
	Excess cortisol along with excess ACTH contributes to cognitive deficits	HD patients	NA	Heuser et al., 1991
	High serum level of cortisol and enlarged adrenal glands	R6/2 mice	Late stage	Björkqvist et al., 2006
	Abnormal elevation of ACTH leads to derangement of the stress response and adrenal-related genes expression specifically in female R6/1 mice	R6/1 mice	Early stage	Du et al., 2012
	Reduced level of gonadotropin-releasing hormone and serum testosterone with ovary weight loss. Estrogen receptor activation improves depression-like behaviors under stress.	R6/1 mice	Early stage	Du et al., 2015
	Progressive memory impairment in response to stress hormones	R6/1 mice	Early stage	Mo et al., 2014
	Restraint-mediated stress impacts olfactory function	R6/1 mice	Early stage	Mo et al., 2014

Because females appear less susceptible to certain deleterious effects of stress hormones (Mo et al., 2014b), it has been proposed that female sex hormones may protect from certain aspects of HD progression (Bode et al., 2008). However, female R6/1 mice are more susceptible than males to depression, suggesting that female sex hormones may contribute to develop this HD-associated symptom, which is regulated by the HPA axis (Du et al., 2015; Keller et al., 2017). In female R6/1 mice, it was found that the hypothalamic expression of gonadotropin-releasing hormone and the serum levels of testosterone decrease in parallel with ovary atrophy (Du et al., 2015). Gonadectomy surgery reduced HPA-axis activity in female mice but had no effect on depression-like behaviors, which were rescued by pharmacologic estrogen receptor activation (Du et al., 2015). Altogether, more studies are needed to define the interconnection of sex hormones with the HPA axis and HD pathogenesis.

3.5. Sleep disorders and circadian abnormalities.

Sleep disorders and circadian abnormalities are well-known symptoms of neurodegenerative diseases including HD which may contribute to systemic metabolic dysfunction (Iranzo, 2016). Sleep-wake cycles are mainly regulated by the suprachiasmatic nucleus (SCN) in the hypothalamus in response to signals from peripheral tissues and extrinsic factors such as light-dark visual cues (Hastings et al., 2018). Clinical evidence indicates that early HD patients have higher night activity and poor sleep quality (Diago et al., 2017; Morton et al., 2005). Melatonin is a key sleep regulator that is synthesized by the SCN and secreted at night. Delayed onset of the diurnal melatonin rise and lower plasma levels are found in HD patients compared to healthy controls (Aziz et al., 2009a; Kalliolia et al., 2014). Moreover, by MRI analysis, the grey matter volume of the hypothalamus is lower in pre-HD carriers compared to controls (Bartlett et al., 2019). Besides that, reduced sleep efficiency and increased awaken time are observed in pre-HD carriers. Moreover, a postmortem study indicated that the levels of two regulatory neuropeptides, vasoactive intestinal polypeptide and arginine vasopressin, are lower in the SCN of HD patients compared to controls (van Wamelen et al., 2013).

Age-dependent circadian dysfunction is found also in BACHD, zQ175, and R6/2 mice (Fisher et al., 2013; Loh et al., 2013). In R6/2 mice, besides an increase in daytime activity (Morton et al., 2005), the mRNA levels of three circadian genes (mPer2, mBmal1, and mPK2) were altered in the SCN and may explain circadian defects (Kudo et al., 2011). Additionally, reduced sleep time and reduced SCN neuronal activity with enhanced potassium current was found in BACHD mice, and these phenotypes can be rescued by treatment or injection with NMDA (N-methyl-d-aspartate) (Kuljis et al., 2018). More recently, in R6/2 mice, reduction of the number of intrinsically photosensitive retinal ganglion cells (ipRGC) was found to influence SCN due to ipRGC gene downregulation by mutant HTT-polyQ and impairment of the Tbr2 transcription factor in ipRGC cells (Lin et al., 2019b). To summarize, these results demonstrate that circadian dysfunction is a component of HD that may have profound impact on systemic homeostasis and body functions (Table 9).

Table 9.

Defects in circadian behaviors are found in HD. Abnormalities in the SCN including the alteration in melatonin concentration may contribute to sleep disorders and circadian rhythm disturbances in HD patients.

Circadian behaviors	Phenotype	Model	Stage of HD	Reference
Sleep disorders	Impaired sleep quality, higher blood pressure	HD patients	Early stage	Diago et al., 2017
	Increased night activity	HD patients	NA	Morton et al., 2005
	Alteration in mean melatonin concentration	HD patients	Early stage	Aziz et al., 2009
	Reduced the level of melatonin and mean melatonin concentration	Pre-HD carriers HD patients	Asymptomatic Stage I to III	Kalliolia et al., 2014
	Reduced grey matter volume in the hypothalamus, reduced sleep efficiency, increased awaken time	Pre-HD carriers	Asymptomatic	Bartlett et al., 2019
	Lower level of vasoactive intestinal polypeptide and arginine vasopressin in the SCN, no change in the mRNA level of melatonin	Postmortem HD patients’ brain	Grade II	van Wamelen et al., 2013
	Increased daytime activity, alterations of three circadian genes, mPer2, mBmal1, and mPK2	R6/2 mice	Middle to late stage	Morton et al., 2005
	No changes in circadian genes, age-dependent circadian dysfunction, alteration in body temperature	BACHD mice	Early to late stage	Kudo et al., 2011
	Abnormal non-rapid eye movement sleep period and rapid eye movement sleep period, alteration in body temperature	R6/2 mice	Late stage	Fisher et al., 2013
	Affected daytime sleep, age- and dose- dependent circadian dysfunction	zQ175 mice	Early to late stage	Loh et al., 2013
	Reduced sleep time and SCN neuron activity	BACHD mice	Early stage	Kuljis et al., 2018
	Reduction in the number of intrinsically photosensitive retinal ganglion cells (ipRGCs), HTT-polyQ aggregates, downregulation of Tbr2, downregulation of ipRGC-enriched genes, changes in pupillary light reflex and pupil constriction	R6/2 mice	Middle stage	Lin et al., 2019b

3.6. Retina.

Retinal dysfunction is an early symptom in HD and perhaps contributes to the abnormal perception of the visual stimuli that regulate the circadian cycle. HD patients have higher increment thresholds for a foveal blue test light, which can be used as a noninvasive diagnosis for HD (Paulus et al., 1993). Recently, analysis of a pre-HD carrier found lower retinal response, as assessed with electroretinograms (ERG) (Knapp et al., 2018). However, another study found a higher ERG response in HD patients (Pearl et al., 2017). Despite these conflicting results, retinal dysfunction is an established feature of HD.

HTT-polyQ aggregates and retinal dysfunction are found in both R6/1 and R6/2 mice (Helmlinger et al., 2002). R6/2 mice display age-dependent reduction in the ERG (Helmlinger et al., 2002) whereas R6/1 mice experience age-dependent reduction in cone opsin and in the number of cone cells (Batcha et al., 2012). In addition, HD-associated retinal dysfunction is characterized by higher retinal stress and neuronal remodeling, which is a phenotype that commonly occurs in retinal pathologies. These results suggest that mutant HTT gradually causes retinal dysfunction and degeneration (Table 10).

Table 10.

HTT-polyQ-related phenotypes are observed in the retina. Retinal degeneration and dysfunction are early events in HD.

Retina	Phenotype	Model	Stage of HD	Reference
	Higher increment thresholds	HD patients	Early stage	Paulus et al., 1993
	Higher electroretinogram response	HD patients	Early stage	Pearl et al., 2017
	Reduction of electroretinogram response	Pre-HD carriers	Asymptomatic	Knapp et al., 2018
	Age-dependent reduction of cone opsin and loss of cone cells, higher retinal stress and neuronal remodeling	R6/1 mice	Middle stage	Batcha et al., 2012
	Age-dependent retinal dysfunction, HTT-polyQ aggregates	R6/2 mice	Middle stage	Helmlinger et al., 2002

4. SYSTEMIC EFFECTS OF PATHOGENIC HUNTINGTIN VIA THE BLOODSTREAM

4.1. Circulation.

Blood cells are among the most accessible cells in the organism, so the wide range of effects of pathogenic huntingtin on different blood cells such as erythrocytes and leukocytes has been extensively investigated (for a review of this work, see (Sassone et al., 2009)). Recently, microarray and RNA-seq analyses done from HD blood and brain samples have indicated that the gene expression changes induced in the brain by HTT-polyQ are similar to those found in blood cells, which suggests that the blood can be used to investigate HD in a non-invasive manner in humans (Mina et al., 2016; Moss et al., 2017) and monkeys (Clever et al., 2019). Moreover, other blood-based biomarkers have been established for HD. For example, the neurofilament light (NfL) protein found in the blood of HD patients can be used to predict the disease severity (Byrne et al., 2018; Johnson et al., 2018). Similarly, shorter telomeres and DNA damage signatures in peripheral blood cells are also used as biomarkers to distinguish premanifest from manifest HD mutation carriers (Castaldo et al., 2019). Moreover, inflammatory markers, CRP and IL-6, are higher in the blood of HD patients whereas significantly lower levels are found for thioredoxin reductase-1, thioredoxin-1, and myeloperoxidase (Sánchez-López et al., 2012).

Importantly, bone marrow transplantation has been found to partially improve HD-associated behavioral deficits in YAC128 and BACHD mouse models, indicating that deletion of peripheral cells that express mutant HTT can influence HD pathology in the central nervous system (Kwan et al., 2012). Altogether, these studies indicate that blood cells and the plasma are profoundly affected and may contribute to influence HD.

In the following paragraphs, we will discuss specific examples of how pathogenic huntingtin can impact blood cells and the circulation. Specifically, we will examine the outcome of pathogenic huntingtin activity on platelets and on the blood-brain barrier (BBB).

4.2. Platelets.

Platelets seem to be particularly affected in HD patients. Specifically, there are more HTT-polyQ aggregates (presumably indicative of overall HTT levels) in platelets than in leukocytes and red blood cells, and these HTT-polyQ inclusions arise in a stage-dependent manner, i.e. there are more HTT-polyQ aggregates inside platelets in late stage HD patients (Denis et al., 2019). Although there are no changes in platelet number in HD, platelets appear hyperactive. Specifically, R6/2 mice bleed less and for shorter periods of time and are prone to thrombosis. Moreover, dysregulation of nitric oxide synthase (NOS) was observed in platelets of patients with advanced HD (Carrizzo et al., 2014). Concerning the secretion of signaling factors by platelets, pathogenic HTT was found to associate with platelet alpha granules, suggesting that it may influence the release of factors there stored. Interestingly although the levels of angiogenic factors (ANG-2, HGF, bFGF) in platelets of HD patients did not change, their release decreased upon platelet activation with thrombin and collagen although the release of Pf4 did not change (Denis et al., 2019). Moreover, there are higher serotonin levels in dense granules of platelets from HD patients but its release upon platelet activation is reduced (Denis et al., 2019; Diez-Ewald et al., 1980). In another study, it was found that platelets from HD patients show decreased mitochondrial complex I activity and increased activity of mitochondrial complex IV (Silva et al., 2013). Furthermore, HTT-polyQ aggregates are found also in the mitochondria fraction of platelets. Altogether, these results indicate that pathogenic huntingtin impairs many normal functions of platelets (Table 11).

Table 11.

HTT-polyQ-related phenotypes are observed in platelets and the BBB. HTT-polyQ aggregates in platelets impair the physiological function and mitochondrial activity of platelets. Likewise, HTT-polyQ aggregates in endothelial cells and blood vessels result in decreased levels of tight-junction proteins and poor BBB properties.

Blood	Phenotype	Model	Stage of HD	Reference
Platelets	HTT-polyQ aggregates in platelets, impaired secretion of serotonin and angiogenic factors	HD patients	Stage I to V	Denis et al., 2019
	HTT-polyQ aggregates in cell membranes, alpha granules, and open-canalicular system within platelets	zQ175 mice	Late stage	Denis et al., 2019
	Decreased mitochondrial complex I activity and citrate synthase activity, increased adenosine monophosphate production	Pre-HD carriers	Asymptomatic	Silva et al., 2013
	Decreased activity of the mitochondrial complex I and increased activity of complex IV, HTT-polyQ aggregates in the mitochondria fraction	HD patients	Symptomatic (no score)	Silva et al., 2013
	Dysregulation of the nitric oxide synthase (NOS) pathway, impairment of phosphorylation of endothelial NOS	HD patients	Stage I to IV	Carrizzo et al., 2014
	Lower release of serotonin	HD patients	NA	Diez-Ewald et al., 1980
Blood-Brain Barrier	Smaller blood vessels, decreased tight junction-related proteins, HTT-polyQ aggregates in blood vessels	HD patients	Stage I to V	Drouin-Ouellet et al., 2015
	HTT-polyQ aggregates in blood vessels and endothelial cells, decreased tight junction-related proteins	R6/2 mice	Late stage	Drouin-Ouellet et al., 2015
	Decreased mRNA and protein levels of tight junction-related proteins	R6/2 mice	Middle stage	Di Pardo et al., 2017
	Impaired angiogenesis and poor BBB properties due to Wnt/β-catenin pathway activation	HD patients-derived iPSC, postmortem brain tissues	Grade 2	Lim et al., 2017
Other blood cells	Higher concentration of neurofilament light protein	HD patients	Stage I to II	Byrne et al., 2018
	Shorter telomeres and DNA damage	Pre-HD carriers	Asymptomatic	Castaldo et al., 2019
	Higher level of CRP and IL-6, decreased levels of thioredoxin reductase-1, thioredoxin-1, and myeloperoxidase/white blood cell ratio	HD patients	Late stage	Sánchez-López et al., 2012

4.3. Blood-Brain Barrier.

The BBB is a highly selective semipermeable barrier of endothelial cells that separates the blood from the brain, and that filters the entry of non-essential substances from the blood to the brain and vice versa. A clinical study suggests that there is BBB leakage in HD patients, and that this is possibly due to an increase in smaller blood vessels and a decrease in tight junction components (Drouin-Ouellet et al., 2015). Similar results are also found in the R6/2 mouse model. Specifically, pathogenic HTT-polyQ aggregates in endothelial cells lead to decreased assembly of intercellular tight junctions and to interruption of BBB filtering functions. Moreover, HTT-polyQ aggregates are found in endocytic vesicles that mediate transcytosis in BBB endothelial cells, which suggests that a leaky BBB may help the extracellular spread of HTT-polyQ aggregates between the blood and the brain. Moreover, by employing R6/2 mice, tight junctions were found to be perturbed due to lower mRNA and protein levels of tight junction-related proteins, which resulted in BBB impairment (Di Pardo et al., 2017).

In another study, the BBB was modeled in vitro by obtaining endothelial cells from HD patient-derived induced pluripotent stem cells (iPSC). This study indicated that HD iPSC-derived endothelial cells display impaired angiogenesis and poor BBB properties due to Wnt/β-catenin pathway activation (Lim et al., 2017). In detail, higher migratory capacity and deficits in tight junctions and transcytosis were found for HD iPSC-derived endothelial cells compared to control, and these phenotypes were rescued by Wnt signaling inhibition. Altogether, these studies indicate that pathogenic huntingtin perturbs BBB filtering functions.

Interestingly, defects in BBB permeability are increasingly recognized not only as a component of HD but also as a therapeutic opportunity for HD treatment. Specifically, higher BBB permeability may facilitate the delivery of neurotrophic cytokines to the brain and hence promote neuroprotection and neuronal cell proliferation. In R6/2 mice, focused ultrasound could temporary open the BBB and facilitate the delivery of a liposome-encapsulated GDNF-encoding DNA plasmid to the brain, which resulted in GDNF overexpression, decreased apoptosis and oxidative stress, and improvement of motor function (Lin et al., 2019a).

5. SYSTEMIC INTER-TISSUE CROSSTALK IN HUNTINGTON’s DISEASE

5.1. Inter-tissue transmission of mutant huntingtin.

As we have explained above, mutant HTT is expressed ubiquitously, i.e. not only in the CNS but also in peripheral tissues. Therefore, mutant HTT may directly impact the function of cells in peripheral tissues by locally altering protein quality control in these tissues in a cell-autonomous manner. However, additional possible mechanisms involved in the systemic manifestation of HD may include: 1) spread of HTT-polyQ from one tissue to another tissue; and 2) modulation of expression and/or secretion of signaling factors by peripheral tissues in response to therapeutic regimens for HD (such as exercise) or in response to local activity of mutant HTT in peripheral tissues (Figure 2).

There is fairly extensive evidence that HTT can spread in the extracellular space. One of the first observations consisted in the finding that aggregates of pathogenic HTT can be detected in fetal grafts years after they were transplanted into the brain of HD patients (Cicchetti et al., 2014). Subsequently, several studies have reinforced the notion that HTT-polyQ can spread across different areas of the brain. For example, implantation into the cerebral ventricles of fibroblasts derived from HD patients and that express HTT-polyQ led to the cell non-autonomous transmission of HTT-polyQ in wild-type mice. In turn, this induced major HD features, i.e. motor and cognitive deficits, loss of striatal medium spiny neurons, and increased inflammation and gliosis (Jeon et al., 2016). Similar results were obtained with the injection of homogenates of postmortem brains from HD patients, although lower propagation than with Htt-polyQ fibrils was observed (Gosset et al., 2020; Masnata et al., 2019). Additionally, studies in Drosophila have also established transcellular spreading of human huntingtin throughout the brain (Babcock and Ganetzky, 2015).

Concerning the inter-tissue spread of HTT-polyQ, this may occur via secretion into the bloodstream or via axonal transport in neurons (Kim et al., 2017; Lee et al., 2010; Tang, 2018). Indeed, it has been suggested that circulating HTT-polyQ may spread from/to the brain, gut, blood, muscle, and other tissues in humans and rodents (Li et al., 1993; Sassone et al., 2009), as observed for other pathogenic proteins linked with neurodegeneration. For example, injection of preformed α-synuclein fibrils into the gut of wild-type mice led to endogenous α-synuclein propagation to the brain via the vagus nerve and caused Parkinson’s disease-like pathology (Kim et al., 2019). These phenotypes and α-synuclein transmission were rescued by vagotomy, indicating that the vagus nerve is a possible route for transmission of pathogenic proteins (presumably also HTT-polyQ) from the gut to the brain, an vice versa.

Evidence for inter-tissue transmission of HTT-polyQ was found between skeletal muscle and neurons in C. elegans, and this was linked with neurodegeneration and reduced lifespan (Kim et al., 2017). Specifically, exon 1 of HTT was fused with polyQ and with either N-terminal or C-terminal fragments of the Venus fluorescent protein, and expressed specifically in pharyngeal muscles and neurons, respectively. Subsequently, bidirectional transmission between pharyngeal muscles and neurons was observed by using bimolecular fluorescent complementation technology (Kim et al., 2017), i.e. Venus fluorescence is detected only when the N-terminal and C-terminal fragments are in close proximity within the same cell, which in this case indicates transmission of HTT-polyQ from one cell type to the other. Interestingly, this study determined that pathogenic HTT-Q97 is transmitted more efficiently than wild-type HTT-Q25, and that inter-cellular exchange of HTT-Q97 gradually increases with aging, suggesting that inter-tissue spread of HTT-polyQ is age-regulated (Kim et al., 2017).

Another study reported inter-tissue transmission of HTT-polyQ in N171-82Q mice. In this model, HTT-polyQ is expressed specifically in CNS neurons and in the heart. However, soluble HTT-polyQ proteins were also found in skeletal muscle, indicating spreading of HTT-polyQ from neurons and/or the heart to skeletal muscle (Corrochano et al., 2018).

Lastly, a recent study reported that mutant HTT can spread via the bloodstream in mice (Rieux et al., 2020). In this study, parabiosis was used to join the circulation of wild-type and HD mice (zQ175). Subsequently, mutant HTT was detected in the plasma and circulating blood cells of WT mice. Furthermore, post-mortem analyses revealed the presence of HTT aggregates in the liver, kidney, skeletal muscle, and brain, and this was accompanied by brain vascular abnormalities (Rieux et al., 2020).

Altogether, there is evidence for inter-tissue spreading of HTT-polyQ in animal models. However, it remains unknown whether this is a regulated process or whether HTT-polyQ is released into the extracellular environment upon cell death, and subsequently uptaken via endocytosis by receiving cells. Because HTT can be degraded via the autophagy-lysosome system (Cortes and La Spada, 2014; Kegel et al., 2000), it is possible that once it is within this system it can avoid degradation and be released into the extracellular space via secretory autophagy and lysosomal exocytosis (Buratta et al., 2020).

Alternative means of intercellular HTT-polyQ transport may include long cell protrusions (also known as tunneling nanotubes) and extracellular vesicles such as exosomes (Demontis, 2004; Demontis and Dahmann, 2007; Tang, 2018). For example, it has been recently shown that mutant HTT-polyQ but not normal HTT can be transported by nanotubes via a mechanism that requires the SUMO E3-like protein Rhes (Sharma and Subramaniam, 2019). Although nanotubes are normally involved in transmission between nearby cells, mutant HTT could be transported across longer distances via vesicles such as exosomes (Jeon et al., 2016; Ren et al., 2009).

Altogether, more studies are needed to determine the quantitatively most important mechanisms and routes of HTT inter-tissue transmission. However, it must be noted that the physiological significance of such HTT-polyQ inter-tissue spreading remains unclear as HTT is widely expressed and therefore an influx of mutant HTT from another tissue may not significantly modify protein quality control in target cells that already express mutant HTT. However, it is possible that certain cell types or subpopulations express lower levels or better tolerate mutant HTT (Saudou and Humbert, 2016): in that case, an influx of external HTT may contribute to the collapse of protein quality control and facilitate HD pathogenesis in such cells.

5.2. Signaling factors induced by mutant huntingtin or by therapeutic interventions (exercise).

Another hypothesis for systemic inter-tissue crosstalk in HD is that there could be signaling factors that are produced and/or secreted in response to therapeutic interventions such as exercise or, conversely, in response to HTT activity in peripheral tissues. Such signaling factors may in turn impact whole-body processes and possibly even the activity of pathogenic HTT in the brain. In agreement with this model, parabiosis experiments indicate that HD mice (zQ175) display reduced pathology when their circulation is joined with that of wild-type mice (Rieux et al., 2020). Specifically, reduced mitochondrial dysfunction is found in peripheral organs whereas vessel diameter and expression of key neuronal proteins (DARPP32) is restored in the central nervous system of these HD mice. Altogether, this study pinpoints a key role for circulating factors in modulating HD pathology in peripheral tissues and in the brain (Rieux et al., 2020).

The source tissue(s) of such protective circulating factors remains unexplored but it is possible that skeletal muscle contributes to such pool. The rationale for this hypothesis is that it is well known that exercise protects premanifest HD carriers and manifest HD patients from disease progression, leading to an improvement in cognitive performance, motor function (Busse et al., 2013; Khalil et al., 2013; Quinn et al., 2016), and white matter microstructure (Metzler-Baddeley et al., 2014).

In addition, exercise improves cognitive performance and motor function also in HD mouse models, with a reduction of neuropathology in R6/1 mice (Harrison et al., 2013) and of HTT-polyQ levels in CAG140 mice (Stefanko et al., 2017), and by rescuing corticostriatal synaptic disconnection in R6/2 mice (Cepeda et al., 2010). Motor deficits and spatial working memory were rescued in R6/1 mice presumably at least in part via increased expression of BDNF in the striatum of R6/1 mice (Pang et al., 2006). Moreover, wheel running from a juvenile age delays onset of some motor deficits in R6/1 transgenic mice (van Dellen et al., 2008). Similarly, regular swimming training improved mitochondrial respiration, survival, the health of the intestine, and protected against HD in C. elegans (Laranjeiro et al., 2019). However, endurance exercise was found to exacerbate HD progression in another disease model (Corrochano et al., 2018), suggesting that some forms of exercise could be deleterious for HD.

Together, these studies indicate that certain exercise programs preserve cognitive and motor functions in HD (Table 12), which may indicate a role for inter-tissue signaling between contracting skeletal muscle and the brain in HD. Presumably, muscle-secreted factors (myokines and myometabolites) known to be induced by exercise (Demontis et al., 2014; Demontis et al., 2013; Pedersen, 2019; Pedersen et al., 2007; Rai and Demontis, 2016) may contribute to such systemic benefits of exercise on HD pathogenesis.

Table 12.

Improvement and enhancement of HD pathology by exercise. In most of conditions, exercise improves HD pathology.

Effect of exercise on HD	Improvement	Model	Stage of HD	Reference
	Motor function	Symptomatic HD patients	Early-Mid stage	Busse et al., 2013
	Motor function	R6/1 mice	Early stage	Van Dellen et al., 2008
	Cognitive performance but no rescue of motor coordination	R6/1 mice	Early stage	Pang et al., 2006
	Cognitive performance	Symptomatic HD patients	Mid stage	Khalil et al., 2013
	Fitness improvement and motor function	Symptomatic HD patients	Mid stage	Quinn et al., 2016
	Cognitive improvement and alteration of white matter microstructure	Symptomatic HD patients	Early-Late stage	Metzler-Baddeley et al., 2014
	Motor function	R6/1 mice	Middle stage	Harrison et al., 2013
	Non-motor behavior improvement and reduced HTT-polyQ aggregates	CAG140 knock-in mice	Early stage	Stefanko et al., 2017
	Rescued corticostriatal synaptic disconnection and motor function	R6/2 mice	Early stage	Cepeda et al., 2010
	Mitochondrial respiration and survival	C. elegans	Specific expression of HTT-polyQ in neurons	Laranjeiro et al., 2019

Terminology (relevant to all tables):

HD stages in HD patients: TFC stages are defined by TFC scores from stage I (least severe) to stage V (most severe). (References by Paulsen et al., 2010; Shoulson & Fahn, 1979).

UHDRS scores, from 0–124, higher scores indicate a higher level of motor function impairment. Early stage: <25, middle stage: 25–50, late stage: >50. (Reference by Kieburtz et al., 2001).

Postmortem brain tissues are scored from grade 0 (no abnormality) to grade 5 (most severe). (Reference by Vonsattel et al., 1985).

HD stages in R6/1 mice: early stage: <14 months, middle stage: 15 to 21 months, and late stage: >22 months. (Reference by Mangiarini et al., 1996).

HD stages in R6/2 mice: early stage: <6 weeks, middle stage: 6 to 11 weeks, and late stage: >12 weeks. (References by Mangiarini et al., 1996; Di Pardo, A et al., 2017).

HD stages in BACHD mice: early stage: <6 months, middle stage: 6 to 11 months, and late stage: >12 months. (Reference by Gray et al., 2008).

HD stages in N171-82Q mice: early stage: <8 months, middle stage: 8 to 16 months, and late stage: >17 months. (Reference by Schilling et al. 1999).

HD stages in CAG140 knock-in mice: early stage: <4 months, middle stage: 4 to 11 months, and late stage: >12 months. (Reference by Menalled et al., 2003).

HD stages in zQ175 mice: early stage: <4 weeks, middle stage: 5 to 11 weeks, and late stage: >12 weeks. (Reference by Menalled et al., 2012a).

HD stage in Hdh150 knock-in mice: early stage:<8 months, middle stage: 8 to 14 months, and late stage: >15 months. (Reference by Lin et al. 2001).

NA: not applicable

Drosophila HD models: Tissue specific Gal4 drivers were used, in combination with Gal4-responsive transgenes, including UAS-Httex1-polyQ-GFP lines, which are used to express GFP-tagged pathogenic human huntingtin (first 16 amino acids of exon 1) with polyQ of different lengths (Q25, Q46, Q72, and Q103).

C. elegans HD models: P_mec-3htt57Q128 was used to express the first 57 amino acids of human Huntingtin protein fused with polyQ.

Although such factors that influence brain HD pathogenesis have not yet been identified, there is growing evidence for muscle-to-brain signaling via myokines (Delezie and Handschin, 2018; Demontis and Perrimon, 2010; Lourenco et al., 2019; Rai and Demontis, 2016; Robles-Murguia et al., 2020). On this basis, it is possible that these and/or other myokines may provide an avenue for muscle-to-brain signaling also in the context of HD, and that they may thus influence HD pathology in the central nervous system.

6. CONCLUSIONS

Beyond the well-established mechanisms of HD progression in the central nervous system, here we have reviewed growing evidence indicating that also peripheral tissues are affected in HD and impact whole body functions. In addition, systemic signaling originating from peripheral tissues can influence HD progression in the brain.

It remains largely unknown whether peripheral pathology is a cause or a result of brain neurodegeneration, and associated cognitive, psychiatric and motor symptoms in HD. Presumably, expression of mutant HTT in a given tissue is the primary cause for HD-associated defects locally seen in that tissue. For example, HD-associated cognitive, psychiatric and motor symptoms are presumably primarily driven by changes in the brain. However, based on the studies here reviewed, we propose that some aspects of local HD pathogenesis in a given tissue/organ are likely influenced by crosstalk of such tissue/organ with other tissues. In this model, HD pathogenesis in the brain is influenced by signals from peripheral tissues. Specifically, it is possible that peripheral changes could modulate brain dysfunction, and thus alter onset and/or progression of cognitive, psychiatric, and motor symptoms. Likewise, HD pathology in a peripheral tissue may also be influenced by signals from the brain and from other peripheral tissues.

Although much remains to be learnt on inter-tissue signaling in HD, the studies here reviewed suggest that treatment strategies for HD should include the amelioration of HD symptoms in peripheral tissues. Moreover, harnessing signaling between peripheral tissues and the brain may provide a means for reducing HD progression in the central nervous system.

Highlights:

• Mutant HTT expression in peripheral tissues impacts systemic whole-organism functions

• HTT expression in peripheral tissues induces systemic body wasting (cachexia) and dysfunction in metabolic homeostasis

• Signaling factors produced and/or secreted in response to therapeutic interventions or HTT may modify HD progression.

• Harnessing signaling between peripheral tissues and the brain may provide a means for reducing HD progression.