Polyglutamine diseases are a family of nine neurodegenerative diseases that includes spinal and bulbar muscular atrophy (SBMA); Huntington’s disease (HD); dentatorubral pallidoluysian atrophy (DRPLA); and spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, and 17 [
1,
2]. Polyglutamine diseases are caused by expansions of the cytosine-adenine-guanine (CAG) trinucleotide repeat in the exons of specific genes. These genes code for unrelated proteins, that is, androgen receptor (AR), huntingtin (HTT), atrophin-1, ataxin-1, ataxin-2, ataxin-3, α1a-subunit of the P/Q voltage-dependent calcium channel (
CACNA1A), ataxin-7, and the TATA-box binding protein (TBP). CAG expansions result in the production of proteins with aberrantly expanded polyglutamine tracts. All polyglutamine diseases are autosomal dominant, except SBMA, which is X-linked. Polyglutamine diseases belong to the family of brain misfolding diseases, which are a large group of neurodegenerative disorders that also includes Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and many others [
3]. Brain misfolding diseases represent a major health burden for the entire world with an estimated number of more than 30 million patients in the next 50 years [
4,
5]. Brain misfolding diseases share several commonalities, such as being late-onset and progressive diseases. Symptoms typically manifest around the third to fifth decade of life, except for the juvenile forms observed in patients with very long repeats [
6,
7]. Another key feature is neuronal loss, and neurons seem to be extremely vulnerable in these conditions despite the fact that, very often, the disease-related proteins have ubiquitous expression and housekeeping functions in the cells [
8,
9]. One such example is TBP, which is the universal basal transcription factor expressed in all cell types and controlling the expression of nearly all genes.
2. Clinical Presentation and Disease Pathogenesis of Polyglutamine Diseases
Polyglutamine expansions cause neurodegeneration mainly through toxic gain-of-function mechanisms. It is worth noting that patients may also show symptoms that partially overlap with loss-of-function mutations of the polyglutamine protein-encoding genes. This scenario is particularly evident in SBMA, thus indicating that loss-of-function mechanisms also contribute at least in part to disease pathogenesis.
HD is characterized by the dysfunction and loss of cortico-striatal neurons [12], with clinical manifestations spanning from cognitive to psychiatric and motor (chorea) symptoms. Disease progression is associated with body weight loss and progressive skeletal muscle weakness, wasting, and atrophy. HTT is widely expressed in the body, including skeletal muscle. At the subcellular level, HTT localizes to the nucleus, cytosol, endoplasmic reticulum, Golgi apparatus, and mitochondria. In the neurons, HTT has also been detected in the synaptic compartment. HTT has been implicated in several processes, from the synthesis of brain-derived neurotrophic factor (BDNF) to vesicular trafficking, ciliogenesis, and others.
SBMA is the only polyglutamine disease that is X-linked and male-specific [13,24]. The reason for the sex bias of SBMA is that males have higher circulating androgen levels than females, and polyglutamine-expanded AR requires androgen binding to exert most of its neurotoxic effects. SBMA is characterized by the loss of lower motor neurons and primary involvement of skeletal muscle.
DRPLA is characterized by the degeneration of neurons in the dentate nucleus of the cerebellum and pallidum [
14]. Patients present with cerebellar ataxia, chorea, epilepsy, and dementia. Atrophin-1 localizes mainly to the cytosol, but it is also present in the nucleus and is involved in protein trafficking and degradation.
Polyglutamine ataxias are characterized by the primary loss of Purkinje cells [
28]. Particularly interesting is the case of SCA17, as TBP regulates the expression of genes transcribed by the three eukaryotic RNA polymerases in the nucleus.
Within the family of polyglutamine disease-causing proteins, AR and TBP are the most well-known proteins in terms of structure and function; they are both transcription factors. TBP is composed of two domains, the amino-terminal domain that is intrinsically disordered, and the carboxy-terminal domain that is highly conserved and ordered into beta-sheets motifs. TBP binds to TATA-box or is recruited by basal transcription factors to the TATA-less promoters and plays a fundamental role in the process of initiation of transcription. AR is composed of three domains. Similar to TBP, AR has an intrinsically disordered amino-terminal domain, a DNA-binding domain (two zinc fingers,) and the ligand-binding domain that forms 12 alpha-helices and two beta-sheets. How polyglutamine expansions affect TBP and AR function is not known.
3. Muscle Pathology in Polyglutamine Disease Patients
Polyglutamine-expanded proteins cause damage to several tissues through cell-autonomous and noncell-autonomous mechanisms [29,30]. Peripheral symptoms are important components of HD manifestations, highlighting the relevance of peripheral toxicity of mutant HTT in the onset and progression of disease [31]. It is interesting to note that CAG expansions in HD patients were reported to be higher in skeletal muscle compared to lymphocytes, which may suggest why skeletal muscles are more prone to or are a primary site of the disease [32]. HD patients undergo progressive severe body mass loss, which is mainly due to skeletal muscle wasting [33]. HD patients show skeletal muscle weakness and signs of peripheral motor pathology, such as defects in eye movements and swallowing, gait abnormalities, reduced lower limb muscle strength, and dysarthria [34,35,36]. Changes in body composition and muscle wasting were not detected in a cohort of HD patients with early-to-moderate symptoms [37]. However, reduction of lean and fat body mass was reported in other HD patient cohorts as an early phenomenon [38].
SBMA represents the only neuromuscular disease within the family of polyglutamine diseases with selective degeneration of lower motor neurons resulting in skeletal muscle atrophy [42]. SBMA patients show fatigue, muscle weakness and atrophy, fasciculations, and reduced contractility [43]. SBMA muscles are characterized by neurogenic signs, such as fiber atrophy and fiber-type grouping, and myopathic changes, such as the presence of centronucleated fibers and fiber splitting and degeneration [44]. SBMA patients have very high levels of serum creatine kinase, much more than observed in patients suffering from neurogenic atrophy [45], even years before the appearance of symptoms [46]. Interestingly, in SBMA patients, the severity of peripheral symptoms directly correlates with the length of the pathogenic CAG repeat [47,48]. Although high androgen levels are responsible for disease manifestations in SBMA, an intriguing observation was that muscle strength positively correlates with testosterone levels in patients [49]. Thus, in SBMA patients, muscle atrophy is likely to result from the combination of toxic gain-of-function and loss-of-function mechanisms.
4. Skeletal Muscle Pathology Is Recapitulated in Mouse Models of Polyglutamine Diseases
Many of the peripheral symptoms observed in patients suffering from polyglutamine diseases are well recapitulated in R6/2 mice, which express a portion of the human HD gene under human gene promoter elements (1 kb of 5 UTR sequence and exon 1 together with ~120 CAG repeats) and represent a valuable model of HD, and knock-in mice expressing HTT-150Q [
58,
59], SCA17 [
60], and SBMA [
61,
62,
63]. HD mice develop diabetes and metabolic syndrome, motor dysfunction, mitochondrial abnormalities, skeletal muscle weakness and wasting, and body weight loss [
64,
65,
66]. Some of these symptoms, namely glucose intolerance and insulin insensitivity, dyslipidemia, muscle wasting, and body weight loss, have been described also in knock-in and transgenic SBMA mice [
41,
67], as well as in knock-in mice with either pan- or muscle-specific expression of mutant TBP modeling juvenile forms of SCA17 [
60]. HD R6/2 transgenic mice with a pathologically expanded CAG tract show motor abnormalities starting early and progressively enhancing in terms of severity and manifestations. Motor dysfunction is associated with muscle denervation, neuromuscular junction (NMJ) abnormalities, defects in muscle contraction and calcium dynamics, and loss of muscle regenerative capacity [
64,
68]. Different from SOD1-linked ALS, HD, SBMA, and SCA17 mice do not develop paralysis. This is consistent with a lack of structural denervation. Rather, polyglutamine disease models show mild-to-severe functional denervation, with severe structural abnormalities at the NMJ occurring at the late stage of disease and not associated with motor neuron loss, as reported in several SBMA mice [
61,
62]. Defects in neuromuscular transmission are associated with skeletal muscle hyperexcitability in knock-in and transgenic SBMA mice [
69,
70].
In the case of SBMA, the development of multiple animal models for conditional expression of polyglutamine-expanded AR has allowed establishing that skeletal muscle is a primary target of toxicity. Overexpression of polyglutamine-expanded AR in all tissues but skeletal muscle prevented the development of symptoms in transgenic mice, indicating that expression of the disease protein is necessary for triggering disease manifestations at least in mice [
71]. Conversely, overexpression of non-expanded AR solely in skeletal muscle elicited a phenotype resembling SBMA, indicating that dysregulation of AR signaling is toxic to muscle [
72].
5. Pathological Processes in the Skeletal Muscle in Polyglutamine Diseases
Aggregation and inclusion body formation A hallmark of neurodegenerative diseases is the deposition of mutant proteins into inclusion bodies, which sequester cellular proteins, such as components of the ubiquitin-proteasome system and autophagy degradation machineries causing alterations of the cellular proteome. Inclusion bodies are present in the central nervous system of HD patients and animal models of disease. Importantly, inclusion bodies have been detected not only in neurons but also in skeletal muscle fibers and other peripheral tissues of R6/2 mice and knock-in mice expressing HTT-150Q [
58,
76,
77], as well as in primary myotubes derived from skeletal muscle biopsies of HD patients [
56]. Similarly, inclusions have been detected in the skeletal muscle of mouse models of SBMA [
61,
63,
78,
79]. Inclusions positive for mutant HTT and AR are found in the cytosol and nucleus as a single structure in most cases, and their appearance correlates with the onset of muscle atrophy.
Mitochondrial pathology. Body weight and skeletal muscle function and homeostasis are tightly linked to mitochondrial function. HD muscles are characterized by mitochondrial dysfunction with defects in oxidative phosphorylation and ATP production in patients and mouse models [
84,
85,
86]. These deficits are detected in presymptomatic patients, indicating that they occur early and are possibly causative of muscle atrophy and energy unbalance. The relevance of skeletal muscle in body homeostasis is underlined also by the observation that the R6/2 mice present with elevated energy expenditure, reduced body weight, and increased adiposity, all defects not due to reduced food intake [
56]. HD myofibers show mitochondrial depolarization, reduced oxygen consumption, cytochrome c release, oxidative stress, and induction of apoptosis [
56]. ATP production in skeletal muscle mitochondria is reduced not only in HD patients but also in DRPLA patients, indicating impaired energy metabolism [
65]. Mitochondrial pathology is a key component of SBMA muscle pathology, as reported in many cellular and animal models of SBMA [
27,
41,
87,
88,
89,
90].
Altered muscle metabolism and fiber-type switch. Both SBMA [
41,
67] and HD [
64,
66,
94,
95] muscles undergo a glycolytic-to-oxidative metabolic switch associated with fast-to-slow fiber-type change. Loss of type IIb fibers and switch to type I fibers was also reported in a juvenile DRPLA patient [
40]. The glycolytic-to-oxidative switch precedes signs of denervation and neuropathy in SBMA knock-in mice [
41] and transgenic mice [
61]. These metabolic alterations are associated with fiber-type changes, with progressive loss of type II glycolytic fibers and concomitant increase in type IIa and IIx fibers as the disease approaches advanced stages. Consistent with these metabolic alterations, SBMA muscle is characterized by an early down-regulation of expression of glycolytic genes and a concomitant up-regulation of oxidative genes, again long before the appearance of neurogenic signs of muscle pathology.
Activation of anabolic and catabolic pathways in the skeletal muscle of polyglutamine diseases. Skeletal muscle homeostasis is maintained by the balance between anabolic and catabolic pathways. Expression of polyglutamine-expanded AR and polyglutamine-expanded HTT results in increased protein synthesis and activation of protein kinase B (also known as Akt)/mechanistic target of rapamycin (mTOR) pathway in skeletal muscle [
41,
98]. Activation of these anabolic pathways likely occurs to compensate for increased energy demand. At the same time, activation of catabolic pathways leads to protein degradation via proteasome and autophagy. SBMA muscle is indeed characterized by the upregulation of expression of the atrogenes, genes that are induced upon muscle damage and atrophy and that are involved in protein degradation [
99]. At the same time, genes involved in autophagy are upregulated, likely with the involvement of transcription factor EB (TFEB) [
41], which is dysregulated in polyglutamine diseases [
100]. Chronic activation of these pathways is associated with activation of caspases that result in cell death via apoptosis, inflammation, and atrophy [
99].
Gene expression dysregulation. Polyglutamine expansions affect gene expression. Interestingly, gene expression changes are similar in the central nervous system and skeletal muscle of transgenic and knock-in HD mice [
94]. Importantly, gene expression changes have been detected in skeletal muscle not only in HD mice but also in patients [
66]. Among the genes dysregulated in HD and SBMA transgenic and knock-in mice, several genes code for proteins involved in skeletal muscle contractility and reactivation of expression of denervation markers associated with the loss of motor units [
41,
62,
67,
101,
102]. Importantly, it was reported that HD muscles were characterized by early loss of expression of muscle chloride CIC-1 and potassium Kv3.4 channels, resulting in muscle hyperexcitability [
103,
104].
6. Modeling Skeletal Muscle in a Dish
With the exception of SBMA, analysis of skeletal muscle pathology in patients suffering from polyglutamine diseases is limited by the fact that muscle biopsies are not available. Due to the severity of these conditions, muscle biopsies are not routinely performed unless recommended by the physician. Muscle biopsies give the possibility to grow primary satellite cells that can be differentiated into myoblasts and myotubes for further analysis [
57,
106]. An alternative approach is represented by the use of induced pluripotent stem cells (iPSCs) derived from skin biopsies of patients and healthy controls. These cells can be differentiated into satellite cells [
107,
108], in two-dimensional (2D) cell cultures [
109], and in three-dimensional (3D) human microphysiological systems that mimic the key structural and functional properties of skeletal muscle [
110,
111,
112]. The iPSCs obtained from patients can be differentiated into several cell types, such as motor neurons, astrocytes, microglia, medium spiny neurons, neural precursors, and retinal photoreceptors [
113,
114,
115,
116,
117,
118,
119]. SBMA, HD, and SCA iPSC-derived neural cells exhibit defects that recapitulate the principal pathological features observed in mouse models, suggesting that these cells are an extremely relevant model to study biochemical/molecular pathways and to screen drugs with beneficial effects. Polyglutamine iPSC-derived muscle cells will give researchers much information regarding the onset of disease, progression, and cell-autonomous mechanisms.
7. Conclusions
The relevance of skeletal muscle homeostasis in neurodegenerative diseases is underlined by the fact that this tissue is a valuable therapeutic target. Intervention to ameliorate skeletal muscle metabolism and reduce atrophy is effective in animal models of polyglutamine diseases. Pharmacologic inhibition of myostatin ameliorates muscle atrophy and body weight loss, indicating that it has beneficial effects on the phenotype of HD mice [
132]. Ghrelin administration to HD mice ameliorates muscle atrophy and pathology, further supporting the idea that skeletal muscle is important in HD pathogenesis and is a key target tissue for therapy development [
133]. Pharmacologic strategies to reduce polyglutamine-expanded AR expression in skeletal muscle ameliorates disease in knock-in SBMA mice [
74]. Genetic and pharmacologic strategies to stimulate insulin-like growth factor 1 signaling ameliorate the phenotype of transgenic SBMA mice [
23,
134]. Moreover, treatment of SBMA mice with the beta-agonist, clenbuterol, attenuates symptoms [
75], an approach that has benefits also in SBMA patients [
135,
136]. Therefore, strategies to delay or attenuate skeletal muscle westing and atrophy and improve metabolism may have beneficial effects on body metabolism and central nervous system function.
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