Understanding the potential causes of gastrointestinal dysfunctions in multiple system atrophy

Multiple system atrophy (MSA) is a rare, progressive neurodegenerative disorder characterised by autonomic, pyramidal, parkinsonian and/or cerebellar dysfunction. Autonomic symptoms of MSA include deficits associated with the gastrointestinal (GI) system, such as difficulty swallowing, abdominal pain and bloating, nausea, delayed gastric emptying, and constipation. To date, studies assessing GI dysfunctions in MSA have primarily focused on alterations of the gut microbiome, however growing evidence indicates other structural components of the GI tract, such as the enteric nervous system, the intestinal barrier, GI hormones, and the GI-driven immune response may contribute to MSA-related GI symptoms. Here, we provide an in-depth exploration of the physiological, structural, and immunological changes theorised to underpin GI dysfunction in MSA patients and highlight areas for future research in order to identify more suitable pharmaceutical treatments for GI symptoms in patients with MSA.


Introduction
Multiple system atrophy (MSA) is a rare, progressive neurodegenerative disorder characterised by autonomic, parkinsonian, pyramidal, and/or cerebellar dysfunction.The estimated incidence of MSA worldwide is 0.6-0.7 cases per 100,000 person-years (Gilman et al., 2008) and onset typically occurs between 55 and 60 years of age (Wullner et al., 2007).MSA affects males and females equally and has a 7-to 9-year mean survival time from symptom onset (Schrag et al., 2008).
The causes of MSA remain unknown, however, the pathophysiological basis is believed to centre around the involvement of oligodendroglial cytoplasmic inclusions containing intracellular protein aggregates of α-synuclein (α-syn) (Papp et al., 1989;Papp and Lantos, 1994), which drive neuronal loss within the central nervous system (CNS).Broadly, MSA is subdivided into two clinical phenotypical categories: cerebellar (MSA-C) or parkinsonian (MSA-P); however several overlaps can be observed with autonomic failure seen in both subtypes, which often includes postural hypotension, urogenital dysfunction, incontinence, temperature dysregulation, and sleep disturbances (Parikh et al., 2002).In addition, gastrointestinal (GI) dysfunctions are a common feature of disease and include dysphagia (difficulty swallowing), abdominal pain and bloating, nausea, gastroparesis (delayed gastric emptying), faecal incontinence, and constipation (Mishima et al., 2022;Tanaka et al., 2012;Taniguchi et al., 2015;Thomaides et al., 2005).
Functional changes in the GI tract have been widely studied in both Parkinson's disease (PD) and MSA using various non-invasive methodological approaches.However, unlike PD, studies on the structural changes that occur in the GI tract that may contribute to disease-related dysfunctions are limited in MSA, with most research focusing on the gut microbiome (Engen et al., 2017;Tan et al., 2018;Wan et al., 2019).Although the gut microbiome is known to play a critical role in maintaining GI physiology, and likely contributes to functional deficits in the GI tract, this review aims to explore other potential pathophysiological mechanisms that could contribute to gut dysfunctions in MSA and highlights potential areas for therapeutic intervention that may prove successful in alleviating GI deficits.

Gastrointestinal symptoms in MSA
Individuals with MSA have been shown to exhibit varying symptoms of GI dysfunctions including dysphagia, abdominal pain and bloating, nausea, gastroparesis, faecal incontinence, and constipation (Garg et al., 2020;Mishima et al., 2022;Sakakibara et al., 2004;Stocchi et al., 2000;Tanaka et al., 2012;Taniguchi et al., 2015;Thomaides et al., 2005).Whilst characterisation of GI deficit phenotypes in patients with MSA has been largely successful, variability in the methodology used to assess GI function, limitations with self-reporting, variations in patient sample sizes, and interference of medications, have collectively made it difficult to draw conclusions on the severity of GI deficits and at what disease stage deficits appear (Table 1).Moreover, most studies present MSA as a general disorder and do not differentiate between the two subtypes, which may also hamper our understanding of what drives GI dysfunctions in MSA.However, it should be noted that some studies have found significant differences between MSA subtypes, with the use of laxatives being more prominent in MSA-P than MSA-C patients, and a higher frequency of individuals with MSA-P suffering from constipation than MSA-C (Garg et al., 2020;Mishima et al., 2022;Zhang et al., 2017).However, whether these differences can be attributed to pathophysiology remains unclear.

Central causes of GI dysfunction in MSA
There is evidence of GI dysfunction across various regions of the GI tract in patients with MSA, however, the anatomical pathways and pathophysiology that contribute to GI symptoms remain unclear and are believed to be multifaceted.In the CNS, MSA has been shown to affect multiple physiologically important areas that are critical in facilitating neural control of GI function (Browning and Travagli, 2014;Sato et al., 2007).
Patients with MSA-C predominantly show degeneration in specific regions of the cerebellum, which includes the cerebellar vermis and hemispheres, dentate nucleus, inferior olivary nuclei, pontine basis and pontocerebellar fibers (Jellinger, 2014).Neuronal loss in these regions may be associated with GI dysfunction given the role of the cerebellum in the modulation of gut function.For instance, the neurons of the cerebellum have been found to be activated following gastric distention in both human (Ladabaum et al., 2001) and rodents (Min et al., 2011).Moreover, the cerebellum can modulate GI function by way of indirect inputs to the brainstem (Lisander and Martner, 1975;Manchanda et al., 1972), where stimulation of the cerebellum triggers vagally dependent and independent pathways, which lead to intestinal and gastric motility, as well as gastric acid secretion (Lisander and Martner, 1975;Manchanda et al., 1972).Indeed, gastric emptying has been shown to be a feature of MSA-C (Tanaka et al., 2012).However, individuals with MSA-P (Thomaides et al., 2005) and PD (Soliman et al., 2021), where neuron loss and damage in the CNS is more widespread (Mirdamadi, 2016), also suffer from gastroparesis indicating other structures of the central and peripheral nervous system are likely implicated.
The locus coeruleus (LC), located in the dorsal pons of the brainstem, also undergoes neuronal degeneration in both subtypes of MSA (Benarroch et al., 2008), which may have functional implications within the GI tract.The LC is the primary source of the neuromodulator norepinephrine (NE) in the brain (Benarroch, 2009).Neurons of the LC have extensively branched axons that project throughout the brain and provide the only source of NE to the neocortex, cerebellum, hippocampus, and most of the thalamus (Benarroch, 2009).In relation to GI physiology, the LC possesses efferent connections, including reciprocal connections with GI regulation centres in the CNS, such as the brainstem and hypothalamus (Browning and Travagli, 2014;Cunningham Jr. and Sawchenko, 1988;Kobayashi et al., 1974).Anatomical and physiological studies have revealed activation of LC neurons in a pressuredependent manner following distention of the stomach or colon, with colonic-distention-inducing an increase in LC neuronal activity likely dependent on the release of corticotropin releasing factor (CRF) from Barrington's nucleus (BN) (Valentino et al., 1995).Interestingly, microinjection of CRF into the LC of healthy rats stimulates and accelerates colonic motility and transit time (Monnikes et al., 1996;Tack et al., 2006).Although showing no influence on motility and emptying of the stomach, microinjection of CRF into the LC was found to inhibit gastric acid secretion, which may occur via spinal pathways as vagotomy shows no effect upon CRF (Monnikes et al., 1996).Thus, it could be speculated that the delayed gastric emptying observed in patients with MSA (Tanaka et al., 2012;Thomaides et al., 2005) may reflect or be exacerbated by the above-mentioned pathological feature of MSA.
Like PD, the dopaminergic neurons of the substantia nigra (SN) are also affected in MSA and could contribute to GI dysfunction.Experimentally induced degeneration of dopaminergic neurons in the SN using injection of 6-hydroxydopamine (6-OHDA) can result in GI dysmotility (Blandini et al., 2008;Pellegrini et al., 2016).GI motility perturbations may reflect changes in enteric neurotransmission as many enteric neurotransmitters involved in motility, such as dopamine, nitric oxide, and vasoactive intestinal peptide, are altered following central 6-OHDA administration (Fornai et al., 2016;Zhu et al., 2012).For instance, injection of 6-OHDA into the medial forebrain bundle of rats induces impaired excitatory neurotransmission characterised by loss of myenteric neuronal choline acetyltransferase positivity, a decrease in acetylcholine release, abnormal smooth muscle motor activity, and decreased colonic transit rate (Fornai et al., 2016).In addition, unilateral substantia nigra administration of 6-OHDA has been shown to delay gastric emptying and induce constipation, which is speculated to relate to an increase in GI tyrosine hydroxylase (catalyst for dopamine precursor) and neuronal nitric oxide synthase in the gastric antrum and proximal colon (Zhu et al., 2012).
Other regions of the basal ganglia, including the dorsolateral caudal putamen and the caudate nucleus, which are susceptible in individuals with MSA, may contribute to GI symptoms in these patients.The putamen and the globus pallidus are also involved in colonic motility as stimulation of the putamen and the globus pallidus through either centrally administered dopamine D1/D2 receptor agonists or electrical stimulation can evoke colonic contractions likely via BN (Bueno et al., 1992;Valentino et al., 1999).Thus, lesions of the basal ganglia may be involved in prolonged colonic transit time (CTT) in the rectosigmoid segment and total colonic transit, which are observed in patients with MSA (Sakakibara et al., 2004).Indeed, constipation has been reported to be higher in patients with MSA-P when compared to MSA-C (Mishima et al., 2022;Zhang et al., 2017) where greater involvement of the basal ganglia is reported (Kass-Iliyya et al., 2015).
Control of pelvic-perineal muscles, which play a central role in erection, urinary/faecal continence, and ejaculation, are largely facilitated through Onuf's nucleus (ON) (Schellino et al., 2020).In humans, ON is located within the ventral horn of the upper sacral segment at position S2-S3 (rarely S1).ON shows regulatory roles over lower abdominal striated musculature, such as the sphincter urethrae membranaceous muscle, external urethral sphincter muscle, the levator ani, and the external anal sphincter (Gerrits et al., 1997;Mannen, 2000;Schellino et al., 2020).An important pathological hallmark found is MSA-P is neurodegeneration of ON (Schellino et al., 2020;Wenning et al., 1997).Neurogenic changes in ON are not apparent early in MSA disease course, but are observed at later stages with increasing incontinence and detrusor muscle over-activity (Yamamoto et al., 2005;Yamamoto et al., 2014).It has been proposed that degeneration of ON may be responsible for denervation of the external urethral sphincter and external anal sphincter (EAS) leading to the progressive loss of urinary and bowel function (O'Sullivan et al., 2008;Yamamoto et al., 2005).Indeed, MSA patient sphincter electromyographs have shown denervation of motor unit potentials of up to 75 to 100% (Bannister, 1992;Campese et al., 2021;Palace et al., 1997;Sakakibara et al., 2004).
MSA patients appear to demonstrate dysfunction of the recto-anal inhibitory reflex, which in healthy subjects is described by the temporary and involuntary relaxation of the internal anal sphincter (IAS) in response to distention of the rectum (Zbar et al., 1998).This enables the upper anal canal to discriminate between flatus or faecal material facilitating the process of anal sampling (Zbar et al., 1998).Consistent with degeneration of ON, this reflex is found to be impaired in MSA patients as both rectal and anal pressures are observed to increase (Edwards et al., 1994;Sakakibara et al., 2004;Stocchi et al., 2000), but whether this is subtype specific remains unclear.It has also been speculated that paradoxical anal sphincter contraction on defecation (anismus), which occurs in patients with PD and MSA, could arise following degenerative lesions in the spinal tract.Resting anal pressure is primarily facilitated by the sympathetic nervous system, which provides innervation via intermediolateral (IML) columns and autonomic ganglia (Browning and Travagli, 2014).Patients with MSA are seen to have low resting anal pressure, which may suggest the presence of degenerative lesions in the IML columns, autonomic ganglia, or both (Bethlem and Den Hartog Jager, 1960;Stocchi et al., 2000).Additionally, the occurrence of anismus has been observed in patients with spinal cord injury or anaesthetic block at the thoracic 12 to lumbar 2 spinal level suggesting the cause may be localised in the suprasacral descending pathway to the external sphincter (Frenckner and Ihre, 1976;Lynch et al., 2001;Sakakibara et al., 2011).Fig. 1 provides a summary of the neuroanatomical regions in the CNS that are potentially involved in causing GI deficits in MSA.
Although there are limited studies regarding the contribution of CNS damage to GI dysfunction in MSA, evidence indicates that the CNS plays an important role.To date, many MSA studies use PD as a template, despite evidence that the two pathologies differ greatly when it comes to both CNS and GI phenotype.Thus, more studies are needed to better  understand the role of the CNS in MSA-related GI dysfunctions.Schematic diagram of central and peripheral neuronal tracts and regions thought to be implicated in MSA-related GI dysfunctions.Central regions regulated by Barrington's nucleus (BN), and regions that include the cerebellum, basal ganglia (BG), locus coeruleus (LC) and dorsal medial vagus (DMV) are often lesioned in patients with MSA.Similarly, peripheral spinal pathways implicated in gut function, such as the intermediolateral spinal (IML) tract and Onuf's nucleus (ON), also show evidence of degeneration in patients diagnosed with MSA.ON plays an integral role in coordinating contraction and relaxation of the internal (IAS) and external anal sphincters (EAS), which appear to be dysregulated in patients with MSA.

The enteric nervous system in MSA-related GI dysfunction
The enteric nervous system (ENS) is a collection of nerve cell bodies, their processes, and enteric glial cells embedded within the wall of the GI tract.Broadly separated into two ganglionated plexuses within the anatomical layers of the GI tract, referred to as the myenteric and submucous plexuses (Furness, 2012), these plexi work in unison to modulate GI function.The myenteric plexus is located between the longitudinal and circular layers of the muscularis externa and extends from the proximal oesophagus to the internal anal sphincter of the GI tract.The myenteric plexus is primarily involved in the initiation and control of inherent myogenic activity of the longitudinal and circular muscle layers, which facilitates gut motility (Furness et al., 2015), while absorption, secretion, and motor control of smooth muscle is primarily controlled by the submucosal plexus, which is located between the smooth muscle layers and mucosa (Furness et al., 2015).
The presence of enteric neuropathy could be suggested by impaired colonic motility and transit time observed in MSA patients (Sakakibara et al., 2004).Slowed CTT reflects decreased contraction of colonic smooth muscle, which is a major cause of decreased bowel frequency in MSA patients (Sakakibara et al., 2004).Lower GI tract motility is primarily facilitated by slow waves that are identified in the myenteric and submucosal plexi, where interstitial cells of Cajal (ICC) exist (Sanders et al., 2016).ICCs are known as the pacemaker cells of the GI tract, giving rise to slow waves that are generated by ICCs and conducted to adjacent smooth muscle cells through gap junctions enabling generation of phasic contractions (Sanders et al., 2016).Previous studies in other GI disorders have shown that reduced numbers of ICCs and changes in the number of neurons in the myenteric plexus that express the excitatory neuropeptide substance P are associated with slow-transit constipation (He et al., 2000;Tzavella et al., 1996).Additionally, slowed CTT may reflect pathology of extrinsic autonomic nerves, which are known to induce or modify the pacemaker activity of ICCs (Huizinga et al., 2021).Whilst impairments in ICC function may be implicated in GI dysfunction, no studies to date have investigated their role in MSA.
Recent studies have indicated alterations in the ENS of patients with MSA, which could provide a new therapeutic direction for MSA-related GI dysfunctions.A study by Ozawa and colleagues revealed shrinkage of myenteric neurons alongside preservation of submucosal neurons in small intestinal biopsies from patients with MSA.The authors speculated that given the preservation of submucosal neurons, large sized myenteric neurons such as Dogiel type I neurons may be more vulnerable in MSA (Ozawa et al., 2019).Three broad classes of Dogiel type 1 neurons exist within the ENS and include muscle motor neurones, secretomotor neurones, and neurons innervating enteroendocrine cells (Hansen, 2003).Chronic idiopathic constipation and oesophageal sphincteral achalasia have been shown to be attributed to the loss or malfunction of inhibitory motor neurons (Hansen, 2003;Wood et al., 1999).Given this, plus the expansive role of Dogiel type 1 neurons and the propensity for loss of Dogiel type 1-like neurons in MSA, it is feasible that they may play a role in MSA associated GI dysfunctions.However, the presence and type of neuron loss in MSA is uncertain and further research in this area is warranted.It should be noted that a limitation of the Ozawa et al.
study was that participating MSA patients underwent a gastrostomy, a surgical procedure for dysphagia.The insertion of a feeding tube resulted in patients consuming a liquid diet for years prior to the study (Ozawa et al., 2019).Thus, the authors could not discern whether the change in diet or MSA pathology caused the alterations to myenteric neurons in the ENS.
Enteric glial cells have also been implicated in GI dysfunction.For example, in PD, which shares some GI symptom similarities to MSA, enteric glial cell density and size have been shown to increase, suggestive of enteric gliosis (Emmi et al., 2023).This increase is thought to be linked to pathogenic intestinal α-syn, as suggested by inoculation studies where α-syn fibrils induce reactive gliosis (Challis et al., 2020).Although the role of enteric glial cells in MSA-related GI dysfunctions has not been clearly established, expression of the glial marker glial fibrillary acidic protein (GFAP) has been shown to be significantly reduced in biopsies from patients with MSA when compared to healthy controls (Clairembault et al., 2014) (Fig. 2).Enteric glial cells facilitate bidirectional communication between enteric glia and neurons allowing regulation and coordination of enteric reflexes (Delvalle et al., 2018b;McClain et al., 2015), and communication between extrinsic and intrinsic neurons that innervate the GI tract (Gulbransen et al., 2010;Peterson et al., 2010).Furthermore, enteric glial cells are known to contribute to disease processes in which glia can acquire inflammatory and tumorigenic phenotypes (Delvalle et al., 2018a;Selgrad et al., 2009).Given GFAP expression was significantly lower in biopsies from patients with MSA (Clairembault et al., 2014), further studies are needed in both human biopsies and animal models of MSA to determine the cause and/or impact this may have on the ENS and the GI tract overall.

Intestinal α-syn
While the presence of pathological α-syn has been established in the CNS of MSA patients, studies investigating α-syn deposition in the ENS are limited.Studies in patients with PD and associated animal models have indicated a role of α-syn in GI dysfunction and pathogenesis.Autopsied PD patients have revealed α-syn aggregates in various regions of the GI tract such as the rectum, colon, salivary glands, lower parts of the oesophagus, and stomach (Fayyad et al., 2019;Lebouvier et al., 2008;Wakabayashi et al., 1988).However, unlike PD, α-syn aggregation in the GI tract and associated nerves of patients with MSA appears to play a lesser role.A study by Pouclet et al. (2012) detected phosphorylated α-syn aggregates in the submucosal plexus of the descending colon in 1 of 6 MSA patients and in 5 of 9 PD patients (Pouclet et al., 2012), which may suggest a lesser degree of severity of α-syn invasion in MSA (Fig. 2).Interestingly, Nakamura et al. and Ozawa and colleagues failed to find phosphorylated α-syn deposits in intestinal tissue from patients with MSA (2015; Ozawa et al., 2019).The lack of α-syn seen in these studies may reflect the absence of myelinated oligodendrocytes within the peripheral nervous system.However, the presence of aggregated phosphorylated α-syn within the cytoplasm of Schwann cells (Schwann cell cytoplasmic inclusions, [SCCIs]) was observed in 12 out of 14 patients with MSA (86%) (Nakamura et al., 2015).
Analysis of SCCIs revealed a similar composition to oligodendroglial cytoplasmic inclusions with a immunohistochemical profile consisting of aggregated α-syn, ubiquitin, and p62, a ubiquitin-proteasome systemrelated protein (Nakamura et al., 2015).In relation to GI tract innervation, the anterior nerves of the sacral cord revealed the highest frequency of SCCIs (69%) and tended to be more frequent in the anterior rather than the posterior nerves at each level (Nakamura et al., 2015).This observation may explain the autonomic dysfunction observed in the GI tract as sacral preganglionic parasympathetic fibers exit from the sacral cord and travel to the terminal ganglia of the pelvic plexuses, and terminate in the ENS.Further, SCCIs were observed in the glossopharyngeal-vagus nerves (46%) and hypoglossal (9%) nerves, which could be responsible for dysphagia symptoms seen in MSA (Taniguchi et al., 2015;Thomaides et al., 2005).Furthermore, this study C.F. Craig et al. hinted at the possibility of neuronal propagation of α-syn fibrils through anterograde axonal transport as SCCIs tended to appear more prevalent in the proximal rather than the distal spinal nerve roots (Nakamura et al., 2015).The presence of SCCIs in visceral organs were found in 2 of 14 patients with MSA (14%), the subserosal nerves of the stomach in one patient, and the adrenal gland and urinary bladder in one other patient (Nakamura et al., 2015).Moreover, SCCIs were most prevalent in the anterior nerve of the sacral cord, cranial nerves, and the spinal and sympathetic ganglia (Nakamura et al., 2015).
Whilst our understanding of α-syn transmission between the gut and brain in MSA is incomplete, recent evidence has emerged elucidating other possible transmission routes of α-syn between the periphery and the brain .Ding et al. observed the presence of pathological α-syn in the nerve terminals of the detrusor and external urethral sphincter of patients diagnosed with MSA (Ding et al., 2020).Interestingly, injection of α-syn-preformed fibrils into the external urethral sphincter or detrusor sphincter of α-syn expressing transgenic mice (A53T TgM83 +/− ) triggered the transmission of pathological α-syn from the urogenital tract to the brain (Ding et al., 2020).Further, injection of α-syn fibrils into the urinary bladder of mice over expressing oligodendroglial α-syn showed motor deficits and α-syn pathology in the CNS (Peelaerts, 2023).The seeding and eventual propagation of α-syn pathology from the bladder of MSA patients has been speculated to arise following repeated urinary tract infections (UTI) however, a causal relationship is yet to be confirmed.Interestingly, an epidemiological nested-case control study in the Danish population found an association between UTIs and future diagnosis of MSA following infection, and this relationship was confirmed in animal studies where induction of repeated UTIs caused by Escherichia coli infection caused synucleinopathy with oligodendroglial involvement, which was thought to be driven by innate immune cells (Peelaerts, 2023).

Gut barrier dysfunction, inflammation, and the immune response
The gut is exposed to a vast array of different antigens from luminal commensal bacteria, diet, and pathogens, and as such requires both the innate and adaptive immune systems to maintain intestinal homeostasis and intestinal barrier integrity (Hou et al., 2021).The intestinal innate immune system includes innate lymphoid cells, dendritic cells, macrophages, mast cells, and eosinophils (Montalban-Arques et al., 2018;Mowat and Agace, 2014).Other cells with immune function include intestinal epithelial cells, Paneth cells (Mowat and Agace, 2014), microfold cells (Garrett et al., 2010), and T and B cells in the adaptive segment of the GI tract.
The intestinal immune system and the intestinal epithelial barrier strongly interact with each other as evidenced in GI inflammatory disorders (Diefenbach et al., 2020).Intestinal immune dysregulation is observed in several disorders of the GI tract such as inflammatory bowel disease (Michielan and D'Inca, 2015), celiac disease (Cardoso-Silva et al., 2019), colorectal cancer (Stidham and Higgins, 2018), and irritable bowel syndrome (Piche et al., 2009), all of which are often associated with intestinal inflammation and dysfunction, as well as GI symptoms such as constipation and incontinence (Cardoso-Silva et al., 2019;Hou et al., 2021;Michielan and D'Inca, 2015;Piche et al., 2009).
The integrity of the intestinal barrier relies on paracellular protein junctional complexes including tight junctions (TJs), adherens junctions, and desmosomes (Lee et al., 2018).TJs provide a structural boundary between the basolateral and apical membranes and are comprised of transmembrane proteins, the most studied of which are occluden, claudins, and zonula occludens (ZO).These TJ protein complex's restrict paracellular entry of large hydrophilic molecules into systemic circulation (Lee et al., 2018).A study in colon biopsies taken from MSA patients revealed a significant disruption of the TJ protein ZO-1 (Engen et al., 2017).In healthy controls, immunofluorescence staining for ZO-1 of the colonic sigmoid mucosa showed a typical smooth and organized distribution of staining restricted to the epithelial lining of the colonic lamina propria and epithelial apical cell junctions of the crypts.In contrast, MSA subjects had complete disruption, or very minimal immunofluorescence, of ZO-1 at the apical surface of the crypts and the epithelial lining of the lamina propria (Engen et al., 2017).Thus, a compromised intestinal barrier may facilitate entry of luminal immunogenic molecules into the lamina propria triggering intestinal inflammation and subsequent dysfunction.
Few studies have explored whether GI inflammation contributes to MSA-related GI deficits.
Recently, a genome-wide association study revealed genetic overlap between MSA and inflammatory bowel disease (IBD) at 3 shared genetic loci including an intron of the C7 gene and in leading variants upstream of the DENND1B and RSP04 genes (Shadrin et al., 2021).DENND1B is involved in clathrin-mediated endocytosis (Wu et al., 2011;Yang et al., 2016) and has been associated with down modulation of surface T-cell receptors and modulation of TH2 signalling and function (Yang et al., 2016).Evidence indicates dysfunctions in synaptic vesicle endocytosis (Hansen et al., 2011;Lee et al., 2008;Valera and Masliah, 2018;Zou et al., 2021) and immune signalling and regulation (Williams et al., 2020;Contaldi et al., 2022) in the pathogenesis of MSA.However, whether DENND1B variants contribute to the pathogenesis of MSA is yet to be determined.
Altered C7 expression has been observed in the midbrain of the proteolipid protein PLP-α-syn transgenic model of MSA-P (Refolo et al., 2018).Thus, it can be inferred that a shared genetic etiology between MSA and inflammatory bowel disease exists with an important role of the C7 gene in both diseases.It could be argued that such polygenic overlap between MSA and IBD could indicate enhanced risk of developing intestinal inflammation in patients with MSA.
Evidence of intestinal inflammation in MSA patients has been found by Engen and colleagues who observed a significant upregulation of the transmembrane protein toll-like-receptor-4 (TLR-4) in the lamina propria of the colonic sigmoid mucosa (Engen et al., 2017) (Fig. 2).TLR-4 belongs to the family of pattern recognition receptors, which recognize pathogen-associated molecular patterns, and are the first line of defence against infections such as bacterial lipopolysaccharide.
TLR-4 is normally expressed at low levels by different intestinal cell types, including epithelial and lamina propria mononuclear cells (Abreu, 2010;Otte et al., 2004).In the ENS, TLR-4 expression is observed within the myenteric and submucosal plexus in both neurons and glial cells (Barajon et al., 2009;Caputi et al., 2017); however, Engen et al. noted that very few TLR4-positive cells expressed GFAP, indicating that other cells, such as leukocytes, could be responsible for TLR-4 expression (Engen et al., 2017).This may reflect a similar inflammatory response as seen in inflammatory bowel disease where circulating leukocytes are trafficked into the inflamed intestinal mucosa following antigen induced mucosal expression of chemoattractants, chemoattractant receptors, and adhesion molecules (Arseneau and Cominelli, 2015;Habtezion et al., 2016).
Although TLR4 is upregulated in MSA patients, the consequences of this remains unknown (Engen et al., 2017).Elevation of TLR4 expression is observed in the intestinal mucosa of ulcerative colitis and Crohn's disease patients (Brown et al., 2014) and is believed to be involved in the sustained secretion of inflammatory mediators and development of intestinal inflammation (Dai et al., 2022).Conversely, TLR4 upregulation may promote repair and restore intestinal homeostasis evidenced by TLR4 knockout mouse models which indicate TLR4 signalling is required for protection against epithelial injury, bacterial invasion, and inflammation (Fukata et al., 2005;Rakoff-Nahoum et al., 2004).Whether intestinal TLR4 upregulation in MSA patients facilitates a pathological proinflammatory state or a beneficial reparative response to intestinal injury remains unknown (Engen et al., 2017).
TLR4 is expressed on enteric neurons and evidence indicates that TLR4 signalling is involved in regulating intestinal motility, ENS cell distribution, and neuronal-microbiota interactions 0.Moreover, an increase in TLR4 expression has been shown to negatively correlate with intestinal barrier permeability as well as influence TJ expression and organisation (Dheer et al., 2016;Peterson et al., 2010), suggesting a relationship between TJ proteins and TLR4 expression.Indeed, studies have shown that ligation of TLR4 induces the activation of transcription factors and kinases, which have been shown to redistribute occludin and ZO-1 leading to intestinal barrier dysfunction (Costantini et al., 2009a;Costantini et al., 2009b;Nighot et al., 2017;Peterson et al., 2010).
In summary, only one study has reported GI inflammation and increased intestinal permeability in subjects with MSA (Engen et al., 2017).Moreover, it is unclear which cells upregulate or contribute to TLR4 expression, and whether TLR4 expression facilitates repair or exacerbates intestinal barrier deficits in MSA subjects.Intestinal permeability has only been suggested based on altered ZO1 expression and localisation and should be confirmed using specific GI permeability tests.Thus, future studies should further delve into these topics to either further verify such findings and/or elucidate the impact they have on GI function in MSA.

Gut hormones
GI hormones can be classified as endocrine, paracrine, or neurocrine, based on the method and route by which the molecule is transported to its target cells (Parikh and Thevenin, 2022).Enteroendocrine cells secrete endocrine hormones directly into the bloodstream before being delivered to target cells with receptor-specificity for the hormone (Parikh and Thevenin, 2022).Examples of endocrine hormones include ghrelin, gastrin, cholecystokinin, and motilin (Parikh and Thevenin, 2022;Wierup et al., 2007).Enteroendocrine cells also secrete paracrine hormones such as somatostatin and histamine, which diffuse through the extracellular space and act only on local target tissues (Parikh and Thevenin, 2022).Finally, neurocrine hormones are secreted by postganglionic non-cholinergic neurons of the ENS and include hormones such as gastrin-releasing peptide, vasoactive intestinal peptide, and enkephalins (Fothergill and Furness, 2018;Parikh and Thevenin, 2022).
The gut hormone ghrelin has been found to be reduced relative to its inactive from (unacylated ghrelin) in patients with MSA when compared with control subjects (Ozawa et al., 2013).Importantly, the severity of GI symptoms in MSA correlated with levels of the active form of ghrelin (acylated ghrelin) (Ozawa et al., 2013).
Cholinergic innervation of the GI tract is believed to regulate ghrelin secretion in humans (Maier et al., 2004) with accumulating evidence suggesting that active ghrelin facilitates motility of the colorectum (Shimizu et al., 2006) and gastric antrum (Dornonville de la Cour et al., 2004;Levin et al., 2006;Tack et al., 2006;Tack et al., 2005;Trudel et al., 2002) as well as stimulating gastric acid secretion (Khatib et al., 2018).Gastric and colonic contractions induced by electrical field stimulation in the rat (Dass et al., 2003) and mouse (Kitazawa et al., 2005) are enhanced by the presence of ghrelin.Moreover, ghrelin evokes cholinergically mediated contractions of the rat jejunum (Edholm et al., 2004).Abnormal ghrelin secretion may be due to neurodegeneration of the vagal nuclei (Benarroch et al., 2006), which provides extrinsic cholinergic innervation to the GI tract (Benarroch et al., 2006) and is observed to be degenerated in post-mortem examination of MSA patients (Benarroch et al., 2006).We speculate that perturbations in ghrelin release in patients with MSA may be due to degeneration of vagal autonomic nuclei or other cholinergic neural structures (Fig. 2).
Patients with MSA also have impaired release of gastrin following hypoglycaemia (Polinsky et al., 1988), which is known to stimulate gastrin release.Gastrin release is mediated partly by cholinergic reflexes and sympathetic nervous activity (Dickinson, 2004;Walsh and Lam, 1980).Interestingly, there is evidence that patients have deficits in catecholamine responses to insulin-induced hypoglycaemia, possibly contributing to impaired gastrin release (Polinsky et al., 1982).In addition, impaired ghrelin secretion observed in MSA patients could be in part responsible for delayed gastric emptying and slow CTT seen frequently in MSA patients (Fig. 2) (Sakakibara et al., 2004;Tanaka et al., 2012) and may be a promising therapeutic target in the treatment of MSA GI deficits.Past studies have shown the efficacy of ghrelin and ghrelin receptor agonists in the management of gastroparesis (Murray et al., 2005;Sanger and Furness, 2016) and chronic constipation (Acosta et al., 2016;Naitou et al., 2015;Sanger and Furness, 2016).Future studies should investigate the efficacy of ghrelin and ghrelin receptor agonists in patients with MSA.
There is substantial evidence that alterations in gut microbial communities can cause immune dysregulation and co-morbid health conditions (Wu and Wu, 2012).The relationship between microbiota, inflammation, and GI dysfunction has received growing attention in recent years.Patients with MSA are observed to have gut dysbiosis and it is possible that bacterial metabolites could play a role in both GI dysfunction and disease pathogenesis (Engen et al., 2017;Tan et al., 2018;Wan et al., 2019) (Fig. 2).Alternatively, alterations to the ENS could drive motility changes that cause dysbiosis (Rhee et al., 2009;Singh et al., 2021).
The existence of an increased abundance of Bacteroides and Akkermansia may have deleterious or beneficial effects in MSA patients.Bacteroides and principally Akkermansia are mucin degraders (Derrien et al., 2010), which could trigger disruption of mucin homeostasis (Derrien et al., 2010) and contribute to intestinal barrier disruption in MSA (Engen et al., 2017).Goblet cell derived mucin proteins including MUC2 provide the intestinal mucous layer with viscous properties (Birchenough et al., 2015), which enable luminal mucus to retain antimicrobial proteins such as defensins, cathelicidines, lysosomes, and immunoglobulins (Ig) such as soluble IgG IgA, and IgM (McGuckin et al., 2009).Disruption of mucous layer composition may make an individual more prone to infiltration of luminal antigens triggering intestinal inflammatory events (Hansson, 2012).Akkermansia has also been shown to have proinflammatory properties including upregulation in genes involving IL-4 signalling, complement and coagulation cascades, antigen presentation pathways, and B and T cell receptor signalling (Derrien et al., 2011).The reduction in genus Bifidobacterium seen in MSA patients may also promote a proinflammatory intestinal environment as Bifidobacterium is considered an inflammation-suppressing bacteria as bioactive factors from Bifidobacterium were shown to improve the intestinal epithelial cell barrier and reduce intestinal inflammation (Ewaschuk et al., 2008).
MSA patients show alterations in microbial metabolites, which may impact host energy homeostasis, the immune system, inflammation, and hence contribute to disease progression (Engen et al., 2017;Nicholson et al., 2012;Tan et al., 2018;Wan et al., 2019).For instance, functional analysis based on the Kyoto Encyclopedia of Genes and Genomes database showed a reduction in pathways involving galactose and methane metabolism, and pantothenate and CoA biosynthesis in the faecal microbiome of MSA patients (Wan et al., 2019); all of which relate to energy production, cofactors, and vitamin metabolism, respectively.MSA patients also had significantly lower levels of short-chain fatty acids (butyrate, propionate; and acetate propionate) (Tan et al., 2018).This reduction may induce deleterious effects on the intestinal epithelium as colonic epithelial cells utilise short-chain fatty acids as energy substrates (Russell et al., 2013).For instance, butyrate has been suggested to prevent the development of inflammatory bowel disease as it facilitates the regeneration of colonocytes which facilitate intestinal barrier integrity (Cao et al., 2014;Louis and Flint, 2009).
The implications of gut dysbiosis on MSA disease progression and GI dysfunction remains unclear.Future studies in this area are needed as commensal bacteria could possibly be utilized as novel biomarkers in clinical diagnosis.Implementation of pre− /probiotics to rebalance gut microbiota dysbiosis may also be a novel therapeutic target for MSA GI deficits.
Disruption of intestinal barrier integrity in patients with MSA is observed with evidence of reduced expression of the epithelial tight junction protein Zonula occludens-1 (ZO-1).Impaired intestinal barrier integrity may facilitate translocation of luminal immunogenic microbiota and/or antigens into the mucosa.This may trigger an inflammatory response as implied by the upregulation of Toll-like receptor 4 (TLR-4) in the mucosa of MSA patients, which may be facilitated by leukocytes or epithelial cells.In addition, evidence of reduced expression of the glial marker GFAP and shrinkage of myenteric plexus (MP) neurons (most likely Dogiel Type 1) is observed in the gut of MSA patients.Reduced ghrelin circulation in MSA patients may have deleterious effects on gastrointestinal contractions mediated by the enteric nervous system.Unlike PD where α-syn aggregation in the ENS is firmly established, reports of alpha-synuclein (α-syn) invasion in the submucosal plexus (SMP) of MSA patients is contradictory.

Conclusions
It is clear from the limited studies that individuals with MSA suffer from GI symptoms.However, our understanding of symptom severity and onset has been hindered by the varying methods used to identify GI symptoms and the lack of appropriate controls.Moreover, reducing MSA to a general disorder and not recognising the distinct subtypes hinders our understanding of disease symptomology and potential treatment.Nevertheless, possible pathophysiological areas of interest which contribute to GI symptoms have been identified, and may lie within the central, peripheral, and intestinal environment.Patients with MSA are observed to present with degeneration in central anatomical areas believed to regulate GI function including the basal ganglia, LC, cerebellum, and DMV.Degeneration of spinal tracts, nuclei, and nervature may be involved in anal sphincter and GI dysfunctions.At a local intestinal level, there is evidence of atrophy of myenteric neurons intestinal aggregation of α-syn, albeit controversial.Additionally, factors excluding neuropathy such as intestinal inflammation, intestinal permeability, and gut dysbiosis are observed.
Given that GI dysfunctions in patients with MSA have been shown to elevate the likelihood of severe complications ranging from an inability to intake/absorb medication, malnutrition, aspiration pneumonia, and colonic pseudo-obstruction, the need for further research is urgent.Future investigations should broaden research on the intestinal barrier, enteric glial cells, GI inflammation, and GI hormones, which have the potential to be therapeutically targeted.Currently, there are limited effective treatment options for MSA-related GI dysfunctions, and this is most likely due to the underlying mechanisms and pathophysiology being poorly understood.Thus, further studies are needed to better understand the molecular, cellular, and structural changes that contribute to GI symptoms.

Fig. 1 .
Fig. 1.Degeneration of neuroanatomical regions in the CNS that contribute to GI dysfunctions in MSA.

Fig. 2 .
Fig. 2. Schematic representation of the ENS and intestinal pathology that may contribute to GI-related impairments in MSA.

Table 1
Summary of studies assessing GI dysfunctions in MSA patients.
78% MSA-P and 55% MSA-C experienced constipation when compared to control (p < 0.01).No significant difference between MSA-P and MSA-C.Delayed gastric emptying was measured through the (13)C-acetate breath test.Delayed gastric emptying was observed in patients with MSA when compared to healthy controls (p < 0.01).MSA = 22 males and 29 females; mean age of 65.6 ± 10.1 years.Mean disease duration = 4.3 ± 2.7 years.MSA-C detected in 43% of Evaluation of constipation via Rome criteria (Rome III) -a validated and 56.9% of MSA patients experienced constipation; Higher levels of constipation in MSA-P group vs. MSA-C (continued on next page) C.F. Craig et al.