Propagation of tau and α-synuclein in the brain: therapeutic potential of the glymphatic system

Most common neurodegenerative diseases are characterised by hallmark accumulation of protein deposits either inside or outside cells in the brain. These intra/extracellular accumulations are composed largely of a particular protein as its major component, the identity of which varies between neurodegenerative disease pathologies. Many of these proteins can enter an ‘amyloid state’, in which they form elongated fibres with spines consisting of many-stranded β-sheets which run in parallel with the filament axis [8, 9]. Among the most studied proteins in this group are amyloid-β plaques in AD; tau neurofibrillary tangles in frontotemporal dementia with parkinsonism, and AD; α-synuclein Lewy bodies and neurites in PD, and dementia with Lewy bodies; huntingtin inclusions in Huntington’s disease; TDP-43 inclusions in amyotrophic lateral sclerosis; and Prion protein (PrP) inclusions in prion disease (spongiform encephalopathies) [8, 10].

Although the identity of the initial trigger for amyloid assembly in neurodegenerative disease is unknown, several in vivo conditions that promote the energetically unfavourable amyloid formation have been implicated in disease aetiologies. For example, mutations that destabilise the native form of the protein; increased concentrations of amyloid-prone proteins or de novo generation of amyloid species through native protein cleavage [8]. Notably, dominantly inherited neurodegenerative diseases are often associated with mutations in the gene encoding the protein that forms amyloid structures, such as mutations in MAPT, the tau gene, in inherited forms of frontotemporal dementia with parkinsonism; and mutations in SNCA, the α-synuclein gene, in familial forms of PD. Although many proteins associated with neurodegenerative diseases are capable of forming amyloid aggregates, in this review we will be focusing on intracellular proteins, namely, the two most prevalent proteins tau and α-synuclein, which are thought to drive neurodegeneration, yet their biological relationship with the glymphatic system is only starting to be explored.

In prion disease, the accumulation of amyloid aggregates of PrP throughout the brain leads to gross neurodegeneration and cerebral atrophy. However, what is most commonly associated with this neurodegenerative disorder is its transmissibility. The infectious protein ‘seed’, or ‘proteinaceous infectious particle’ (abbreviated to ‘prion’ by Stanley Prusiner in his seminal 1982 work [13]) can transmit between individuals, and cause its own amplification and replication by recruiting the endogenously expressed PrP^C protein in the brain into the pathological PrP^Sc form, inducing pathological amyloid aggregation and neuronal toxicity [14]. Indeed, this seeded amplified aggregation of PrP can be replicated in vitro, by incubating minute amounts of PrP^Sc ‘seeds’ with excess concentrations of PrP^C [15]. Although prion disease is classically considered as an infectious disease, 99% of cases are sporadic or genetic in origin [16], raising the intriguing suggestion that the trigger of seeded accumulation in the brain can also begin endogenously.

There is limited evidence indicating that the more common neurodegenerative diseases, such as AD and PD, are transmissible between organisms [17], yet they too present with amyloid state aggregates composed of different proteins in the brain, progressive neurodegeneration, and sporadic as well as genetic origins. With that in mind, given the parallels between the amyloid state accumulations in prion disease and these other neurodegenerative disorders, rigorous research efforts have been placed in the last decade on the possible ‘prion-like’ action of other amyloid-prone protein species in the brain, a term first used by Adriano Aguzzi in 2009 [18]. Notably, tau and α-synuclein have demonstrated significant degrees of prion-like action in various experimental settings, as we discuss later [19–23].

Prions are proposed to be released by affected ‘donor’ cells through several different mechanisms, the simplest being direct release into the extracellular space through either exosomal release (Fig. 2c) or leakage through damaged cell membranes (Fig. 2d). Indeed, studies have shown that treatment of neurons with compounds that interfere with exosome release mechanisms result in significant changes in the extent of extracellular release of amyloid-prone protein species [26, 27]. Further, release of protein species can also be achieved through passive leakage across the damaged plasma membrane, a process exacerbated by the presence of fibrillar protein constructs themselves, e.g. fibrillar α-synuclein [28, 29]. These ‘naked’ prion constructs in the extracellular space, in turn, become internalised through numerous mechanisms, including direct pinocytosis, a process thought to occur both clathrin-dependently (Fig. 2f) and independently (Fig. 2g) [30], and attributable to monomer endocytosis only [31]. Oligomers and short fibrils, on the other hand, can be internalised through receptor-mediated endocytosis (Fig. 2f) [32]. For example, lymphocyte-activation gene 3 (LAG3) has been proposed to mediate the internalisation of α-synuclein [33], which is then released intracellularly (Fig. 2j). Recent studies, however, have disputed the role of LAG3 in α-synuclein internalisation in neurons, as it has been demonstrated that this gene is not expressed in neuronal cells, but only in microglia and macrophages [34]. Similarly, heparan sulphate proteoglycans (HSPGs), transmembrane and lipid-anchored cell-surface receptors that interact with a variety of ligands, have also been shown to mediate the internalisation of prions [35, 36]. This process is known as HSPG-mediated micropinocytosis (Fig. 2i), an endocytic process characterized by binding of the prion ligand to surface-bound HSPG, actin-driven membrane ruffling, internalization of extracellular fluids, and formation of large intracellular vacuoles [35]. Prion structures can also be released into the extracellular space as either exosomes [37] (Fig. 2a) or ectosomes [38] (Fig. 2b), which can in turn be internalised by ‘recipient’ cells through vesicular fusion (Fig. 2h). Lastly, protein species have also been shown to be exchanged directly between ‘donor’ and ‘recipient’ cells via tunnelling nanotubes (Fig. 2e), which are F-actin-containing membranous bridges that connect the cytoplasm of remote cells with one another [39, 40]. This phenomenon has been observed to not only occur between neurons, but also between astrocytes, pericytes and also between cell types [40, 41]. Once inside the new host cell, prion-like amyloid-prone proteins can act as templates for their own replication (see Fig. 1b). This process leads to the formation of an initial segment of a new amyloid spine, which, in turn, can break and release new amyloid seeds and propagate to surrounding unaffected cells (Fig. 2k, l).

A number of these mechanisms of transcellular prion propagation involve the release and uptake of pathological seed components into/from the extracellular space, highlighting this part of propagation cascade as a potential therapeutic target. Correspondingly, although the mechanisms that facilitate tau intercellular propagation through secretion and uptake are not fully understood, multiple studies in recent years have taken an immunotherapeutic approach, to aid clearance of extracellular tau and α-synuclein in an attempt to block their propagation throughout the brain [42–45]. Though the precise mechanisms of immunotherapeutic clearance of these proteins from the extracellular space are still not well defined, it has been proposed that antibodies could promote clearance from these spaces, affect brain clearance, or even promote intraneuronal clearance [46].

The concentrations of extracellular proteins such as tau and α-synuclein in the interstitial fluid (ISF) are maintained based on a balance between the extent of cellular release into the space, and extracellular clearance from the space. Tau, for example, has been found to have a particularly low level of extracellular turnover (~ 11–17-day half-life [47]), making it prone to propagation if misfolded in a seed-competent manner. Acceleration of endogenous mechanisms which clear the extracellular ISF of these protein species could therefore potentially hold therapeutic promise against their propagation in the brain. Notably, the glymphatic pathway has been shown to have a critical influence on elimination of extracellular molecules, such as amyloid-β, tau and α-synuclein [48–52]. This pathway may thus have novel therapeutic potential against prion-like propagation in the raft of neurodegenerative disease states.

Studies proposing the existence of a system in the brain, equivalent to the peripheral lymphatic system which drains fluid and clears out waste, date back centuries [53]. Anatomists Rudolf Virchow and Charles Robin were among the pioneers to describe the existence of spaces around blood vessels, including in the brain, in the 1850s, which are now referred to as components of a perivascular system composed of ‘Virchow-Robin’ spaces [54]. Today, the term ‘perivascular space’ is widely accepted to describe the space surrounding the blood vessels which can communicate with the subarachnoid space, where CSF circulates [55]—though the communication between these two spaces is still disputed by some authors [56, 57]. Shortly after the initial descriptions of the perivascular spaces in the brain, in 1869, Gustav Schwalbe provided experimental evidence to show that dyes injected into the cranial subarachnoid spaces of different laboratory species could enter the lymphatic vessels and lymph nodes of the head and neck [58]. These experiments provided a clear link between the early observations of Virchow and Robin to the possible existence of a lymphatic drainage-like system in the central nervous system (CNS). Subsequent studies further demonstrated that injection of a dye either into the CSF or directly into the subarachnoid space could enter the perivascular space, suggesting communication between the CSF and the ISF in the brain [54]. Collectively, these studies provided strong evidence pointing to the existence of a fluid-mediated clearance system in the CNS.

Astroglial cells are proposed to form a key link between the para-arterial CSF influx and the para-venous ISF efflux pathways of the glymphatic system [49]. According to the initial model put forward in 2012, astrocytes, known to be in close physical contact with the brain vasculature, facilitate the otherwise unfeasible movement of fluid and solutes through the brain parenchyma, from the periarterial to the perivenous spaces [49, 60]. A crucial feature of these specialised supporting glial cells is the expression of the protein aquaporin-4 (AQP4), a water-specific channel present on the astrocytic endfeet which ensheath the brain’s vasculature (Fig. 3a) [60, 61]. Notably, AQP4 is considered the main water channel expressed in the brain, and unlike its choroid plexus-localised counterpart AQP1, it is thought not to play a role in epithelial fast water transport [61]. In their description of the glymphatic system, Iliff et al. proposed that AQP4 is the key channel which allows the directional flow of CSF, which is driven by pressure gradients and convective transport, through a network of glial projections [49]. Indeed, when CSF tracer influx was imaged ex vivo, tracer movement into the brain parenchyma was significantly reduced in AQP4-knockout mice compared to wild-type controls [49, 60, 62]. Importantly, by injection of radiolabelled mannitol into the striatum, this study also showed that the rate of fluid and solute clearance from the brain’s interstitial spaces was significantly suppressed in AQP4-knockout animals, further demonstrating the pivotal role of AQP4 in the glymphatic clearance system [49, 60, 62]. Adding to these findings, the study also proposed a crucial role for the glymphatic system in the context of neurodegenerative disease in its clearance of cytotoxic waste products, namely amyloid-β, from the brain [49]. This was followed up by other studies, also implicating the clearance of tau from the brain by this pathway [48, 50], and also α-synuclein [51, 52]. Many additional studies have also shown that the astrocytic AQP4 is essential for fast glymphatic transport in different systems and disease models [48, 62–67], further emphasizing the central role of astroglial cells in this CNS solute clearance system.

Ageing appears to pose a significant risk factor to glymphatic misfunction. Scientific evidence points out that ageing represents multiple threats to the normal function of the glymphatic system, as astrocytic, CSF production and sleeping pattern differences are commonly found in older individuals. One of the pioneering pre-clinical studies looking into the possible changes in glymphatic function during ageing, compared this system among young, middle-aged and old rodents [68]. Results showed a striking decrease in interstitial CSF influx measured using cisternal delivery of fluorescent tracers, with middle-aged animals showing approximately half as much CSF influx compared to young mice, and a reduction of nearly 80% in old compared to young animals [68]. But what could be driving this loss of CSF influx? The same study showed that this change was associated with loss of polarisation of AQP4 in astrocytic endfeet, despite no overall change in AQP4 expression levels, but a marked increase in the number of astrocytes, particularly surrounding larger vessels in the brain [68]. With ageing, AQP4 became localised to astrocytic parenchymal processes rather than endfeet, therefore dysregulating water transport driven by these cells [60, 68]. Notably, the authors observed that certain areas of the brain, such as the striatum and the hippocampus, appeared to have a greater decrease in AQP4 polarisation in older animals [68]. Further, studies in different mammalian species have also found a direct correlation between age and decrease in both production and pressure of CSF within the brain [60, 69–71], reinforcing the hypothesis that glymphatic activity declines with age. In humans, a compelling relationship has also been observed between impairment of the glymphatic pathway and ageing [72]. Using magnetic resonance imaging to evaluate glymphatic flow in the aged, Zhou and colleagues demonstrated an impairment in glymphatic circulation as well as in meningeal lymphatic outflow in older subjects, with the latter appearing to be downstream of the glymphatic pathway [72, 73]. Adding to this, sleep is crucially important for glymphatic clearance, which will be discussed later, and it is well established that sleep quality decreases with age, with insomnia, interrupted and superficial sleep as well as shorter sleep duration being common among older adults, causing a decline in nocturnal brain waste clearance [74]. Although further studies are necessary to understand whether the failure of the glymphatic system in ageing contributes to the accumulation of harmful proteins, whether these protein waste products accelerate the effects of age on the glymphatic system, or if a concomitant, collaborative process is at play, is yet to be established.

It is important to note, however, that the glymphatic system does not work in isolation to remove soluble waste products from the brain; there are various overlapping clearance systems which work in concert to achieve and maintain parenchymal homeostasis (for reviews see [75, 76]). Waste solutes in the brain can be enzymatically degraded, cleared directly into the blood at the blood–brain barrier, or transported into the CSF by any number of converging mechanisms, glymphatic clearance being one. Indeed, since its initial description in 2012, the glymphatic pathway has received much attention and interest, and rekindled research efforts into many of these clearance pathways, CSF dynamics and fluid flow in the brain. It also sparked some controversy [77, 78]. For example, in their examination of perfusion-fixed tissues, Carare and Weller suggest that the perivascular fluid movement takes place along the basement membranes of the smooth muscle cells of the cerebral arteries only, rather than in the perivascular space of both veins and arteries, and antiparallel rather than parallel to vessel blood flow [79]. This has been re-challenged; Mestre et al. [80] suggested that this observation was artefactual as a result of vessel and perivascular space collapse due to perfusion fixation. Further, based on experimental results, Smith et al. [81] questioned and contradicted the then newly proposed glymphatic pathway, arguing that the transport of solutes in the brain parenchyma occurs in a diffusive fashion, rather than by convective transport as proposed by Iliff and colleagues [49]. Importantly, the authors of this study provided evidence to suggest that AQP4 gene deletion does not impair the transport of solutes from the subarachnoid space to the parenchyma in mice or rats, and dismissed the central role of these water channels in solute transport within the brain. These findings have been re-challenged; a recent study, carried out by a consortium of laboratories across the world, re-evaluated the role of AQP4 in the glymphatic clearance pathway [77]. This rebuttal study extensively examined the role of AQP4 in glymphatic transport using five genetically modified AQP4 mouse lines, including the line used by Smith and colleagues. The group concurred that the CSF influx was higher in wild-type mice than in each of the AQP4 knockout lines tested [77]. Importantly, the study highlighted the central role not only of AQP4, but also of its membrane-binding complex, and suggested that invasive procedures, such as intracerebral injections, may themselves disrupt and impair glymphatic system function [77]. In addition, the authors, along with others, propose that experimental variables such as anaesthesia, age, tracer delivery methods and perfusion-fixation could explain the discrepancy in the findings [77, 80], further reinforcing the pivotal role of AQP4 and the perivascular space in the function of the glymphatic system in the rodent brain. Given the controversy surrounding the glymphatic system, in recent years the pioneering lab (led by Maiken Nedergaard) has published opinion articles reviewing the current areas of controversies, debating data and posing questions that remain outstanding regarding the system [78, 82, 83]. More recently, this has been followed up with an exhaustive Physiological Reviews paper, which brings together the current state of the field of fluid transport in the brain, providing an all-encompassing review on where the glymphatic system fits in the brain’s physiological mechanisms for fluid influx and efflux and importantly providing in-depth discussion and point-by-point evaluation of the debated areas of contention [76]. Readers should therefore refer to this work for more expansive discussion and explanation of the controversies surrounding this area of brain physiology.

Controversy aside, the glymphatic system of fluid exchange in the brain has been shown to have a critical influence on clearance of the extracellular space and elimination of molecules prone to aggregation in neurodegenerative diseases, namely amyloid [49] and tau [48, 50]. Like tau, α-synuclein also causes neuronal cell death through intracellular aggregation, and the neuron-to-neuron and region-to-region patterns of propagation of these proteins in the brain suggest that the extracellular space acts as an essential transport conduit for their spread [51, 52]. As has been described above, age, and associated changes in neuroinflammation have been shown to alter glymphatic function [68–70, 72]. So too has neurodegeneration, which is described below. In addition, genetic variation, in the form of single nucleotide polymorphisms in the AQP4 gene which encodes the water channel central to glymphatic function, has been shown to be associated with differences in sleep quality [84], amyloid-β deposition in the brain [85], and altered rates of cognitive decline after AD diagnosis [86]. Taken together, these findings suggest that the glymphatic system may be present as a novel therapeutic target for neurodegenerative pathologies (Fig. 3). Below we will review the evidence showing the propagation-prone nature of these two intracellular proteins in the context of neurodegeneration. Further, we summarise what is known on the involvement of the glymphatic system in their clearance, advocating future studies aimed at therapeutically modulating this system for the treatment of tauopathies and synucleinopathies.

The first evidence supporting the theory that tau behaves in a prion-like manner, came from human post-mortem studies [99–101]. Results of a cross-sectional study of multiple brains of patients at varying stages of clinical progression showed that tau follows a stereotypical pattern of spread, which can be classified into different stages. This pattern starts initially at the entorhinal regions and locus coeruleus (stages I–II), progressing to encompass large portions of the neo-cortex (stages V–VI) (Fig. 3d) [97, 100, 101]. Named after the authors of the work, Heiko and Eva Braak, this progression and spread of tau throughout the brain was called the Braak staging system [97, 100]. Despite more recent cross-sectional and imaging studies arguing that tau spread might not be as orderly and spatially restricted as previously proposed [94], the Braak staging system is still the most accepted concept within the field, and serves as the foundation for many studies looking at propagation mechanisms in tauopathies.

Based on this initial hypothesis, more evidence in support of tau’s prion-like propagation came after the studies by Braak & Braak in 1991, using an in vitro system [22]. The research showed that, in cultured cell lines, extracellular tau aggregates can transmit their misfolded state from the outside to the inside of a cell, demonstrating that cells treated with tau aggregates can internalise this protein, leading to intracellular tau fibrilization [22]. Importantly, the authors also showed that aggregated intracellular tau transferred between co-cultured cells, further suggesting that the propagation of misfolded species through the brain would reflect a combination of neuronal proximity and perhaps connectivity [22]. Consistent with this concept, a follow-up study further demonstrated that misfolded preformed tau fibrils could spontaneously be up-taken by cells, proposing the likely cellular mechanism underlying this phenomenon to be endocytosis [102]. These studies were not only crucial to support the idea that tau can spread to neighbouring cells and across the neuronal circuitry, but also made ground-breaking steps in helping to identify the mechanisms of tau seeding and spread. Furthermore, the proposed mechanism of tau uptake implies that downregulation of endocytosis could be a potential mechanism to tackle the spread of tau in the brain and therefore a therapeutic target for tauopathies.

Soon after the proposal of recruitment and propagation of tau in cultured cells, the first evidence that tau could propagate in a more physiological system came to light. Clavaguera and colleagues demonstrated that injection of brain extract from mutant tau-expressing mice into the brains of transgenic wild-type-tau-expressing animals, induced assembly of tau into filaments [19]. Importantly, the study also showed that the spread of pathology started at the original site of injection, subsequently reaching neighbouring brain regions over time. The study postulated that the formation of tau aggregates in a single brain cell and their subsequent translocation and spread may occur at the origin of sporadic tauopathies. Furthermore, the authors speculated that similar to tauopathies, other neurodegenerative diseases such as PD may have a similar mechanism of prion-like spread [19].

Soon enough, the connectome hypothesis of transmission of pathological tau was validated in vivo [103–107], giving further evidence of the cell-to-cell transmission and seed aggregation pathway of tau. However, these studies were conducted mostly in transgenic animals, where mutations were artificially enhancing the fibrillation of tau protein in vivo. Added to this, the vast majority of tauopathies are sporadic in nature, raising the question as to the relevance of such models to better understand the underlying mechanisms of tauopathies. To address these points, a study looked into the spread of tau fibrils when injected into non-transgenic animals [93]. The authors demonstrated that a single intrahippocampal injection of insoluble tau fibrils purified from human AD brains, into non-transgenic mice, led to abundant propagation of tau pathology [93]. Their results showed that the pattern of tauopathy in these animals started from the site of injection and spread to its anatomically connected regions, in an extremely rapid fashion, occurring as early as 3 months post-injection [93]. Further, given the existence of different isoforms of tau, the study also investigated and compared the capacity of seeding of distinct tau conformational features and demonstrated that the seeding activity of human brain-derived tau fibrils was much higher than that of synthetic tau fibrils, which are largely used for the study of this protein [93]. Since these initial studies in non-transgenic mice, work has continued aiming to further characterise the patterns of spread. Recently, Cornblath and colleagues injected tau from AD brains into the brains of non-transgenic mice, and showed with network modelling analysis that diffusion through the connectome is the best predictor for tau pathology pattern, corroborating previous findings from others in the field [108]. This study also showed that retrograde, rather than anterograde spread, seems to dominate the spread of tau in the mouse brain [108].

Examination of tau propagation has also been further pursued, but this time investigating the seeding efficiency of different human-derived tau species. Narasimhan and colleagues showed that, like their previously published results, tau extracted from post-mortem brains of patients with different tauopathies, injected into different brain regions of wild-type mice, could induce endogenous mouse tau aggregation and propagation beyond the site of injection [109]. Interestingly, these follow-up findings showed that tau extracted from different human conditions, i.e. AD, progressive supranuclear palsy, and cortical basal degeneration, seeded with different potencies and resulted in different cell-type specificity of tau aggregate transmission [109]. Importantly, the authors also showed that what determined the pattern of pathological spread was the neuronal connectome, rather than the strain of tau itself [109]. These findings provided further evidence in support of the connectome propagation theory in tauopathies, presented a new relevant model system to study the mechanisms of sporadic tauopathies, and shed light on the physiological relevance of the currently available mouse models used to study disease processes in the field. However, given the limitations of some of these models and their translatability to the clinic, it would be prudent to verify how similar and relevant these studies are for patients with these tauopathic disorders. Although a recent work studying the seeding efficiency of α-synuclein in non-human primates has been published [110], as we will discuss later, to date tau propagation is yet to be demonstrated in higher species. Furthermore, despite some recent evidence coming from post-mortem brains of patients suggesting that tau seeding may also occur in humans [111], further studies to confirm whether tau pathology can also be ‘transmitted’ and seeded in the human brain is necessary. As such, in recent years computational modelling studies have started to appear which attempt to simulate tau’s spread throughout the human brain connectome [112], some crucially incorporating tau-PET tracer data in an attempt to validate and substantiate the connectome hypothesis of tau transmission [113, 114], or even predict future tau burden in patients [115]. Given the obvious absence of any experimental seeding data in the human brain, the aforementioned studies will be vital in order to define the translatability of those findings from pre-clinical animal experiments.

Is the glymphatic system involved in the clearance of extracellular tau protein which is prone to propagation in the brain? As discussed above, tau is a neuronal protein, expressed intracellularly, but there is substantial evidence to suggest that it can be secreted and taken up by both neurons and glia [116, 117]. In addition, it is thought that tau secretion to the extracellular space plays an important role in the development and spread of intracellular tau pathology [8, 48, 118, 119] (Fig. 2). Given this evidence, it could be hypothesised that the glymphatic system plays a role in clearing away the secreted extracellular tau that otherwise would remain and become prone to uptake by neighbouring cells. Indeed, in the context of traumatic brain injury (TBI), which is an established risk factor for the early development of dementia and aggregation of tau protein, it has been shown that the glymphatic system is associated with the brain’s vulnerability to neurodegeneration [48]. It was demonstrated that TBI in mice leads to decreased glymphatic influx and impaired clearance of the brain’s interstitium, which can last up to a month post-injury, promoting tau aggregation, neurodegeneration and persistent neuroinflammation in the post-traumatic brain [48]. Furthermore, it has been shown that loss of function of AQP4 impairs the clearance of interstitial solutes in the mouse brain, including the clearance of tau along the glymphatic pathway, resulting in intracellular tau aggregation and neurodegeneration, and exacerbated neurocognitive function after TBI [48].

In the context of tauopathy, we recently examined the relationship between glymphatic inflow and clearance of tau from the cortex of a transgenic mouse model of frontotemporal dementia [50]. We demonstrated that in the transgenic mouse brain, glymphatic function was impaired and the level of AQP4 polarisation to astrocytic endfeet was similarly reduced. We also showed that the degree of cortical tau deposition negatively corresponded to the level of glymphatic function in the healthy mouse cortex, alluding to the inverse relationship between the extent of glymphatic function and the degree of tau deposition in the rodent brain. We went on to show that pharmacological blockage of AQP4 resulted in the disruptions of glymphatic inflow and clearance of tau from the brain [50], strengthening the link between AQP4, glymphatic function and tau clearance. Together, these data give strong evidence to suggest that reducing the glymphatic clearance of tau may potentiate the neurodegenerative disease process, perhaps by encouraging the cell-to-cell propagation of tangle-susceptible tau protein in the brain. These results denote that AQP4 could be a powerful target for therapy in the context of tauopathic dementias.

Although the cause of sporadic PD is not known, its progression appears to have a pattern of spread. Following the proposed stages of tauopathies previously discussed in this review, Braak and colleagues also traced the course of α-synuclein pathology in incidental and symptomatic PD cases [145]. In this unique study, the brains of over 100 α-synuclein-positive individuals were examined post-mortem [145]. The authors proposed that PD-related intraneuronal pathology and neuronal damage do not happen randomly, but appear to follow a recognizable topographical pattern in the brain [145]. According to their model, there are six main stages in the evolution of the pathology, starting at the lower brainstem in the dorsal motor nucleus of the vagus nerve (DMVN) and anterior olfactory structures (stage I), then affecting the midbrain and substantia nigra (stage III), and eventually reaching the neocortex (stage VI) (Fig. 3e) [146, 147]. This study was followed by a subsequent publication where the same authors proposed that the disorder might originate outside the CNS, caused by an unknown pathogen that is capable of passing the mucosal barrier of the gastrointestinal tract and entering the CNS along the projections of the vagus nerve [146]. Led by observations that PD patients can experience several gastrointestinal dysfunctions, in addition to studies reporting the presence of LBs and LNs in the enteric nervous system (ENS) as well as in the vagus nucleus, Braak and colleagues put forward the hypothesis that α-synuclein can behave and propagate in a prion-like manner, spreading from the ENS to the DMVN via vagal pathways; the so-called gut-brain hypothesis [146, 148–150]. Their proposal postulates that α-synuclein can be transneuronally and retrogradely transported from the periphery and in this manner gain entry into the CNS, initially in the brainstem (DMVN), then reaching the substantia nigra via the nucleus tractus solitarius, and from there spreading to other areas of the CNS [146, 148–150]. Furthermore, the authors added to these remarks and proposed that the neurotropic pathogen could also enter the brain via the nasal route, where a nasal infection would give direct access to the olfactory nerve, transporting the pathogen in an anterograde direction, and allowing its invasion and progression into the temporal lobe [148]. Independent of the ‘infection route’, the observations from the Braak studies of sporadic PD highlight that the development of increasing degrees of Lewy pathology not only follows a predictable topographical distribution, but most importantly the predicted areas are anatomically interconnected, leading to the suggestion of a connectome hypothesis of α-synuclein propagation, similar to that described in the tau propagation field. These remarkable observations were the basis for the identification of mechanisms at the molecular and cellular levels that we know of today, which show that physical contacts, i.e., trans-synaptic and trans-neuronal transmission between vulnerable neurons, are central in α-synucleinopathies [151]. Perhaps due to the absence of a robust α-synuclein PET ligand for minimally invasive assessment of α-synuclein aggregation in humans, clinical research on the spread of α-synuclein through the brain’s connectome is hampered, and research to date has focused largely on animal [152] and mathematical [153] models.

Following the proposed Braak stages, early evidence suggested that α-synuclein could be found in intracellular vesicles, and when newly synthesized could be secreted from cells through an exocytotic pathway [154], adding to the hypothesis that this protein could be transmitted in a prion-like manner. Several in vitro studies followed, demonstrating that the extracellular α-synuclein can be internalized by cells, many reporting the cytotoxic effects of extracellular α-synuclein when added to the culture medium [30, 31, 155, 156]. Importantly, later studies have indeed validated this theory, by demonstrating that α-synuclein can be up-taken from the extracellular space and also transmit between cells [21, 157]. Using an in vitro system, the first study of this kind showed that when neuronal cells overexpressing human α-synuclein were co-cultured with different neuronal cells, they could act as donors of α-synuclein to the other cells [21]. This in vitro study demonstrated that exposure to mutated cells can lead to the accumulation of transmitted α-synuclein in host cells, forming Lewy-like inclusions which could lead to neurotoxicity and apoptosis [21].

But how could some sparse extracellular manipulated molecules accumulate intracellularly in a neighbouring cell? To address this question, an elegant study by Luk and colleagues looked at the effects of monomeric and fibrillar α-synuclein in culture [157]. Although the study could not reproduce some of the observations described in previous studies (e.g. [21]), the authors demonstrated that introduction of exogenously assembled α-synuclein fibrils could recruit endogenous soluble α-synuclein protein, converting it into insoluble inclusions [157]. This study was the first indication that exogenous α-synuclein fibrils could act as a catalyser of intracellular α-synuclein, which in turn forms aggregates and structures that closely resemble LBs present in the brains of patients with synucleinopathies [157]. Importantly, the results of this study also showed that neither monomeric nor oligomeric forms of α-synuclein were capable of efficient recruitment or conversion of soluble endogenous proteins into a misfolded state, making evident the crucial importance of a molecular entity capable of corrupting the endogenous protein [157]. Together with later reports further demonstrating that exogenous α-synuclein can lead to pathological hallmarks which resemble PD in vitro [158, 159], these studies were pioneering in demonstrating the seeding property of α-synuclein and its intrinsic spreadable, prion-like nature, in a dish.

Cell-to-cell transmission of pathologic α-synuclein also occurs in vivo. Evidence in support of such a mechanism came from post-mortem studies of patients who received striatal grafts of foetal human midbrain neurons. Upon post-mortem examination, it was reported that patients who had long-term survival of transplanted foetal mesencephalic dopaminergic neurons (> 10 years) developed α-synuclein–positive LBs in grafted neurons, suggestive of propagation from neighbouring α-synuclein-laden host cells [160–163]. These data, along with the strong evidence for the seeding nature of α-synuclein in vitro, led to revolutionary studies investigating the effects of intracerebral inoculation of α-synuclein in mice.

In an initial study, young α-synuclein-mutant transgenic mice, displaying no synucleinopathy phenotype, were given a striatal injection of brain homogenates derived from older mice exhibiting α-synuclein pathology [164]. The study showed that intracerebral inoculation of α-synuclein induced pathological propagation throughout the CNS, to regions far beyond the initial injection sites, initiating PD-like LBs/LNs across different regions of the brain as early as 30 days post-injection [164]. Importantly, the authors reported that inoculation of fibrils assembled from recombinant human α-synuclein induced identical consequences, accelerating and increasing accumulation of α-synuclein pathology in the brain, as well as dramatically reducing the survival of the animals [164]. Although these experiments were pioneering in demonstrating the prion-like cascade nature of synucleinopathies in vivo, it remained to be investigated whether this protein would behave in the same manner in a non-genetically manipulated model system.

To address this question, a follow-up study injected wild-type non-transgenic mice with synthetic α-synuclein fibrils and longitudinally characterised their phenotype [165]. In this innovative study, the authors showed that a single intrastriatal inoculation of α-synuclein fibrils could lead to the cell-to-cell transmission of pathologic α-synuclein and Parkinson’s-like Lewy pathology [165]. Strikingly, their histological results showed accumulation of α-synuclein in the brain from day 30 after the inoculation, spreading through anatomically interconnected regions, as well as reaching the contralateral hemisphere, although fairly modestly, after 30 days [165]. The study also highlighted that progressive accumulation of α-synuclein led to a noticeable loss of dopaminergic neurons, particularly in the substantia nigra, resulting in the development of motor deficits in the animals, resembling the classical pathology and symptoms of PD patients [165]. Further, it has since been demonstrated that, when seeded, the concentration of fibrils controls inclusion formation and neurodegeneration phenotypes in vivo, and that different wild-type rodent strains can show distinct phenotype outcomes when the same concentration of α-synuclein is used for seeding [166]. Although it is still unclear why inclusion burden, rate of spread and dopaminergic neurodegeneration differ, the data highlight how strain effects could potentially influence results and their interpretation in an experimental setting. Subsequent in vivo studies in rodents have further demonstrated the intrinsic prion-like spread of α-synuclein, including a study where homogenates from the brains of patients with LBs were used as the initial pathological seed [23, 158, 167]. Further, it has been demonstrated more recently that substantial inter-case variability in the propensity of patient-derived α-synuclein fibrils also exists [168], alluding to the complex nature of the prion-like propagation of α-synuclein in the brain.

Further to the observations in rodents, a recent study uniquely evaluated the effects of intrastriatal injection of α-synuclein fibrils in non-human primates, in a year-long longitudinal study [110]. Using non-invasive in vivo imaging methods, the authors showed a significant alteration in dopamine transporter binding in the brain areas immediately connected to the region injected with α-synuclein fibrils, a change that became increasingly more prominent over the course of the study [110]. Importantly, the study also showed that α-synuclein fibril injection in the brains of monkeys resulted in Lewy pathology in the substantia nigra, accompanied by a significant reduction of dopaminergic neurons in this area, as observed in early PD [110]. Collectively, these findings were pioneering in demonstrating the prion-like cascade in synucleinopathies in vivo and across species. Together with the early proposals by Braak, these studies solidified the concept of cell-to-cell transmission and propagation of misfolded α-synuclein, which is thought to underlie the CNS spread of LBs/LNs. Furthermore, by demonstrating that α-synuclein fibrils are sufficient to initiate PD-like pathology and to transmit the disease in vivo, these findings provide further evidence that this phenomenon could also occur in humans, as previously proposed.

Could the glymphatic system contribute to the clearance of cytotoxic protein in α-synucleinopathies? To date, studies of glymphatic clearance mechanisms in neurodegenerative diseases have largely focused on amyloid-β, but more recent studies showed that proteins prone to intracellular accumulation can also be subject to glymphatic clearance—as discussed above, in the case of tau. Indeed, mounting evidence indicates that α-synuclein can also be cleared by the glymphatic system, with the first indication coming from studies on MSA, a rare progressive neurodegenerative disease marked by aggregated α-synuclein in glia [169]. Post-mortem histological examination of brains of MSA patients revealed that phosphorylated α-synuclein accumulates at the astrocytic endfeet, suggesting the perivascular glymphatic clearance route of α-synuclein in the brain [170]. Further, injection of the MSA brain tissue-derived α-synuclein into the brains of mice induced an increase in widespread astrogliosis, but more interestingly the authors of this study observed an inverse relationship between the extent of astrocyte activation and the distance from α-synuclein inclusions [171]. Adding to this evidence, histological examination of brain tissues from PD patients showed a negative correlation between α-synuclein deposition and astrocytic AQP4 expression [172], implying a close relationship between glymphatic function and α-synuclein accumulation. These studies were the primary indication of a relationship between α-synuclein accumulation, AQP4 expression and polarisation in the brain. Furthermore, it highlighted that similar to tau, the glymphatic clearance pathway is also likely to play a role in the removal of CNS α-synuclein, and in disease progression of synucleinopathies such as PD.

New data from PD animal models further support the relationship between the glymphatic system and PD pathology progression. To investigate the possible involvement of meningeal lymphatic drainage and α-synuclein clearance, Zou and colleagues surgically blocked the deep cervical lymph nodes of young A53T mutant α-synuclein transgenic mice [51]. Their results showed that at 6 weeks following surgery, there was a dramatic decrease in meningeal lymphatic drainage as a result of drainage blockade, which resulted in a significant increase of α-synuclein deposition in the brains of these animals [51]. Of note, since publication of this mouse study, it has been shown that a similar scenario of impaired meningeal lymphatic drainage occurs in patients with idiopathic PD [173], adding even greater credence to initial experimental work in mice. The acceleration of the pathology observed in the mice was reflected by an increase in dopaminergic neuronal loss in the substantia nigra, which in turn led to severe behavioural motor impairments [51]. Interestingly, the study also reported a dramatic increase in the number of activated glial cells, which was accompanied by a substantial rise in the secretion of inflammatory factors [51]. However, in contrast to previous findings in patients that α-synuclein deposition was inversely proportional to astrocytic AQP4 expression and the distance from α-synuclein inclusions [171, 172], Zou and colleagues reported the opposite. Their data showed a striking increase in the level of expression of AQP4 and a decrease in its polarization, together with dense accumulation of AQP4 protein surrounding α-synuclein-positive cells in the midbrain of affected mice [51]. The discrepancies in the findings might be explained by differences in the stage of the pathology and/or area of brain examined, the technique used to evaluate AQP4 protein localisation, and most importantly, the species examined (mice as opposed to human tissue). For example, the proportion of AQP4 polarisation to glial endfeet itself is known to differ between humans and mice; therefore, it may be that the mouse brain reacts differently to such pathological challenges, i.e., attempts to clear α-synuclein by upregulating AQP4 in surrounding glia, a capability that may be deficient in human disease. Greater research focus is required to address such hypotheses and understand the relationship between glymphatic function and α-synuclein, but a very recent publication has gone some way to unequivocally linking the two. Cui et al. [52] showed that hemizygous AQP4 knockout mice which received a bi-lateral intrastriatal injection of α-synuclein pre-formed fibrils presented an accelerated pathologic deposition of α-synuclein, which in turn, facilitated the loss of dopaminergic neurons and consequent motor-behaviour impairments. Together, these findings further suggest that impaired glymphatic function, through decreased AQP4 expression, is capable of aggravating PD-like pathology in the brain, warranting further investigation of this pathway, or at least this water channel, as a therapeutic target in α-synucleinopathies.

Although research on the influence of the glymphatic system on α-synucleinopathy is in its infancy, it is clear that the above studies provide evidence for the involvement of the glymphatic system in the phenomenon of neuron-to-neuron propagation and clearance of α-synuclein from the brain. Undeniably, more studies aiming to elucidate the interplay between this novel clearance pathway and α-synuclein pathology will be necessary to understand what mechanisms are underlying the progression of the disease. This exciting and promising new area of research may potentially unveil new therapeutic targets for neurodegenerative α-synucleinopathies such as PD.