Infections in the Etiology of Parkinson’s Disease and Synucleinopathies: A Renewed Perspective,Mechanistic Insights,and Therapeutic Implications

An infectious etiology of parkinsonism

An infectious etiology for synucleinopathies has been discussed for decades. Early observations of viral epidemics followed by an outbreak of parkinsonism have led to a long-held hypothesis that parkinsonism could be of infectious origin, and it has long been discussed whether viruses could be the initiating etiology of primary PD or if viruses could be causative for secondary parkinsonism.

A notable example and a historic conception of a potential infectious etiology of PD is the postencephalitic parkinsonism pandemic in the years following the Spanish flu. The Spanish flu, caused by a severely virulent H1N1 influenza A virus, affected millions of people worldwide and resulted in a global pandemic. After the outbreak of the Spanish flu, in 1918–1919, many patients subsequently developed encephalitis lethargica, a condition that is characterized by high fever, delayed physical and mental response and lethargy.6,7, 6,7 Although these symptoms from encephalitis lethargica only occurred sometime after infection it was clear that the onset pattern of encephalitis lethargica inherently followed the one of the influenza pandemics.

During this early phase of encephalitis lethargica, other neurological manifestations concurrently developed as patients experienced phases of delirium or episodes of psychotic manifestations. 8 Encephalitis lethargica could almost be seen as a precursor of neurological disease as a bout of encephalitis was almost invariably followed by postencephalitic parkinsonism. 8 Here, the classical signs of parkinsonism would include a mask-face like appearance, paralysis agitans, tremor, rigidity of all the muscles, and shuffling gait. 7 These symptoms could develop in all infected individuals, including young adults. 9 In the following decades, it was thus speculated that a link between viral respiratory infections and neurological diseases such as PD might exist. 10 However, these links became muddled because of a profusion of clinical signs and the long time between infection and the onset of neurological symptoms in which the association between parkinsonism and influenza was simply forgotten.

Nevertheless, pathological examinations of patients with postencephalitic parkinsonism, some of which were done more recently,11–14 revealed that PD-related brain regions of patients infected with the Spanish flu were indeed severely affected. In infected cases, a remarkable hyaline degeneration with loss of blood vessels was observed in the striatum and more generally in the basal ganglia,6,15, 6,15 Vascular subacute inflammation was sometimes seen, especially in the brainstem, but in the absence of signs of chronic or active neuroinflammation elsewhere in the brain 16 . From these historic cases, the most notably affected region was the substantia nigra, a region typically affected in synucleinopathies, showing loss of pigmented dopaminergic neurons and gliosis.11,12,17, 11,12,17 Maybe most striking was that this neurodegeneration within the substantia nigra and the basal ganglia was accompanied by the presence of rounded or oval viral inclusions bodies but most often there were numerous cellular neurofibrillary tangles.11–14,18, 11–14,18 Importantly, the presence of αSyn positive inclusions in these cases was investigated, with recent immunohistochemical techniques and αSyn-specific antibodies, but αSyn pathology could not be shown.11–13 In cases of postencephalitic parkinsonism, and in the absence of synucleinopathy, it seems that based on these findings, there is more evidence for a tauopathy-related type of parkinsonism after influenza infection rather than predominantly synucleinopathy-related PD. It also suggests that based on its underlying biological mechanisms, postencephalitic parkinsonism might represent a unique entity in the biological classification of PD (Fig. 1) (we further discuss other implications of infections on the clinical classifications of PD below).

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Fig. 1

A role for infections in the etiology of synucleinopathies. The environmental exposure to infectious pathogens can trigger inflammatory cellular pathways involved in disease pathogenesis. Parkinsonism following post-encephalitic infection with viruses arises from neuroinflammatory processes that cause degeneration of dopaminergic neurons in the substantia nigra. Genetic risk variants in mitochondrial or lysosomal genes (PINK1, Parkin, and LRRK2) may exacerbate or precipitate motor and non-motor symptoms by modulating immune responses to infections. This happens via a process that is independent of αSyn. For people with PD that are αSyn negative, PD is likely not a synucleinopathy. Here, the biology of disease would be fundamentally different from αSyn-positive PD. In contrast, PD-related genetic risk factors can lead to increased αSyn aggregation and exaggerated immune reactions, resulting in enhanced inflammatory and neuroinflammatory responses leading to neuronal damage and the onset of new symptoms. For people with PD that are αSyn positive, PD is a synucleinopathy. Interactions between genetic and inflammatory pathogens contribute to the onset and progression of different subtypes of PD, emphasizing the complex biology and the interplay between genetics, environmental insults, and neuroinflammatory pathways.

The development and onset of neurological symptoms after influenza infection varied. The onset of parkinsonism after influenza infection ranged from days to a more prolonged onset with cases that would show neurological symptoms not until decades after infection 8 . Symptoms also developed when the infected person was considered to have fully recovered from the infection. The establishment of infection-related encephalitis lethargica or postencephalitic parkinsonism as a clinical entity thus rests on the abundant epidemiological, clinical, and pathological descriptions during that time. Of note, the delayed onset of neurological symptoms is in line with the natural history of PD, where a long prodromal phase precedes the occurrence of the more typical cardinal symptoms.

Along those lines, case reports of infection with other viruses have also been reported to cause postencephalitic dopamine-responsive parkinsonism with alterations in the basal ganglia and neurodegeneration of the substantia nigra.19–21 Several of these case reports include infections with SARS-CoV-2,22–25 enterovirus,26–29 Japanese encephalitis virus,30–32 St. Louis encephalitis, 33 West Nile virus,34–37 HIV,38–40 and dengue viral infections.19,41, 19,41 Importantly, this spectrum of viruses demonstrates that development of neurological symptoms and brain pathology after infection does not require the virus to enter brain tissue or to replicate in neurons as some of these viruses are not neurotropic.

An infectious etiology of idiopathic Parkinson’s disease

Although viral post-encephalitic cases show many clinical similarities with PD, these cases of viral encephalitis with parkinsonism are etiologically distinct from idiopathic PD. The mechanisms leading to selective degeneration of the basal ganglia and the substantia nigra in the absence of overt synucleinopathy are in stark contrast to the natural history of PD and the progression of pathology in idiopathic cases.

Nevertheless, links between various microbial infections as causative or precipitating triggers of idiopathic PD have recently received renewed attention, especially in the wake of recent epidemiological studies. In a systematic and population-based case control study from Denmark, various types of infections were examined for their potential link with PD. An association was shown between the onset of PD and infection with influenza when the infection occurred more than 10 years before clinical diagnosis. 42 Similar findings were observed in a second and independent systematic population-based study. Here, cases from Finland and the UK were studied. 43 A significant association was again found between infection with influenza and the subsequent development of PD if the time between infection and diagnosis was 5 years. 43

Another notable association from these studies is the link between PD and urinary tract infections. 42 Aside from influenza infections, urinary tract infections were found to be the only other type of infection associated with an increased risk of PD more than 10 years after infection. 42 Interestingly, similar findings were found for urinary tract infections and MSA. 44 Urinary tract infections are common in PD but also in MSA, especially in the years before diagnosis. Even though MSA has a shorter prodrome, urinary tract infections are associated with increased risk of MSA when the infection happens 8 years before the appearance of cardinal symptoms. 44 In the general population, urinary tract infections are prevalent, especially in women. However, in PD and in MSA, urinary tract infections affect men and women equally.44,45, 44,45 The reasons for this are not fully understood, and it remains unclear how central or peripheral pathology of synucleinopathies might contribute to increased susceptibility to urinary tract infection, which in turn could exacerbate neurodegenerative processes, as further discussed below. This requires more research, as these infections are linked with the onset of synucleinopathies.

Other epidemiological results are less clear as to when they occur in relation to the prodrome and how they might contribute to synucleinopathies. Some epidemiological studies focused on the presence of pathogen-specific antigens present in the plasma or brain tissue of people with PD by using ELISA, PCR or immunohistochemical methods. Therefore, it is not known if these infections would have occurred before or after the onset of disease. Other studies also relied on self-reporting or used a questionnaire-based methodology to examine potential associations with PD, 46 which can potentially introduce a recall bias and an under- or overestimation of infection.

Nevertheless, from these studies it is apparent that multiple pathogens might increase the risk of developing synucleinopathy or unmask underlying pathogenesis during disease progression. As such, notable links have been established between infections with hepatitis C virus, herpes virus, Helicobacter pylori and other types of pathogens that are further summarized in Table 1.

Prevention and vaccination

Studies have reported that vaccination may decrease the association between influenza or herpes zoster infection and the development of PD after vaccination.46,60, 46,60 However, in one prospective study in people older than 60 years there was no association found between influenza vaccination and risk of PD. 61 Antivirals for the treatment of chronic hepatitis also result in a reduction of associated PD risk. 47,58,59, 47,58,59 Vaccinations against these viruses could potentially reduce the risk of infection and, subsequently, lower the risk of developing PD. Individual factors such as age, overall health, and genetic risk factors could be taken into consideration when making decisions regarding vaccination and to determine the most appropriate vaccination strategy for individuals at risk of developing PD.

Vaccines are available for most of the viruses listed in Table 1; however, vaccination rates remain relatively low, even for people that already have PD. It has been estimated that vaccination coverage for influenza is approximately only half of all PD patients 62 whereas for SARS-CoV-2 vaccination rates in people with PD are even lower than that of the general population. 63 A reason for this could be restricted access for people with PD to primary healthcare 64 but efforts are needed to improve the number of people with PD or related disorders with healthcare access.

Before as well as after diagnosis of synucleinopathies, it could thus be important for people at risk to get vaccinated for several reasons. PD can weaken the immune system, making individuals more susceptible to infections. 65 Infections and illnesses can sometimes exacerbate disease symptoms or trigger temporary worsening. By avoiding illness through vaccination, individuals with PD may manage their symptoms more effectively. Particularly in MSA, or in patients with autonomic dysfunction, patients may be more vulnerable to developing complications from infections such as pneumonia or influenza. Vaccinations against these diseases can reduce the risk of serious complications and hospitalization.

It could thus be hypothesized that infectious pathogens might be associated with an elevated risk of developing a synucleinopathy. However, as we will also discuss in the next section, these associations do not necessarily imply causality, as these observations, although from multiple systematic and observational studies, cannot definitively establish a causal relationship. On the other hand, the association of these risk factors warrant their involvement in synucleinopathies, regardless of the directionality of these associations. Infections could precipitate a preclinical condition and thus accelerate or trigger the onset of synucleinopathy which needs to be further investigated.

A vicious cycle of infections and synucleinopathies?

These historical and epidemiological observations are a good example to illustrate the complexity of pathogen-host interactions: how do we study the relationship between a potential causative agent and a disease that is only diagnosed following a long asymptomatic period? The prodromal phase in synucleinopathies can range from approximately 5–10 years in MSA to 15–20 years in PD.66–69 If infections would act as triggers of disease, they would therefore need to occur around the time when the asymptomatic period would start. An interaction would at best support the contention that an association might exist, but it cannot prove causality.

The protracted prodrome in synucleinopathies now brings us to a second challenge: if an infection happens after the start of the prodrome, how can we be certain about the direction of causation? If an infection happens too close to the time of diagnosis, this would raise concerns about the causative role of the infection since some of the prodromal symptoms can be risk factors for infection. For these cases, the infection might then be a consequence of the disease rather than a trigger of the disease. However, this does not preclude infections from accelerating disease progression or aggravate symptoms.

One of these early cardinal symptoms of PD and MSA, which in some cases manifests during the prodrome, is autonomic dysfunction.68,70–72, 68,70–72 The autonomic nervous system controls several crucial functions such as heart rate, blood pressure, swallowing, digestion, sweating, temperature, or urinary voiding—among others. In PD and especially in MSA, autonomic dysfunction can be severe. Autonomic dysfunction can occur with the loss of peripheral innervation,73,74, 73,74 leading to desensitization.

Dysfunction of the autonomic nervous system can lead to a higher susceptibility to infections via several mechanisms. Swallowing difficulties or dysphagia can lead to aspiration pneumonia, whereas constipation can disrupt the composition of microbiota or lead to fecal retention and an increased risk of urinary tract infections (via translocation of bacteria). Similarly, loss of bladder control, can lead retention of volume in the urinary bladder, which again can increase the risk of urinary tract infections.

Another important function, that is impaired early in MSA, is thermoregulation.75,76, 75,76 Upon infection, an increase in body temperature or sweating would normally indicate signs of infection, but in MSA these functions are impaired. Impaired sweating can create an environment conducive to the growth of bacteria and increase the risk of skin infections in PD and MSA.57,77, 57,77 In the absence of these signs, this can create a dangerous situation where signs of an infection will be muted. Therefore, in some cases, it is possible that the normal signs of infection are missed in which a condition can worsen, causing neurological symptoms, such as confusion or delirium, which require urgent medical treatment.

There is also a more direct way via which the autonomic nervous system is important in the host response against infections. Neurotransmitters of the autonomous nervous system, such as acetylcholine or norepinephrine, can bind to immune cell receptors, which will lead to activation or proliferation of these cells.78,79, 78,79 Alternatively, via the release of neurotransmitters immune cells that are stored in lymphoid organs can be recruited. 80 The loss of autonomic innervation can result in changes in neuroimmune interactions and potentially impair the ability of the immune response to fight off infections. 81

Clearly, due to the protracted nature of synucleinopathies, and their long prodromal phase, there are significant challenges in studying how infections might influence the initiation or the progression of these diseases. Nevertheless, there is growing epidemiological evidence that infections play a role in both disease initiation and disease progression and that infections might have a causative and reciprocal role in the pathogenesis of these diseases.

Converging cellular signaling pathways during infection and synucleinopathy

Several cellular signaling pathways have been shown to be affected in synucleinopathies, including impaired proteostasis, vesicular transport and mitochondrial dysfunction. 153 Many of these affected cellular pathways are altered during neuroinflammatory states, where glial cells and neurons respond to a peripheral or central insult. Interestingly, such cellular pathways are also impaired by pathogens during infection. Some pathogens have the capacity to hijack the protein synthesis and autophagy machineries so that they can replicate and avoid the host immune response.82,154, 82,154 Mitochondria are also central to the immune response as these organelles participate in several inflammatory signaling pathways. This includes an innate immune mechanism that detects the presence of cytoplasmic DNA within cells. This pathway plays a crucial role in the body’s defense against infections, particularly viral infections, but it can also sense mitochondrial DNA. This pathway involves cyclic GMP-AMP synthase (cGAS) and the transmembrane protein stimulator of interferon genes (STING). 155 The cellular responses to inflammation can trigger deleterious events, including oxidative stress and cytokine-receptor-mediated apoptosis, which might eventually lead to neuronal loss.

The ability to identify foreign DNA is essential for fending off bacterial and viral infections. Cytosolic DNA is a crucial DAMP, capable of activating the innate immune system through PRRs. Cyclic GMP-AMP synthase (cGAS), is a PRR that can detect foreign DNA from pathogens and self-DNA released during cellular damage, activating stimulator of interferon genes (STING), which will trigger a type I interferon (IFN-I) immune response. In recent years, the cGAS-STING pathway has emerged as a critical player in neurological disorders characterized by chronic neuroinflammation. Viral encephalitis after HSV-1 infection activates the cGAS-STING pathway leading to microglial-mediated IFN-I response. 156 STING-deficient mice are more susceptible to HSV-1 encephalitis after peripheral infection. 156 Moreover, the activation of IFN-I signaling has been observed in postmortem PD human samples and in several mouse models.157,158, 157,158 Impaired mitophagy in PD animal models triggers the activation of the cGAS-STING pathway with functional effects on neuroinflammation, dopaminergic neuron loss and motor function.158,159, 158,159 Inhibition of this signaling pathway alleviates the functional effects and even pathological αSyn accumulation in an αSyn preformed fibrils mouse model. 160

Another innate immune response signaling pathway that is activated upon infection is the inflammasome. Inflammasomes are a family of protein complexes that can sense environmental and cellular danger signals. 161 One of the most studied inflammasome proteins is the NLR family pyrin domain containing 3 (NLRP3)-inflammasome, the apoptosis-associated speck-like protein containing a caspase activating recruitment domain (ASC) and caspase-1. 161 Upon activation, inflammasomes produce and release pro-inflammatory cytokines IL-1β and IL-18 through pyroptotic pores. 161 Markers of activation of this pathway are elevated in the brain and in blood samples from PD.162–164 Activation of the NLRP3-inflammsome pathway is also present in genetic and toxicological mouse models of PD.162,165–167, 162,165–167 Recent studies suggest that downregulation of NLRP3-inflammasome can ameliorate dopaminergic neuron loss and motor deficits in different mouse models of PD. 168

Affected signaling pathways in synucleinopathies resemble those seen as a response to pathogen invasion or inflammatory processes. These changes often involve cellular stress and a combination of upregulation of defense mechanisms and downregulation of pathways essential for cell homeostasis. These similarities suggest that both synucleinopathies and infections may involve common mechanisms of neuronal damage and dysfunction (Fig. 3).

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Fig. 3

Overlapping cellular pathways involved in infections and synucleinopathy. The immune system is constantly surveying the presence of pathogens and other inflammatory agents. In the brain, immune and non-immune cells use extracellular and intracellular pattern recognition receptors (PRRs) to identify pathogen-associated (PAMPs) and damage-associated molecular patterns (DAMPs) from microbes and dying cells, respectively. In astrocytes, toll-like receptors (TLRs) that recognize microbial-associated molecular patterns are activated by αSyn. TLRs also mediate the internalization of αSyn into astrocytes. In both cases, TLRs activation triggers a pro-inflammatory phenotype in these cells. In neurons, both infections and neurodegeneration are characterized by endoplasmic reticulum stress, autophagy impairment and mitochondrial dysfunction. Mitochondrial dysfunction can contribute to the activation of an interferon immune response via cGAS-STING. During neurodegeneration and infections, αSyn and other pro-inflammatory mediators are released. Some pathogens and pro-inflammatory mediators can damage the neurovascular unit and cause blood brain barrier (BBB) permeability. At the brain border, αSyn can interact with border associated macrophages to facilitate T cell infiltration. These inflammatory conditions are also characterized by the infiltration of peripheral immune cells including T cells and natural killer (NK) cells into the brain parenchyma. Nigral dopaminergic neurons express MHC-I receptors that can present antigens to cytotoxic T cells. Brain macrophages, but in particular microglia, can recognize PAMPs and DAMPs including αSyn using different PRRs like TLRs and NOD-like receptors (NLRs). Upon recognition by PRRs, microglia establish a pro-inflammatory phenotype with cytokine production that can have persistent effects on neuroinflammation.

Infections as triggers of synucleinopathy

Gut triggers

The involvement of the gut in synucleinopathies has been a field of intensive research. Gut symptoms such as bloating and constipation appear early in some people with PD, indicating that the gut might be a starting site of pathology for these cases. This is in conjunction with early observations of αSyn pathology of the enteric nervous system as well as autonomous nervous system.68,110,192, 68,110,192 There is a bidirectional connection between the gastrointestinal tract and the brain, called the gut-brain axis, and it can be influenced by gut infections, dysbiosis, and inflammatory responses.

Changes in the gut microbiota happen early in PD. Multiple studies have shown that intestinal microbiota dysbiosis can be a prominent feature.192–195 Several factors can influence the gut microbiome, such as geographic factors and ethnicity. However, despite these factors, PD related dysbiosis is broadly characterized by a lower relative abundance of anti-inflammatory bacterial genera and an increase of pro-inflammatory and opportunistic pathogens.195,196, 195,196 These can include Escherichia, Klebsiella, and Porphyromonas.195,196, 195,196 Even though the causes for dysbiosis in PD are not fully understood, the gut microbiota can be affected by exposure to pollutants, pesticides, and infections. 197 Research in preclinical PD models has shown that gut microbiota manipulation can influence symptoms and pathology including αSyn aggregation.198–200 More recently, it was shown that a fecal microbiota transplantation can potentially alleviate motor symptoms in early-stage PD patients. 201

Gut dysbiosis and infections can cause pathological changes in the immunoregulatory function of the gut and increase intestinal permeability. Increased intestinal permeability is a characteristic of PD. 202 It is accompanied by changes in gut microbial metabolites, such as bacterial lipopolysaccharides (LPS) and short-chain fatty acids (SCFAs). Inflammatory mediators can be released from commensal and pathogenic gut microbes and trigger unwanted systemic and central effects. It has been proposed that the disruption of the gut epithelium could trigger a feedback loop that alters the microbiome to a more pro-inflammatory profile resulting in an increased permeability, oxidative stress, and aggregation of αSyn in the gut. 203

Experimental and clinical studies have implicated the enteroendocrine cells of the gut epithelium.204,205, 204,205 Enteroendocrine cells can sense the luminal content in the gastrointestinal epithelium, and they play crucial roles in regulating gastrointestinal functions, including digestion, nutrient absorption, and gut motility by producing and releasing hormones. LPS and SCFAs are potent activators of enteroendocrine cells and interestingly, enteroendocrine cells also produce αSyn. In response to inflammatory stimuli, these cells can transiently express and release αSyn.204,206, 204,206 This thus directly links infectious agents and microbial metabolites (LPS or SCFAs) with the production of αSyn in the gut (Fig. 4).

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Fig. 4

Infections cause αSyn aggregation and neuroinflammation. Infections can contribute to the pathological prossess of synucleinopathies via several routes. In the left panel, the cellular mechanisms that directly increase αSyn expression or influence its aggregation are described. Different bacterial and viral proteins can seed the aggregation of αSyn via direct or indirect molecular interactions. Various cells of the peripheral nervous system can express αSyn in response to bacterial and viral infections. Different cell types in the gut (peritoneal neurons and enteroendocrine cells) respond to inflammation by expressing αSyn. These cells further connect to the parasympathetic vagal nerve via which αSyn can transmit. αSyn can be released by the immune system itself, via neutrophils, during the response to bacterial infections. In the right panel, it is described how inflammatory triggers contribute to synucleinopathy. Pathogens and pro-inflammatory mediators can induce dysfunction of the neurovascular unit and cause blood-brain barrier (BBB) permeability. This increased permeability can facilitate the infiltration of pathogens and peripheral immune cells. Pro-inflammatory mediators or other microbial-derived molecules can be released as a reaction to infections in various peripheral organs. Gut dysbiosis and/or infections increase epithelial permeability and as a consequence pro-inflammatory mediators are released into the circulation; including cis-activating microbial byproducts such as SCFAs and LPS; in addition to trans-activating elements including bacterial outer membrane vesicles (OMVs).

Microbiota-derived SCFAs are common bacterial fermentation products. These gut microbial metabolites, such as for instance butyrate, propionate, or acetate, have many physiological functions including reducing inflammation and preserving epithelial integrity. 207 SCFAs-producing bacteria are less abundant, and these metabolites are decreased in the feces of PD patients.208–210 On the other hand, the release of SCFAs from the gut can influence microglial homeostasis. 211 Animal studies have demonstrated that SCFAs have functional consequences by increasing αSyn aggregation and the development of motor dysfunction. 212

LPS is a component of the outer membrane of Gram-negative bacteria and a potent activator of the innate immune response. LPS from the Enterobacteriaceae family is one of the most immunogenic forms and is found abundantly in the gut of PD patients.196,213, 196,213 It has been recently shown that PD patients have elevated levels of LPS in the blood and high levels of LPS binding protein (LBP) during the prodromal phase are associated with an increased risk of PD.214–216 LPS activates the innate Toll-like receptors 2 and 4 and triggers an immune response that promotes the production of pro-inflammatory cytokines, chemokines, and reactive oxygen species. TLRs activation in microglia leads to the production of pro-inflammatory cytokines, the initiation of the adaptive immune response and recruitment of T and B cells. In PD models, LPS induces a neuroinflammatory response that ultimately leads to neurodegeneration.217–220 This is likely caused by the selective vulnerability of dopaminergic neurons of the substantia nigra to LPS insults.217–220 Additionally, LPS administration can trigger αSyn expression and aggregation.221–224 This has been observed in both the gut as well as in the brain, and more specifically in the substantia nigra, of animal models. 221–224

Aside from the direct or cis-acting pathogenic effects of bacteria on αSyn and PD pathogenesis, bacteria can also exert trans-acting effects. This happens via the release of outer membrane vesicles (OMVs) or alternatively, via the release of amyloidogenic proteins. Gram-negative bacteria can release OMVs from the stomach or the gut which can cross the BBB. 225 E coli or Helicobacter pylori OMVs are only a size of 20–200 nm and thus can readily cross the BBB to infect neurons and glial cells. 226 When taken up by astrocytes, this will lead to the expression of complement proteins and cause an inflammatory reaction. 227 OMVs loaded with LPS can also interact with microglia and other PRRs-expressing cells. 225 Some of these bacterial vesicles also transfer different bacterial molecules such as RNAs, endotoxins and toxins. 225 Because of the direct transfer of pathogen metabolites to the cytosol, OMVs can trigger the activation of the intracellular NLRP3 inflammasome pathway resulting in the release of pro-inflammatory cytokines and mitochondrialdysfunction. 228

Next to these potentially pathogenic carrier vesicles, bacteria can release amyloidogenic proteins. One such well-characterized bacterial protein is the amyloid protein curli. Curli is produced by E. coli and the protein can aggregate into fibrils to facilitate biofilm formation and help with cellular adhesion. Curli fibrils play dual roles in promoting bacterial colonization and infection while also triggering the host immune response, contributing to both bacterial persistence and host defense mechanisms. Curli can cross-seed the aggregation of αSyn in vitro and injection of curli fibrils can lead to αSyn pathology in the gut. 229 Pathological transmission of αSyn to the CNS has been observed in mice after transplantation of curli-expressing E. coli. 230

Aside from gut bacteria, viral gut pathogens have also been associated with synucleinopathy. 231 Viral infection with norovirus can induce a persistent increase in αSyn expression in the enteric nervous system. 92 Here, the degree and the duration of viral infection in infected cases correlates with the expression of αSyn in gut biopsies. 92

Bacterial and viral gut infections can thus have prominent effects on the gut and the brain. They influence gut permeability resulting in systemic inflammation and neuroinflammation. Via the gut-brain axis gut infections can influence brain health and act as a trigger of synucleinopathy.

The role of αSyn in the innate immune response

Innate immunity involves the recognition of specific ligands by PRRs and the production of inflammatory mediators to limit infections and initiate tissue repair. TLRs are expressed by immune and non-immune cells, including microglia, neurons, astrocytes, and oligodendrocytes. 238 TLRs recognize a wide range of PAMPs 154 and DAMPs, including αSyn. 135

Increasing evidence thus suggests the involvement of TLRs in the pathogenesis of synucleinopathies. 239 It has been reported that in postmortem PD brain tissue there is an increased expression of the microbial-associated molecular pattern (MAMPs) receptors, TLR4 and TLR2.240,241, 240,241 In neuronal cell cultures, TLR2 activation increases αSyn protein levels. Since αSyn is recognized by TLRs, it could serve as a chemoattractant for the recruitment of T and B cells. 92 In PD patients, neurons with αSyn pathology have increased expression of TLR2 241 , which could make them particularly susceptible to the pro-inflammatory effects of pathogen derived MAMPs. Moreover, αSyn can increase the expression of TLRs in microglia to promote neuroinflammation, 242 Different high molecular weight assemblies of αSyn have varying affinities for TLRs and also activate the immune response with varyingefficiencies.243–245

Besides the effects of αSyn on microglia, αSyn also modulates the astroglia immune response. The intracellular accumulation of αSyn in astrocytes is necessary for the induction of a pro-inflammatory response. While astrocytes internalize αSyn mediated exclusively by TLR2, pro-inflammatory cytokine expression can be mediated by both TLR2 and TLR4.245–247 These observations have led to the hypothesis that astroglial TLR2 could play a dual role in synucleinopathies. On one hand by protecting neurons by the internalization of potentially toxic extracellular αSyn, and on the other hand by triggering neuroinflammation. 238

In the periphery, αSyn has similar effects on leukocytes via stimulation of TLRs. αSyn stimulates the maturation and activation of dendritic cells through TLR4. 92 In mice, αSyn is required for both a normal pro-inflammatory response to bacterial peptidoglycan and the T cell mediated response to immunization in the peritoneal cavity. 248 Interestingly, neurons that innervate the peritoneum seem to be the source of αSyn as a ligand for TLRs, supporting a mechanistic link between neuronal pathology and the immune response in theperiphery.

The role of αSyn in the adaptive immune response

Adaptive immunity requires the activation of antigen-sensitized cytotoxic T cells and release of cytokines and chemokines in response to foreign molecules or microorganisms. While T cells help to eliminate pathogen infected cells, B cells produce antibodies to protect against extracellular microbes and toxic molecules. 249 T cells can only recognize antigenic peptides displayed by major histocompatibility complex (MHC)-I or MHC-II molecules on the cell surface 249

MHC-I molecules are expressed in all cells to present antigen peptides to CD8 + T cells, which allows them to identify and eliminate infected cells. 249 Under normal conditions CD8 + T cells are tolerant to autologous or self-proteins. However, during infections cells can express microbial genes and present these ‘non-self’ antigenic peptides allowing CD8 + T cells to eliminate these cells. Nigral dopaminergic neurons express MHC-I suggesting that these neurons are potentially susceptible to T cell mediated cytotoxicity 250 . T cell infiltration in areas of the brain with high αSyn burden is seen in PD and DLB patients as well as in the brain of synucleinopathy animal models.147,251–253, 147,251–253 This reinforces the idea that the adaptive immune response is a potential driver of pathogenesis. Furthermore, the infiltration of cytotoxic CD8 + T cells has been seen as an early pathological event that precedes αSyn aggregation and neurodegeneration in the substantianigra. 254

While MHC-I molecules are expressed ubiquitously, MHC-II molecules are expressed on immune antigen presenting cells. MHC-II molecules present peptides from endocytosed molecules to CD4 + T cells to activate them. 249 Genome-wide association studies (GWAS) have found an association between PD risk and the human leukocyte antigen isotype DR (HLA-DR) alleles. HLA-DR is a cell receptor heterodimer expressed on the surface of antigen presenting cells including microglia and macrophages. Reactive microglia and border-associated macrophages positive for HLA-DR are abundant in the substantia nigra of PD cases.143,255,256, 143,255,256 HLA-DR are part of the MHC-II family, and the genetic link between HLA-DR variants and an increased risk of developing PD supports a role of the adaptive immune response in PD pathology.257–259

Within these lines, circulating T cells from PD patients recognize specific αSyn peptides. 260 Pathological αSyn can trigger an immune response and inflammation, producing autoreactive T cells. 261 This loss of self-tolerance could be associated with environmental factors including infections. 262 Here, repeated exposure to αSyn in the context of infection could thereby cause cellular damage. Furthermore, the HLA-DR allele HLA-DRB1*15 : 01 has been linked to an increased risk of PD, and this allele may also be involved in the presentation of αSyn peptides to T cells. 263 Peripherally circulating CD4 + and CD8 + T cells derived from PD patients produce cytokines in response to αSyn, suggesting there may be a chronic memory T cell response in PD. 264 MHC-II expression and T cell infiltration are essential for neuroinflammation and loss of nigral dopaminergic neurons in a mouse model of αSyn overexpression. 265

It is known that infections that activate the immune system may exacerbate existing conditions or trigger new disease activity in autoimmune diseases. For example, urinary tract infections or respiratory tract infections, which are common, have been associated with increased risk of relapses in multiple sclerosis.266,267, 266,267 The immune response to these infections might lead to increased activity of autoreactive T cells that attack the central nervous system. In the case of PD, and given the potential role of infections in synucleinopathies, peripheral infections could trigger autoimmune activation and cause αSyn autoreactive immune cells to attack αSyn presenting cells and contribute to neurodegeneration via a similar mechanism although it needs to be investigated if such links might indeed exist.

An emerging role for the immune response at the brain border

CNS or border-associated macrophages comprise perivascular, meningeal, and choroid plexus macrophages with phagocytic capacity, which support the BBB by scavenging harmful molecules and promoting efficient immune surveillance, including antigen presentation and cytokine production.268,269, 268,269 Leptomeningeal macrophages migrate postnatally into the perivascular space and differentiate into perivascular macrophages. 270 As described above, under disease conditions with alterations of the BBB, such as in synucleinopathies or neuroinflammation 271 peripheral immune cells potentially invade the CNS and encounter the response of border-associated macrophages.272,273, 272,273 Furthermore, studies in PD models indicate that peripheral monocyte infiltration plays a critical role in αSyn-induced neuroinflammation and neurodegeneration. 274

There are only few studies which address the role of border-associated macrophages as potential gatekeepers in such CNS immune cell invasion. A recent study in a model of synucleinopathy has shown that border-associated macrophages, not microglia, are responsible for CD4 + T cell antigen presentation and recruitment necessary for tissue infiltration and cytokine production upon αSyn pathology. 255 Interestingly, αSyn oligomers were detected in perivascular macrophages in synucleinopathy brain tissue. 275 Depletion of border-associated macrophages prevented αSyn-induced neuroinflammation, including microglial reactivity, T cell infiltration, and monocyte recruitment. 255 Whether this has therapeutic implications for synucleinopathies requires further research. 276

If T-cell and CNS macrophage responses are associated with αSyn pathology in synucleinopathies, then infections that cause neuroinflammation could further exacerbate these processes and thereby neurodegeneration. Proliferation of perivascular macrophages was shown to contribute to the development of encephalitic lesions during simian immunodeficiency virus (SIV) infection of adult macaques. 277 Interestingly, border-associated macrophages but not disease-associated microglia show overt long-term transcriptional alterations which persist after resolution of trypanosoma brucei infection in mice. 278 Thus, single infections may alter the brain’s resident immune response at the border. Border-associated macrophages also play a critical role in protecting the brain parenchyma from viruses. In mice infected with lymphocytic choriomeningitis virus (LCMV), depletion of border-associated macrophages prior to infection or conditional deletion of interferon-receptor signaling pathways in myeloid cells resulted in extensive viral spread into the CNS. This appeared to be mediated by MHC-II-positive meningeal macrophages, and low numbers of these cells, as seen upon LPS challenge, correlated with higher viral load. 279

Altogether, available evidence supports the involvement of brain resident and peripheral immune cells driving pathological events that can lead to neurodegeneration in synucleinopathies. Many of these cellular responses could potentially be triggered by infections and further aggravated by genetic risk factors.

Implications for treatment, diagnosis, and prevention

PD is the second most common and the fastest growing neurodegenerative disease affecting more than 4 million people worldwide and its prevalence has doubled in the last 25 years. 67 This increase persists even after correcting for aging suggesting that changing environmental factors could be important players in the pathophysiology of PD. Here, attention has been given to the role of pesticides, but less attention has been given to the role of other inflammatory agents, such as microbial pathogens.

More recently, the neurological symptoms observed after SARS-CoV-2 infection have brought new attention to the long-term neuroinflammatory effects on brain health after infection. The evidence reviewed here suggests that infections could have a role in the etiology of synucleinopathies (Fig. 1). Infections are part of the exposome, and this includes the total environmental exposures that individuals encounter throughout their lives, including factors such as pesticides, heavy metals, air pollution, solvents, and dietary habits. In PD and related synucleinopathies, these exposures may play a role in disease onset or disease development. 305 Studies have linked pesticides, herbicides like paraquat and rotenone, heavy metals such as lead and manganese, air pollution, and certain industrial chemicals (trichloroethylene, perchloroethylene or polychlorinated biphenyls) to an increased risk of PD.305,306, 305,306 When this is further accompanied by aging and immunosenescence (the inability of the immune system to appropriately respond to infection), exposure to environmental factors will lead to increased susceptibility and cause mild or chronic neuroinflammation. 307 Understanding the contribution of different environmental factors is thus crucial for developing preventive strategies and identifying modifiable risk factors for PD.

Because of the clinical heterogeneity in synucleinopathies, subcategorization or stratification of at-risk patients will be key to achieve better therapeutic results. 308 Even though in recent years great progress has been made in the development of molecular diagnosis for synucleinopathies, including protein seeding amplification assays, skin biopsies and new imaging modalities, there are no early diagnostic tests or disease progression markers that could aid in diagnosis or taking preventive measure in the case of modifiable genetic risk factors. Identifying individual risk factors, genetic or environmental, is imperative to perform personalized risk assessment and enable early intervention. It would allow preventive measures to be taken such as lifestyle intervention, diet, exercise or even medication to mitigate risk factors. By understanding an individual’s risk, preventive therapies could have the potential to delay disease onset or improve disease outcomes for those who are at risk of developing PD.

It will also be important to study and consider stratification to evaluate the efficacy of preventive measures. For example, despite the abundant evidence of the link between influenza infections and PD, a recent study based in the UK and Finland found no association with reduced risk of PD and influenza vaccination after 12 years, although a significant association was found between influenza infection and other types of dementia. 43 This suggests that specific host-pathogen interactions might be important in neurological disorders where co-pathologies are present. Recognizing familial or genetic risk factors is vital for making informed decisions about vaccination.

Understanding these risks helps tailor recommendations and emphasizes the importance of vaccination in protecting against potential health threats, especially for those who are predisposed. A downstream consequence of peripheral or systemic infection is neuroinflammation. 309 Neuroinflammatory changes can remain present in the brain long after resolution of infection or even in cases where the pathogen cannot be detected in the brain.181,182,310, 181,182,310 It has been suggested that the neurodegenerative process is driven by a long lasting low-grade neuroimmune response rather than by direct viral neuroinvasion.181,182,310, 181,182,310 Persistent neuroinflammation could help the brain to protect itself from subsequent insults via a primed innate immune system, but at the same time it will aggravate the age-related pathological process.

Much effort has been put into developing therapies that efficiently target neuroinflammation. Several of these approaches include anti-inflammatory medication or the targeted modulation of microglial activity. Another approach is to inhibit the activation of the inflammasome. 311 Here, several compounds are being studied for their potential to inhibit inflammasome activation directly. 312 These include small molecule inhibitors targeting specific inflammasome components.311,312, 311,312 Immunomodulatory therapies, including monoclonal antibodies aim to target specific immune cells or cytokines, which are also being investigated to regulate the immune response 313 . Lifestyle factors, such as diet, exercise, and stress reduction techniques, may help reduce inflammation and oxidative stress since they have been shown to ameliorate motor- and non-motor symptoms in PD.314–319

The recent and increasing popularity of GLP-1 receptor agonists, which are currently used for the treatment of type-2 diabetes, could also help to reduce inflammation in synucleinopathies. GLP-1 receptor agonists are being studied for the treatment of PD as they are potent inhibitors of neuroinflammation via inhibiting NF-kB activation in microglia.320,321, 320,321 In turn, this reduces the conversion of astrocytes into neurotoxic astrocytes. 320 Recent clinical studies have shown that the progression of motor and non-motor symptoms is slowed down or can even be stabilized when the GLP-1 receptor agonists are taken early in the disease course.320–322 While larger trials are ongoing, much research is needed to further evaluate the effects of GLP-1 receptor agonists in the treatment of early PD.

These anti-inflammatory therapeutic approaches could be combined with existing therapies and it is likely that anti-inflammatory therapy might be more successful during early stages. Precision medicine in PD or other synucleinopathies aims to tailor medical treatment to the individual needs of each patient and it should consider factors such as genetic makeup, environmental exposure to risk factors, and lifestyle.308,323, 308,323 We should therefore consider any prior but also future exposure to infectious or inflammatory pathogens as it is an integral part of the patients’ environment and lifestyle. By identifying individuals who may benefit from anti-inflammatory treatments based on their genetic predispositions other tailored anti-inflammatory therapies can then be advised to address their specificneeds.

Implications for research

If infections are indeed involved in the etiology of synucleinopathies, it will necessitate research into the pathogens or type of infections that can contribute to the development of pathology. This will involve studying potential mechanisms by which infections can trigger or exacerbate neurodegeneration. Some of these mechanisms might overlap with the mechanisms involved in toxins, pesticides or other inflammatory molecules that could trigger pathology via environmental exposure.

We would also need to address methodological challenges. The lack of molecular diagnosis tools, the long prodromal phases and the clinical heterogeneity make it especially challenging to evaluate the effect of infections on the disease process and to establish a causal effect in the etiology of synucleinopathies. Not only the type of infection but also the timing of infection might be crucial for when pathology might or might not develop. New animal models that investigate how acute or chronic inflammation caused by infections could shed new light on how these diseases might arise.

It has been proposed that some of PD-linked pathogenic mutations with high prevalence and ancient origin (e.g., LRRK2, PINK, GBA and SNCA) might have provided a survival advantage when life expectancy was shorter and when infections were more common.296,300,305, 296,300,305 Impaired lysosomal function might influence susceptibility to neurodegenerative processes but could potentially have modified immune responses favorably against certain pathogens. Some studies have also suggested that overexpression of αSyn can restrict viral replication or viral assembly. 324 Now, with improved hygiene, and better anti-microbial treatments, these effects are less beneficial and exposure to virulent pathogens could instead lead to age-related neurodegenerative diseases as the same genetic risk factors will act as a disease facilitator at a later age. 280

The notion that these genes provided advantages in the face of infectious diseases is thus supported by their roles in the immune system. This is also seen in other neuroinflammatory diseases, like multiple sclerosis, where age-related and disease associated genetic variants were positively selected because they offered an evolutionarily advantage during times when infections were more common. 325 However, more detailed epidemiological and genetic studies are needed to substantiate these hypotheses and theories. This includes studying how these genetic variants might have been selected in populations when faced with high burdens of infectious diseases. Understanding these mechanisms could provide insights into why these genes are preserved in the human genome despite their association with disease in today’s context. Understanding these risk genes will help us to understand their physiological role and the molecular mechanisms in which they are involved during infection or inflammation. It could identify new potential therapeutic targets or help to devise new strategies for the prevention or treatment of high-risk populations. Importantly, it also warrants the use of novel therapies aimed at these pathways, as inhibiting the cellular pathways important for the immune response could potentially increase the susceptibility towards certain infectious pathogens in a population that is already moreimmunosenescent.