Immune landscape of the enteric nervous system differentiates Parkinson's disease patients from controls: The PADUA-CESNE cohort.

BACKGROUND: Gastrointestinal dysfunction has emerged as a prominent early feature of Parkinson's Disease, shedding new light on the pivotal role of the enteric nervous system in its pathophysiology. However, the role of immune-cell clusters and inflammatory and glial markers in the gut pathogenetic process needs further elucidation. OBJECTIVES: We aimed to study duodenum tissue samples to characterize PD's enteric nervous system pathology further. Twenty patients with advanced PD, six with early PD, and 18 matched controls were included in the PADUA-CESNE cohort. METHODS: Duodenal biopsies from 26 patients with early to advanced stage PD and 18 age-matched HCs were evaluated for the presence of surface markers (CD3+, CD4+, CD8+, CD20+, CD68+, HLA-DR), presence of misfolded alpha-synuclein and enteric glial alteration (GFAP). Correlation of immulogic pattern and clinical characteristic were analyzed. RESULTS: The findings validate that in patients with Parkinson's Disease, the activation and reactive gliosis are linked to the neurodegeneration triggered by the presence of misfolded alpha-synuclein in the enteric nervous system. This process intensifies from the initial to the advanced stages of the disease. The clusters of T- and B-lymphocytes in the enteric system, along with the overall expression of HLA-DR in antigen-presenting cells, exceeded those in the control group. Conversely, no differences in terms of macrophage populations were found. CONCLUSIONS: These findings broaden our understanding of the mechanisms underlying the enteric nervous system's involvement in PD and point to the gastrointestinal system as a potential therapeutic target, especially in the early stages of the disease. Moreover, our results propose a role of T- and B-lymphocytes in maintaining inflammation and ultimately influencing alpha-synuclein misfolding and aggregation.

Sequestosome-1 (SQSTM1/p62) as a target in dopamine catabolite-mediated cellular dyshomeostasis.

Alterations in the dopamine catabolic pathway are known to contribute to the degeneration of nigrostriatal neurons in Parkinson's disease (PD). The progressive cellular buildup of the highly reactive intermediate 3,4-dihydroxyphenylacetaldehye (DOPAL) generates protein cross-linking, oligomerization of the PD-linked αSynuclein (αSyn) and imbalance in protein quality control. In this scenario, the autophagic cargo sequestome-1 (SQSTM1/p62) emerges as a target of DOPAL-dependent oligomerization and accumulation in cytosolic clusters. Although DOPAL-induced oxidative stress and activation of the Nrf2 pathway promote p62 expression, p62 oligomerization rather seems to be a consequence of direct DOPAL modification. DOPAL-induced p62 clusters are positive for ubiquitin and accumulate within lysosomal-related structures, likely affecting the autophagy-lysosomal functionality. Finally, p62 oligomerization and clustering is synergistically augmented by DOPAL-induced αSyn buildup. Hence, the substantial impact on p62 proteostasis caused by DOPAL appears of relevance for dopaminergic neurodegeneration, in which the progressive failure of degradative pathways and the deposition of proteins like αSyn, ubiquitin and p62 in inclusion bodies represent a major trait of PD pathology.

The αSynuclein half-life conundrum.

αSynuclein (αSyn) misfolding and aggregation frequently precedes neuronal loss associated with Parkinson's Disease (PD) and other Synucleinopathies. The progressive buildup of pathological αSyn species results from alterations on αSyn gene and protein sequence, increased local concentrations, variations in αSyn interactome and protein network. Therefore, under physiological conditions, it is mandatory to regulate αSyn proteostasis as an equilibrium among synthesis, trafficking, degradation and extracellular release. In this frame, a crucial parameter is protein half-life. It provides indications of the turnover of a specific protein and depends on mRNA synthesis and translation regulation, subcellular localization, function and clearance by the designated degradative pathways. For αSyn, the molecular mechanisms regulating its proteostasis in neurons have been extensively investigated in various cellular models, either using biochemical or imaging approaches. Nevertheless, a converging estimate of αSyn half-life has not emerged yet. Here, we discuss the challenges in studying αSyn proteostasis under physiological and pathological conditions, the advantages and disadvantages of the experimental strategies proposed so far, and the relevance of determining αSyn half-life from a translational perspective.

Prospective Role of PAK6 and 14-3-3γ as Biomarkers for Parkinson's Disease.

Background: Parkinson's disease is a progressive neurodegenerative disorder mainly distinguished by sporadic etiology, although a genetic component is also well established. Variants in the LRRK2 gene are associated with both familiar and sporadic disease. We have previously shown that PAK6 and 14-3-3γ protein interact with and regulate the activity of LRRK2. Objective: The aim of this study is to quantify PAK6 and 14-3-3γ in plasma as reliable biomarkers for the diagnosis of both sporadic and LRRK2-linked Parkinson's disease. Methods: After an initial quantification of PAK6 and 14-3-3γ expression by means of Western blot in post-mortem human brains, we verified the presence of the two proteins in plasma by using quantitative ELISA tests. We analyzed samples obtained from 39 healthy subjects, 40 patients with sporadic Parkinson's disease, 50 LRRK2-G2019S non-manifesting carriers and 31 patients with LRRK2-G2019S Parkinson's disease. Results: The amount of PAK6 and 14-3-3γ is significantly different in patients with Parkinson's disease compared to healthy subjects. Moreover, the amount of PAK6 also varies with the presence of the G2019S mutation in the LRRK2 gene. Although the generalized linear models show a low association between the presence of Parkinson's disease and PAK6, the kinase could be added in a broader panel of biomarkers for the diagnosis of Parkinson's disease. Conclusions: Changes of PAK6 and 14-3-3γ amount in plasma represent a shared readout for patients affected by sporadic and LRRK2-linked Parkinson's disease. Overall, they can contribute to the establishment of an extended panel of biomarkers for the diagnosis of Parkinson's disease.

SARS-CoV-2-Mimicking Pseudoviral Particles Accelerate α-Synuclein Aggregation In Vitro.

Since the SARS-CoV-2 virus started spreading worldwide, evidence pointed toward an impact of the infection on the nervous system. COVID-19 patients present neurological manifestations and have an increased risk of developing brain-related symptoms in the long term. In fact, evidence in support of the neuroinvasive potential of SARS-CoV-2 has emerged. Considering that viral parkisonism was observed as a consequence of encephalopathies caused by viral infections, it has been already suggested that COVID-19 could affect the dopaminergic neurons and contribute to neurodegeneration in Parkinson's disease (PD), by promoting the formation of amyloid fibrils constituted by the PD-related protein α-synuclein. Here, we observe not only that SARS-CoV-2 viral spike protein and nucleocapsid protein can alone promote α-synuclein aggregation but also that the spike protein organization in a corona shape on the viral envelope may be crucial in triggering fast amyloid fibrils formation, thus possibly contributing to PD pathogenesis.

Site-Specific Ubiquitination of Tau Amyloids Promoted by the E3 Ligase CHIP.

Post-translational modifications of Tau are emerging as key players in determining the onset and progression of different tauopathies such as Alzheimer's disease, and are recognized to mediate the structural diversity of the disease-specific Tau amyloids. Here we show that the E3 ligase CHIP catalyzes the site-specific ubiquitination of Tau filaments both in vitro and in cellular models, proving that also Tau amyloid aggregates are direct substrate of PTMs. Transmission electron microscopy and mass spectrometry analysis on ubiquitin-modified Tau amyloids revealed that the conformation of the filaments restricts CHIP-mediated ubiquitination to specific positions of the repeat domain, while only minor alterations in the structure of the fibril core were inferred using seeding experiments in vitro and in a cell-based tauopathy model. Overexpression of CHIP significantly increased the ubiquitination of exogenous PHF, proving that the ligase can interact and modify Tau aggregates also in a complex cellular environment.

Scavenging neurotoxic aldehydes using lysine carbon dots.

Reactive aldehydes generated in cells and tissues are associated with adverse physiological effects. Dihydroxyphenylacetaldehyde (DOPAL), the biogenic aldehyde enzymatically produced from dopamine, is cytotoxic, generates reactive oxygen species, and triggers aggregation of proteins such as α-synuclein implicated in Parkinson's disease. Here, we demonstrate that carbon dots (C-dots) prepared from lysine as the carbonaceous precursor bind DOPAL molecules through interactions between the aldehyde units and amine residues on the C-dot surface. A set of biophysical and in vitro experiments attests to attenuation of the adverse biological activity of DOPAL. In particular, we show that the lysine-C-dots inhibit DOPAL-induced α-synuclein oligomerization and cytotoxicity. This work underlines the potential of lysine-C-dots as an effective therapeutic vehicle for aldehyde scavenging.

Hsc70 rescues the synaptic vesicle trafficking defects caused by α-synuclein dimers.

Aberrant buildup of α-synuclein is associated with Parkinson's disease (PD) and other neurodegenerative disorders. At synapses, α-synuclein accumulation leads to severe synaptic vesicle trafficking defects. We previously demonstrated that different molecular species of α-synuclein produce distinct effects on synaptic vesicle recycling, and that the synaptic phenotypes caused by monomeric α-synuclein were ameliorated by Hsc70. Here, we tested whether Hsc70 could also correct synaptic deficits induced by α-synuclein dimers. Indeed, co-injection of Hsc70 with α-synuclein dimers completely reversed the synaptic deficits, resulting in synapses with normal appearance. This work lends additional support for pursuing chaperone-based strategies to treat PD and other synucleinopathies.

DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson's disease.

Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson's disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration.

α-Synuclein Toxicity in Drosophila melanogaster Is Enhanced by the Presence of Iron: Implications for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the preferential loss of dopaminergic neurons and by the accumulation of intracellular inclusions mainly composed of α-synuclein (α-Syn). While the etiopathogenesis of the disorder is still elusive, recent experimental evidence supports the involvement of ferroptosis, an iron-dependent cell death pathway, in the pathogenesis of PD. In the present work, using different ferroptosis inducers and inhibitors, we evaluated, in vivo, the involvement of iron in the α-Syn-mediated toxicity. Using a Drosophila melanogaster model of PD based on the selective over-expression of α-Syn within dopaminergic neurons, we demonstrated that the over-expression of α-Syn promotes the accumulation of protein aggregates, which is accompanied by dopaminergic neurodegeneration, locomotor impairment, and lifespan reduction. These pathological phenotypes were further exacerbated by reduced intracellular levels of glutathione or increased concentrations of iron. Coherently, both the use of an iron chelator and the presence of the antioxidant compound N-acetylcysteine exerted protective effects. Overall, our results support the involvement of ferroptosis in the α-Syn-mediated toxicity.

Copper Binding and Redox Activity of α-Synuclein in Membrane-Like Environment.

α-Synuclein (αSyn) constitutes the main protein component of Lewy bodies, which are the pathologic hallmark in Parkinson's disease. αSyn is unstructured in solution but the interaction of αSyn with lipid membrane modulates its conformation by inducing an α-helical structure of the N-terminal region. In addition, the interaction with metal ions can trigger αSyn conformation upon binding and/or through the metal-promoted generation of reactive oxygen species which lead to a cascade of structural alterations. For these reasons, the ternary interaction between αSyn, copper, and membranes needs to be elucidated in detail. Here, we investigated the structural properties of copper-αSyn binding through NMR, EPR, and XAS analyses, with particular emphasis on copper(I) coordination since the reduced state is particularly relevant for oxygen activation chemistry. The analysis was performed in different membrane model systems, such as micellar sodium dodecyl sulfate (SDS) and unilamellar vesicles, comparing the binding of full-length αSyn and N-terminal peptide fragments. The presence of membrane-like environments induced the formation of a copper:αSyn = 1:2 complex where Cu+ was bound to the Met1 and Met5 residues of two helical peptide chains. In this coordination, Cu+ is stabilized and is unreactive in the presence of O2 in catechol substrate oxidation.

Duodenal alpha-Synuclein Pathology and Enteric Gliosis in Advanced Parkinson's Disease.

BACKGROUND: The role of the gut-brain axis has been recently highlighted as a major contributor to Parkinson's disease (PD) physiopathology, with numerous studies investigating bidirectional transmission of pathological protein aggregates, such as α-synuclein (αSyn). However, the extent and the characteristics of pathology in the enteric nervous system have not been fully investigated. OBJECTIVE: We characterized αSyn alterations and glial responses in duodenum biopsies of patients with PD by employing topography-specific sampling and conformation-specific αSyn antibodies. METHODS: We examined 18 patients with advanced PD who underwent Duodopa percutaneous endoscopic gastrostomy and jejunal tube procedure, 4 untreated patients with early PD (disease duration <5 years), and 18 age- and -sex-matched healthy control subjects undergoing routine diagnostic endoscopy. A mean of four duodenal wall biopsies were sampled from each patient. Immunohistochemistry was performed for anti-aggregated αSyn (5G4) and glial fibrillary acidic protein antibodies. Morphometrical semiquantitative analysis was performed to characterize αSyn-5G4+ and glial fibrillary acidic protein-positive density and size. RESULTS: Immunoreactivity for aggregated α-Syn was identified in all patients with PD (early and advanced) compared with controls. αSyn-5G4+ colocalized with neuronal marker ß-III-tubulin. Evaluation of enteric glial cells demonstrated an increased size and density when compared with controls, suggesting reactive gliosis. CONCLUSIONS: We found evidence of synuclein pathology and gliosis in the duodenum of patients with PD, including early de novo cases. Future studies are required to evaluate how early in the disease process duodenal pathology occurs and its possible contribution to levodopa effect in chronic patients. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Exploiting HOPNO-dicopper center interaction to development of inhibitors for human tyrosinase.

In human, Tyrosinase enzyme (TyH) is involved in the key steps of protective pigments biosynthesis (in skin, eyes and hair). The use of molecules targeting its binuclear copper active site represents a relevant strategy to regulate TyH activities. In this work, we targeted 2-Hydroxypyridine-N-oxide analogs (HOPNO, an established chelating group for the tyrosinase dicopper active site) with the aim to combine effects induced by combination with a reference inhibitor (kojic acid) or natural substrate (tyrosine). The HOPNO-MeOH (3) and the racemic amino acid HOPNO-AA compounds (11) were tested on purified tyrosinases from different sources (fungal, bacterial and human) for comparison purposes. Both compounds have more potent inhibitory activities than the parent HOPNO moiety and display strictly competitive inhibition constant, in particular with human tyrosinase. Furthermore, 11 appears to be the most active on the B16-F1 mammal melanoma cells. The investigations were completed by stereospecificity analysis. Racemic mixture of the fully protected amino acid 10 was separated by chiral HPLC into the corresponding enantiomers. Assignment of the absolute configuration of the deprotected compounds was completed, based on X-ray crystallography. The inhibition activities on melanin production were tested on lysates and whole human melanoma MNT-1 cells. Results showed significant enhancement of the inhibitory effects for the (S) enantiomer compared to the (R) enantiomer. Computational studies led to an explanation of this difference of activity based for both enantiomers on the respective position of the amino acid group versus the HOPNO plane.

DJ-1 promotes energy balance by regulating both mitochondrial and autophagic homeostasis.

The protein DJ-1 is mutated in rare familial forms of recessive Parkinson's disease and in parkinsonism accompanied by amyotrophic lateral sclerosis symptoms and dementia. DJ-1 is considered a multitasking protein able to confer protection under various conditions of stress. However, the precise cellular function still remains elusive. In the present work, we evaluated fruit flies lacking the expression of the DJ-1 homolog dj-1ß as compared to control aged-matched individuals. Behavioral evaluations included lifespan, locomotion in an open field arena, sensitivity to oxidative insults, and resistance to starvation. Molecular analyses were carried out by analyzing the mitochondrial morphology and functionality, and the autophagic response. We demonstrated that dj-1ß null mutant flies are hypoactive and display higher sensitivity to oxidative insults and food deprivation. Analysis of mitochondrial homeostasis revealed that loss of dj-1ß leads to larger and more circular mitochondria, characterized by impaired complex-I-linked respiration while preserving ATP production capacity. Additionally, dj-1ß null mutant flies present an impaired autophagic response, which is suppressed by treatment with the antioxidant molecule N-Acetyl-L-Cysteine. Overall, our data point to a mechanism whereby DJ-1 plays a critical role in the maintenance of energy homeostasis, by sustaining mitochondrial homeostasis and affecting the autophagic flux through the maintenance of the cellular redox state. In light of the involvement of DJ-1 in neurodegenerative diseases and considering that neurons are highly energy-demanding cells, particularly sensitive to redox stress, our study sheds light on a key role of DJ-1 in the maintenance of cellular homeostasis.

Neuroprotective Effect of Antiapoptotic URG7 Protein on Human Neuroblastoma Cell Line SH-SY5Y.

Up-regulated Gene clone 7 (URG7) is a protein localized in the endoplasmic reticulum (ER) and overexpressed in liver cells upon hepatitis B virus (HBV) infection. Its activity has been related to the attenuation of ER stress resulting from HBV infection, promoting protein folding and ubiquitination and reducing cell apoptosis overall. While the antiapoptotic activity of URG7 in HBV-infected cells may have negative implications, this effect could be exploited positively in the field of proteinopathies, such as neurodegenerative diseases. In this work, we aimed to verify the possible contribution of URG7 as a reliever of cellular proteostasis alterations in a neuronal in vitro system. Following tunicamycin-induced ER stress, URG7 was shown to modulate different markers of the unfolded protein response (UPR) in favor of cell survival, mitigating ER stress and activating autophagy. Furthermore, URG7 promoted ubiquitination, and determined a reduction in protein aggregation, calcium release from the ER and intracellular ROS content, confirming its pro-survival activity. Therefore, in light of the results reported in this work, we hypothesize that URG7 offers activity as an ER stress reliever in a neuronal in vitro model, and we paved the way for a new approach in the treatment of neurodegenerative diseases.

DJ-1 and SOD1 Act Independently in the Protection against Anoxia in Drosophila melanogaster.

Redox homeostasis is a vital process the maintenance of which is assured by the presence of numerous antioxidant small molecules and enzymes and the alteration of which is involved in many pathologies, including several neurodegenerative disorders. Among the different enzymes involved in the antioxidant response, SOD1 and DJ-1 have both been associated with the pathogenesis of amyotrophic lateral sclerosis and Parkinson's disease, suggesting a possible interplay in their mechanism of action. Copper deficiency in the SOD1-active site has been proposed as a central determinant in SOD1-related neurodegeneration. SOD1 maturation mainly relies on the presence of the protein copper chaperone for SOD1 (CCS), but a CCS-independent alternative pathway also exists and functions under anaerobic conditions. To explore the possible involvement of DJ-1 in such a pathway in vivo, we exposed Drosophila melanogaster to anoxia and evaluated the effect of DJ-1 on fly survival and SOD1 levels, in the presence or absence of CCS. Loss of DJ-1 negatively affects the fly response to the anoxic treatment, but our data indicate that the protective activity of DJ-1 is independent of SOD1 in Drosophila, indicating that the two proteins may act in different pathways.

Water-Soluble Melanin-Protein-Fe/Cu Conjugates Derived from Norepinephrine as Reliable Models for Neuromelanin of Human Brain Locus Coeruleus.

Water-soluble melanin-protein-Fe/Cu conjugates derived from norepinephrine and fibrillar ß-lactoglobulin are reliable models for neuromelanin (NM) of human brain locus coeruleus. Both iron and copper promote catecholamine oxidation and exhibit strong tendency to remain coupled in oligonuclear aggregates. The Fe-Cu clusters are EPR silent and affect the 1 H NMR spectra of the conjugates through a specific sequence of signals. Derivatives containing only Fe or Cu exhibit different NMR patterns. The EPR spectra show weak signals of paramagnetic FeIII in conjugates containing Fe or mixed Fe-Cu sites due to small amounts of mononuclear centers. The latter derivatives exhibit EPR signals for isolated CuII centers. These features parallel the EPR behavior of NM from locus coeruleus. The spectral data indicate that FeIII is bound to the melanic fraction, whereas CuII is bound on the protein fibrils, suggesting that the Fe-Cu clusters occur at the interface between the two components of the synthetic NMs.

Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease.

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80% of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinson's disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knock-in mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular re-localization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

LRRK2 as a target for modulating immune system responses.

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are associated with familial and sporadic cases of Parkinson's disease (PD) but are also found in patients with immune- related disorders, such as inflammatory bowel disease (IBD) and leprosy, linking LRRK2 to the immune system. Supporting this genetic evidence, in the last decade LRRK2 was robustly shown to modulate inflammatory responses at both systemic and central nervous system level. In this review, we recapitulate the role of LRRK2 in central and peripheral inflammation in PD and inflammatory disease models. Moreover, we discuss how LRRK2 inhibitors and anti- inflammatory drugs may be beneficial at reducing disease risk/progression in LRRK2-mutation carriers and manifesting PD patients, thus supporting LRRK2 as a promising disease-modifying PD strategy.

The potential convergence of NLRP3 inflammasome, potassium, and dopamine mechanisms in Parkinson's disease.

The pathology of Parkinson's disease (PD) is characterized by α-synuclein aggregation, microglia-mediated neuroinflammation, and dopaminergic neurodegeneration in the substantia nigra with collateral striatal dopamine signaling deficiency. Microglial NLRP3 inflammasome activation has been linked independently to each of these facets of PD pathology. The voltage-gated potassium channel Kv1.3, upregulated in microglia by α-synuclein and facilitating potassium efflux, has also been identified as a modulator of neuroinflammation and neurodegeneration in models of PD. Evidence increasingly suggests that microglial Kv1.3 is mechanistically coupled with NLRP3 inflammasome activation, which is contingent on potassium efflux. Potassium conductance also influences dopamine release from midbrain dopaminergic neurons. Dopamine, in turn, has been shown to inhibit NLRP3 inflammasome activation in microglia. In this review, we provide a literature framework for a hypothesis in which Kv1.3 activity-induced NLRP3 inflammasome activation, evoked by stimuli such as α-synuclein, could lead to microglia utilizing dopamine from adjacent dopaminergic neurons to counteract this process and fend off an activated state. If this is the case, a sufficient dopamine supply would ensure that microglia remain under control, but as dopamine is gradually siphoned from the neurons by microglial demand, NLRP3 inflammasome activation and Kv1.3 activity would progressively intensify to promote each of the three major facets of PD pathology: α-synuclein aggregation, microglia-mediated neuroinflammation, and dopaminergic neurodegeneration. Risk factors overlapping to varying degrees to render brain regions susceptible to such a mechanism would include a high density of microglia, an initially sufficient supply of dopamine, and poor insulation of the dopaminergic neurons by myelin.