⍺ -Synuclein levels in Parkinson ’ s disease – Cell types and forms that contribute to pathogenesis

Parkinson ’ s disease (PD) has two main pathological hallmarks, the loss of nigral dopamine neurons and the proteinaceous aggregations of ⍺ -synuclein ( ⍺ Syn) in neuronal Lewy pathology. These two co-existing features suggest a causative association between ⍺ Syn aggregation and the underpinning mechanism of neuronal degeneration in PD. Both increased levels and post-translational modifications of ⍺ Syn can contribute to the formation of pathological aggregations of ⍺ Syn in neurons. Recent studies have shown that the protein is also expressed by multiple types of non-neuronal cells in the brain and peripheral tissues, suggesting additional roles of the protein and potential diversity in non-neuronal pathogenic triggers. It is important to determine (1) the threshold levels triggering ⍺ Syn to convert from a biological to a pathologic form in different brain cells in PD; (2) the dominant form of pathologic ⍺ Syn and the associated post-translational modification of the protein in each cell type involved in PD; and (3) the cell type associated biological processes impacted by pathologic ⍺ Syn in PD. This review integrates these aspects and speculates on potential pathological mechanisms and their impact on neuronal and non-neuronal ⍺ Syn in the brains of patients with PD.


Introduction
The crucial role of ⍺-synuclein (⍺Syn) in the pathogenesis of Parkinson's disease (PD) is strongly implied by two facts: familial cases with missense mutations or multiplication of its gene SNCA have autosomal dominant PD, and in both familial and sporadic cases the brain has characteristic proteinaceous aggregations of ⍺Syn (Choong and Mochizuki, 2022).The protein is highly distributed in the central nervous system (CNS), mainly expressed by neurons but also by glia (Zhang et al., 2016).Recently, ⍺Syn was found to be critical to the development and function of hematopoietic cells (Ling et al., 2022;Shameli et al., 2016) and a normal immune system (Alam et al., 2022).This has entrenched the hypothesis that peripheral ⍺Syn may contribute to the neuropathology of PD by altering the immune system or by immune cell involvement in the CNS pathology.
The ⍺Syn protein consists of an N-terminal amphipathic repeat region (1-60), a hydrophobic non-amyloid β-component (NAC) region (61-95), and a negatively charged C-terminal acidic region (96-140) (Bisi et al., 2021).Its physiological conformations are disordered soluble monomers or helically folded ⍺Syn tetramer (de Boni et al., 2022;Li et al., 2022;Mehra et al., 2019) with a 50:50 split in the amounts of these conformations in human brain (de Boni et al., 2022).Most reported SNCA point mutations (A30P, A53T, A53E, E46K, H50Q, and G51D) are located at the N-terminus with a new mutation, E83Q, in the NAC domain (Kumar et al., 2022).The N-terminus of ⍺Syn acquires a helical secondary structure essential for vesicle and membrane binding, whereas post-translational modifications (PTMs) to the N-terminus drive folding toward the helical form and then impact the downstream aggregation (Bartels et al., 2010;Iyer et al., 2016).The NAC amyloid binding core may either be in ⍺-helical form to also anchor vesicles, which is highly sensitive to the lipid composition (Fusco et al., 2016;Fusco et al., 2014), or adopt a β-sheet structure to form protofibrils and amyloid fibrils conferring a key role in misfolding, oligomerization, and aggregation (Bertoncini et al., 2005;Giasson et al., 2001;Mehra et al., 2019).With this flexible structure, the more exposed the N-terminus and the beginning of the NAC region, the more monomeric ⍺Syn is prone to aggregation (Stephens et al., 2020).The NAC domain mutation increases this propensity (Kumar et al., 2022).The unstructured C-terminus tends to present a random coil under physiological conditions (Brodie et al., 2019).Its intermolecular interplay with the N-terminus or NAC is important in maintaining the innate unfolded structure of ⍺Syn, preventing it from conformational changes (Hong et al., 2011).The loss of this interaction due to C-terminus PTMs will expose the N-terminus, causing a stronger binding to membranes (Zhang et al., 2022).
PTMs can either facilitate or inhibit the formation of ⍺Syn aggregation, as previously reviewed (Gadhavi et al., 2022).It is plausible to think that ⍺Syn pathology could be successfully blocked therapeutically by targeting post-translational processing abnormalities to stop its conversion from a physiological to a pathological state.The mostly studied ⍺Syn PTMs include phosphorylation, acetylation, nitration, and truncation, all of which have been reported to be involved in forming toxic forms of the protein (Anderson et al., 2006;Bartels et al., 2010;Iyer et al., 2016).Among these, phosphorylation at Ser129 (pS129) is the most abundant PTM in PD and has therefore been used as a pathologic marker.It is estimated that only 4% of monomeric ⍺Syn is usually phosphorylated at any time, contrasting to the striking 96% of ⍺Syn identified as phosphorylated in Lewy pathology (Anderson et al., 2006;Fujiwara et al., 2002).Besides pSer129, other PTM sites pTyr39, pSer87, pTyr125, pTyr133, pTyr136, and nTyr39 have been reported in neurons in Lewy body diseases (Altay et al., 2023;Paleologou et al., 2010), while more limited PTMs are found in astrocytes in the same cases (only pTyr39 and nTyr39, (Altay et al., 2022), highlighting that astrocytes and neurons harbor different αSyn forms.The nitration at Tyr39, Tyr125, Tyr133, and Tyr136 was shown to stabilize oligomers, and therefore inhibit αSyn fibrillation (Barrett and Timothy Greenamyre, 2015).
Recently the inventory LC-MS/MS analyses have explored the toxicity of pathologic species of ⍺Syn depending on the efficient transmission and amplification, where the phosphorylation and acetylation of soluble ⍺Syn were involved (Zhang et al., 2023).In addition, the C-terminus truncation at a threshold region around residue 121 facilitates ⍺Syn fibrillation and suppresses toxic oligomer formation, which is correlated to the loss of the negative electrostatic potential of ⍺Syn (Farzadfard et al., 2022).
Several studies have shown endogenous ⍺Syn levels as a risk and predictive factor for forming pathological ⍺Syn (Conway et al., 1998;Courte et al., 2020;Vasili et al., 2022).An early in vitro study (Conway et al., 1998) revealed that increased concentrations of ⍺Syn promoted the formation of fibrillar ⍺Syn, whereas mutant forms (A53T and A30P) demonstrated an accelerated fibrillization rate compared to wild-type ⍺Syn.Recent human data shows that destabilization of the helically folded ⍺Syn tetramer into more disordered soluble monomers leads to increased levels of insoluble fibrils in PD (de Boni et al., 2022).These studies suggest a high cellular level of soluble monomeric ⍺Syn beyond a particular threshold may increase inclusion formation in vivo.Indeed, SNCA triplication cases and half of the duplication cases have early onset parkinsonism, with nearly all multiplication cases having widespread cortical ⍺Syn pathology (Konno et al., 2016;Olgiati et al., 2015).Overexpression of ⍺Syn in mice dopaminergic neurons alters neuronal firing properties, calcium dynamics, and dopamine release (Dagra et al., 2021).Vice versa, neuronal hyperactivity increases the nitration of ⍺Syn, its aggregation, and its intraneuronal transmission (Helwig et al., 2022).Similarly, chronic neuromodulation of nigral neurons aggravates motor deficits in a ⍺Syn-based rat model of PD (Torre-Muruzabal et al., 2019).Neuronal activation leads to changes in PTM and subcellular location of ⍺Syn.These data suggest that the activity of neurons changes the level of and PTMs on ⍺Syn, affecting its aggregation.This review will focus on ⍺Syn's level, form, and subcellular location in neurons, astrocytes, microglia, and oligodendrocytes.Given the central role of pathologic ⍺Syn in PD, understanding its pathophysiological impact on different types of neural cells will bring additional insights into the process of ⍺Syn pathology.

⍺-Synuclein in neurons
In PD, ⍺Syn aggregates are typically found in the neuronal cytoplasm as Lewy bodies (LBs) and in neuronal processes as Lewy neurites (LN) (Braak et al., 2003;Spillantini et al., 1997).The protein has different pathological manifestations in PD neurons, particularly changing its solubility to oligomeric and fibril species, which coalesce into diffuse and solid aggregates.Oligomeric species have been considered the most toxic, but recent findings also indicate that fibrils and other mature species of ⍺Syn are toxic, leaving the most toxic forms of ⍺Syn in PD still under debate (Emin et al., 2022;Hijaz and Volpicelli-Daley, 2020;Pacheco et al., 2015;Recasens et al., 2014).

Endogenous ⍺Syn levels in neurons and the conversion to PD pathologic forms
⍺Syn is particularly enriched at presynaptic nerve terminals and is therefore abundant in the brain (Sjöstedt et al., 2020;Taguchi et al., 2019).Both human and mouse neurons have high ⍺Syn expression levels compared to astrocytes (Geertsma et al., 2024;Kon et al., 2023;Zhang et al., 2016).The soluble protein is diffuse but localizes to different cellular organelles, particularly those enriched in lipids, including the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and synaptic vesicles (Hoozemans et al., 2007;Lee et al., 2004;Li et al., 2007;Maroteaux et al., 1988).The biological level of ⍺Syn is around 45 μM in the rat brain synaptic fractionation (Wilhelm et al., 2014).However, the comparative levels of endogenous ⍺Syn in an entire neuron or other neuronal organelles remain to be elucidated, as different neuronal populations with different functions express different biological levels of ⍺Syn.In the mouse brain, regions vulnerable to PD have higher cell body amounts of ⍺Syn (olfactory bulb, enteric neurons, and brainstem catecholaminergic neurons), and excitatory vesicular glutamate transporter-1 synapses have higher levels of ⍺Syn (Geertsma et al., 2024;Taguchi et al., 2019).In contrast, ⍺Syn is relatively absent from cholineacetyltransferase-positive motor neurons (Geertsma et al., 2024;Taguchi et al., 2019).The amount of ⍺Syn in inhibitory synapses differs among brain regions and is not found in inhibitory synapses in the cerebral cortex, hippocampus, subthalamus, or thalamus (Taguchi et al., 2019).
Humans older than 80 years old have twice the protein level of ⍺Syn in the substantia nigra than those younger than 60 years old (Li et al., 2004).Interestingly, the ⍺Syn protein level is not impacted by age in the frontal cortex or caudate nucleus.The increased protein level of ⍺Syn in nigral neurons is associated with decreased tyrosine hydroxylase (TH) levels in humans and monkeys with age (Chu and Kordower, 2007).This implies that, in the elderly without PD, increasing ⍺Syn protein levels in aged dopamine neurons relates to decreased neuronal dopamine metabolism in general, a finding that has been recently linked to fatigue (Scheffer et al., 2021).This age change in nigral neurons is likely to predispose to PD (reduced dopamine and increased ⍺Syn) and may explain why aging is the major risk factor for PD.It has been shown that brain regions and neurons with low levels of ⍺Syn do not develop Lewy pathology (Erskine et al., 2018), consistent with the concept that a certain level/threshold of ⍺Syn protein is required for pathological inclusion formation.
Significant overexpression of wild-type ⍺Syn in mice and monkeys triggers the formation of pathological ⍺Syn and its aggregates in cortical and midbrain neuronal cell bodies and processes that are reminiscent of PD pathology (Kahle et al., 2000;Kirik et al., 2003;Masliah et al., 2000;Oliveras-Salvá et al., 2013;Wagner et al., 2020).Similarly, the conversion of endogenous ⍺Syn to toxic species has been associated with the amount of this protein in neurons in genetic forms of PD (Farrer et al., 2004;Ikeuchi et al., 2008;Ross et al., 2008).For instance, PD patients with SNCA triplication express double the amount of ⍺Syn compared to controls and have significant Lewy pathology in the brainstem and cortex and severe clinical symptoms (Farrer et al., 2004).Homozygous SNCA duplication PD patients present a more severe disease manifestation compared to their heterozygous SNCA duplication parents (Ikeuchi et al., 2008).Although there is more ⍺Syn protein visualised in aging nigral neurons (Li et al., 2004), SNCA mRNA measured with different methods decreases in neurons containing LBs in sporadic PD prior to the complete degeneration and loss of the neurons (Kingsbury et al., 2004;Kon et al., 2023).Also, in regions with and without neuronal loss but with LB formation, there is no substantive increase over aged controls in the amount of soluble ⍺Syn protein in brain extracts in PD using multiple methods (de Boni et al., 2022;Moors et al., 2022;Tong et al., 2010;Zhou et al., 2011).These data show that there is no significant increase in the pool of soluble ⍺Syn when LBs are forming and before cell loss (even if more ⍺Syn protein can be observed in aging neurons).There is some indication that the form of soluble ⍺Syn protein may be destabilised prior to LB formation (de Boni et al., 2022) and, as identified above, the protein undergoes significant PTMs as LBs form prior to neuronal loss.These changes may drive the increased ⍺Syn insolubility and LB fibril formation in sporadic PD that is not observed in controls (Campbell et al., 2001;de Boni et al., 2022;Kahle et al., 2001;Moors et al., 2022;Zhou et al., 2011).However, such PD neuronal changes appear to occur without increased expression or levels of the soluble ⍺Syn protein within vulnerable neurons, and this has been interpreted as a proteinopenia.The loss of normal ⍺Syn protein (Espay and Okun, 2023) and the reduction in SNCA mRNA expression in LB containing neurons (Kon et al., 2023) have been hypothesised to contribute to PD pathogenesis.
Together, these data demonstrate that types of neurons, functional status, and aging are associated with the cellular level of ⍺Syn.Ageassociated ⍺Syn increases in selective brain regions and cell types may predispose these regions to PD pathology, but once the conformation of ⍺Syn becomes pathological, the levels of ⍺Syn mRNA and normal protein appear to reduce in neurons forming Lewy inclusions.This indicates that highly selective molecular changes occur to normal αSyn protein in neurons forming pathological inclusions in sporadic PD, a finding which needs to be assessed and verified further at a molecular level in situ in human tissue (eg.using Nanostring CosMx and/or 10× Xenium technologies or similar).That there is no increase in neuronal αSyn protein that underpins the neurodegeneration in sporadic PD is important to understand further at a molecular level as it has impact on current modelling of the disease.

Post-translational modifications of ⍺Syn in neurons in PD
Large increases in the phosphorylation of ⍺Syn at Ser129 specifically occur with increased neuronal activity that increases ⍺Syn binding to presynaptic membranes (Ramalingam et al., 2023).⍺Syn is subject to a variety of PTMs, including phosphorylation, acetylation, oxidation, nitration, and ubiquitination, which may influence its biology, including oligomerization and aggregate formation (Burré et al., 2018).Higher levels of phosphorylated ⍺Syn correlate with nigral neuron loss in mouse models (Oliveras-Salvá et al., 2013;Vasili et al., 2022).There are a variety of phosphorylation sites on ⍺Syn (pSer87, pTyr125, pSer129, pTyr133, and pTyr136) with many appearing to be pathologically relevant to PD (Altay et al., 2023;Meade et al., 2019;Paleologou et al., 2010).The best studied among these phosphorylation sites is pSer129, which has been confirmed to play a central role in regulating ⍺Syn aggregation and neuronal degeneration in PD (Anderson et al., 2006;Fujiwara et al., 2002;Waxman and Giasson, 2008).This may suggest that increased neuronal activity is involved, as Ser129 is specifically and highly phosphorylated in that context (Ramalingam et al., 2023).Aging also increases the level of pSer129 in nigral dopamine neurons, as shown in squirrel monkeys, where those older than 16 years have a three-fold increase in pSer129-ir neurons compared to those younger than 10 years old (McCormack et al., 2012).These large changes in pSer129 levels with increased activity and in aged dopamine neurons may predispose to PD and also suggest that ⍺Syn dephosphorylation by protein phosphatase 2 A is important for the maintenance of normal ⍺Syn function with high neuronal activity and age.
Although there are many ⍺Syn phosphorylation sites, pSer129 is the most abundant in LBs (Altay et al., 2023;Anderson et al., 2006;Fujiwara et al., 2002;Waxman and Giasson, 2008).Not only is most of the ⍺Syn phosphorylated at Ser129 in LBs, but the level of insoluble pSer129 increases with disease progression in PD brains (Zhou et al., 2011), and more pSer129 is observed in the cortex of PD patients with dementia compared to non-demented PD patients (Swirski et al., 2014).This is consistent with an ongoing increase in LBs in the PD brain over time.Animal models utilizing preformed fibrils (PFF) or brain homogenate injections develop extensive pSer129-enriched inclusions in cortical and nigral neurons (Chu et al., 2019;Luk et al., 2012;Masuda-Suzukake et al., 2013;Recasens et al., 2014;Shimozawa et al., 2017) showing that ⍺Syn fibrils increase Ser129 phosphorylation.While these findings could suggest pSer129 is required for pathological inclusion formation, it was recently found that the molecular modification of pSer129 lessened ⍺Syn seeding and aggregation propensity, as well as attenuated cytotoxicity (Ghanem et al., 2022).This study also pointed out that pSer129 occurs at a late stage in mice injected with PFFs.Together, these new reports suggest that phosphorylation at Ser129 may occur for protection against cytotoxicity and other cytopathological markers occur earlier than pS129.
Nitration, as a consequence of elevated oxidative stress, is another major PTM of ⍺Syn observed in neurons at four tyrosine residues, including Y39, Y125, Y133, and Y136 (Giasson et al., 2000).Increased nY39 has been found in a cellular model of PD (Danielson et al., 2009).When all four sites were nitrated, the formed dimers and polymers induced a dose-dependent cytotoxicity in SH-SY5Y cells (Liu et al., 2011).The administration of nitrated ⍺Syn in the rat substantia nigra caused severe dopamine neuron loss and subsequently led to the upregulation of dopamine D2 receptors in the striatum (Yu et al., 2010).Moreover, abundant nitrated ⍺Syn levels have been observed in cortical and nigral neurons in the brains of PD and other synucleinopathy patients (Giasson et al., 2000).It has been suggested that nitrated ⍺Syn favors an oligomer conformation (Souza et al., 2000), thus propagating the formation of potentially pathologic ⍺Syn.
Together, these data demonstrate that neuronal ⍺Syn undergoes significant PTMs in patients with PD, indicating that a variety of cellular enzymatic processes are increased in vulnerable neurons in PD.Whether PTMs in surviving neurons indicate a detrimental or compensatory modification remains unclear, and whether there are specific PTMs only found in regions with substantial cell loss, indicating potentially greater pathogenicity, has not been determined.

The toxic effects of ⍺Syn on neuronal biological processes in PD
Although ⍺Syn predominantly localizes to presynaptic terminals, it is also abundantly expressed in the cytosol and processes, where pathological deposits are typically found (Li et al., 2007;Spillantini et al., 1997), and recent data suggests that distinct conformations may impact different organelles (Martinez-Valbuena et al., 2022).Together with the known locations in neuronal organelles, pathologic ⍺Syn is predicted to impact multiple cellular biological functions, including synaptic vesicle trafficking, mitochondrial functioning, and autophagy/lysosomal functioning (Kon et al., 2024;Martinez-Valbuena et al., 2022).
Pathologic ⍺Syn manifested as large oligomers can disrupt synaptic vesicle trafficking by binding to synaptobrevin-2, blocking the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, thus inhibiting neurotransmitter release (Choi et al., 2013).Other transgenic PD mouse models also show hippocampal and striatal synapses have impaired neurotransmitter release.In addition, these models had reduced levels of synaptic proteins and redistribution of SNARE proteins in striatal synapses, as observed in PD patient brains (Garcia-Reitböck et al., 2010;Nemani et al., 2010).Similarly, rat hippocampal and dopaminergic neurons overexpressing ⍺Syn have revealed impairments in synaptic vesicle exocytosis (Nemani et al., 2010).In transgenic mice expressing E57K ⍺Syn oligomers are preferentially formed with subsequent synaptic and neuronal loss in the cortex compared to mice expressing wild-type ⍺Syn or non-transgenic mice (Rockenstein et al., 2014).Human iPSC-derived cortical neurons with SNCA mutations (E46K and E57K) show ⍺Syn oligomerization preponderance, dysfunctional axonal transport, and synaptic degeneration (Prots et al., 2018).These findings collectively suggest that the pathologic presynaptic ⍺Syn and its oligomeric forms may be the beginning of cytopathological events, which can induce neural circuit chaos.
Under physiological conditions, ⍺Syn localizes to mitochondrial membranes, as shown in mouse nigral neurons, suggesting its potential pathological role in mitochondrial dysfunction in PD (Li et al., 2007).This implication was first observed in an early study on the mitochondrial respiratory-chain enzyme proteins with post-mortem human brain tissue, which revealed specific impairment of Complex I activity in the substantia nigra of PD patients (Schapira et al., 1990), which has been replicated since (Devi et al., 2008).Furthermore, a cell culture study revealed that the ⍺Syn accumulation in mitochondria of human dopaminergic neurons reduced Complex I activity and increased reactive oxygen species (ROS), which occurred earlier in neurons expressing A53T ⍺Syn than neurons expressing wild-type ⍺Syn (Devi et al., 2008).This impact relied on the N-terminal 32 amino acid region that contains evenly spaced positive residues, a region of ⍺Syn crucial for binding to the outer mitochondrial membrane protein TOM20 inhibiting protein import and where oligomeric αSyn can cause mitochondrial fragmentation (Di Maio et al., 2016;Nakamura et al., 2011).Other A53T mouse primary cortical and midbrain neuronal models further demonstrated increased mitophagy, mitochondrial destruction, and cell loss (Chen et al., 2015;Choubey et al., 2011;Martin et al., 2006).Introducing A53T αSyn to human embryonic stem cell-derived neurons enriched αSyn in the mitochondrial fractions and induced significant mitochondrial transport defects and fragmentation (Pozo Devoto et al., 2017).Importantly, αSyn-mediated mitochondrial fragmentation is linked to αSyn expression levels, with recent work suggesting this dysfunction is associated with faster disease propagation (Martinez-Valbuena et al., 2022).Human iPSC-derived cortical neurons from PD patients carrying the A53T mutation have enhanced oligomeric species linked with mitochondrial dyshomeostasis (Choi et al., 2022).Together, these data demonstrate that different pathological forms of αSyn disrupt mitochondrial functioning, enhancing oxidative stress detrimental to neuronal survival.
αSyn degradation mainly occurs via the autophagolysosomal system and the ubiquitin-proteasome pathway.Mutant forms of αSyn (A53T and A30P) have been reported to inhibit proteasome activity and chaperone-mediated autophagy (CMA) by blocking lysosomal uptake through CMA in cell cultures (Cuervo et al., 2004;Tanaka et al., 2001).Furthermore, rat cortical neurons expressing A53T αSyn demonstrated impaired CMA (Xilouri et al., 2009).CMA proteins are selectively reduced in regions with increasing αSyn accumulation in PD patients (Alvarez-Erviti et al., 2011;Murphy et al., 2015).Human midbrain neurons overexpressing αSyn have aggregations associated with lysosomal dysfunction (Mazzulli et al., 2016).Lysosomes are particularly important for the degradation of fibrillar αSyn internalized from surrounding tissue (Bayati et al., 2022).The recent identification of a proportion of perforated lysosomes, specifically in neurons where internalized fibrillar αSyn accumulates, is suggested to impact further intracellular αSyn accumulation and increase its propagation (Sanyal et al., 2024).αSyn also impacts the ubiquitin-proteasome system (UPS) which is now known to be enriched and regulate proteins at synapses including αSyn (Sun et al., 2023).An in vivo study showed increased αSyn and pSer129 impaired proteasome activity leading to UPS dysfunction in rodent dopaminergic neurons expressing A53T (McKinnon et al., 2020).In PD, there are significant changes in the transcription of proteasome subunits consistent with a decline in its synaptic function (Dick et al., 2023).These studies highlight that pathological αSyn interferes with protein degradation, which may further increase protein levels and toxicity, promoting neuronal death.

Alpha-synuclein in astrocytes
αSyn accumulation in astrocytes is common in PD, distinguishing PD from other parkinsonian disorders (Song et al., 2009).PD post-mortem brains often display astrocytic accumulation of αSyn in regions containing LBs, including the olfactory bulb, brainstem, basal forebrain, limbic and frontal cortices.Astrocytic αSyn can even be seen in the absence of LBs, suggesting that the biological function of astrocytes may be impacted early in the disease (Braak et al., 2007).Astrocytic αSyn also correlates with the loss of nigral dopaminergic neurons in PD.Since many of their biological functions support neuronal survival, astrocytes may play a key role in PD pathogenesis.Much of the literature defines two phenotypic states of astrocytes: a pro-inflammatory A1-type, which is more abundant in post-mortem PD brains, and the immunoregulatory A2-type, which supports neuronal survival.While this is a reductionistic approach neglecting the diversity and regional specificity of astrocytic phenotypes revealed by recent transcriptomic data (Batiuk et al., 2020;Clarke et al., 2021), it simplifies concepts for easy communication.

Endogenous αSyn levels in astrocytes and the conversion to PD pathological forms
While the endogenous expression of SNCA in astrocytes is low, there is some evidence suggesting a physiological function for αSyn in astrocytes (Sorrentino et al., 2019;Tanji et al., 2001).Astrocyte cultures from SNCA-KO mice reveal disruption in fatty acid metabolism (Castagnet et al., 2005).Conversely, cultured astrocytes from healthy mice upregulate αSyn when exposed to oxidative stress or inflammatory cytokines (Cheng and Trombetta, 2004;Tanji et al., 2001).Therefore, while de novo αSyn accumulation within astrocytes is possible and may reflect lipid transport for astrocytic upregulation, it is also likely that astrocytic αSyn accumulation is derived exogenously.Several studies have demonstrated that astrocytes can internalize extracellular αSyn much more rapidly than neurons (Cavaliere et al., 2017;Lindström et al., 2017).Once internalized, αSyn is localized in the endo-lysosomal compartments (Kovacs et al., 2014), upon which it is degraded.Dysfunction of astrocytic lysosomal proteolysis and their capacity to clear toxic damaged proteins initiates neuronal loss in PD models (Morrone Parfitt et al., 2024), and once their capacity to degrade excess αSyn oligomers is exceeded, astrocytes try to maintain homeostasis by transporting αSyn to nearby cells via tunneling nanotubes or exosomes (Abounit et al., 2016;Ngolab et al., 2017;Rostami et al., 2017;Rostami et al., 2021).
These astrocytes with defective lysosomes exchange αSyn for mitochondria to maintain homeostasis (Rostami et al., 2017).As astrocytes exhaust their capacity to process αSyn and become dysfunctional, they spread αSyn in the brain.

Post-translational modifications of αSyn in astrocytes in PD
PTM of αSyn in astrocytes has only recently gained attention.Previously, mass spectrometry had shown a unique interactome between αSyn and astrocyte proteins (Shrivastava et al., 2016).Other studies have also identified truncated αSyn species within astrocytes, which are either cleaved prior to astrocytic phagocytosis or processed by lysosomal cathepsins within the astrocyte (Sorrentino et al., 2019).More recently, Altay et al. (2022) systematically investigated the PTM profile of αSyn in astrocytes.In their study, astrocytic αSyn aggregations were negative for the conventionally used αSyn markers ubiquitin, p62, and pSer129, but positive for phosphorylation and nitration at Y39.Interestingly, nitration of αSyn has been associated with microglial activation (Reynolds et al., 2008), and the authors speculate this PTM may be key to astrocytic signaling of microglia (Altay et al., 2022).Nonetheless, the heterogeneity of αSyn species and regional and phenotypic diversity in astrocytes must be considered in future PD studies.

The toxic effects of αSyn astrocyte biological processes in PD
Internalization of αSyn by astrocytes leads to the conversion of astrocytes to a proinflammatory phenotype (Sonninen et al., 2020).This subsequently leads to an increase in the transcription and release of inflammatory cytokines, mediated by the nuclear factor kappa-B (NF-κB) and P38 mitogen-activated protein kinase signaling cascades (Kim et al., 2018).The level of cytokines produced by astrocytes following αSyn treatment is much lower than that induced by TNF-α, an alternate pathway that facilitates microglial activation of astrocytes (Russ et al., 2021).Interestingly, αSyn triggers surface expression of major histocompatibility complex (MHC)-II in cultured astrocytes, especially if astrocytes are in the vicinity of CD4 + T cells, suggesting they can act as antigen-processing cells (Rostami et al., 2020).Astrocytes can also transfer MHC-II/αSyn deposits to adjacent astrocytes through tunneling nanotubes, further spreading αSyn (Rostami et al., 2020).
αSyn also induces mitochondrial dysfunction in astrocytes.In cultured astrocytes, αSyn oligomers localize to mitochondria, leading to reduced oxygen consumption and altered morphology with decreased ATP levels (Braidy et al., 2013;Lindström et al., 2017).Decrease in mitochondrial respiration has also been demonstrated in iPSC-derived astrocytes exposed to high molecular weight αSyn fibrils (Russ et al., 2021).Beyond mitochondrial dysfunction, αSyn also results in increased ER stress with subsequent activation of the unfolded protein response and disrupts the homeostasis of iron and other metals (further reviewed by (Wang et al., 2021).

Endogenous αSyn levels in microglia and the conversion to PD pathological forms
While mature microglia show some SNCA expression, RNA sequencing studies from mice suggest that SNCA expression is greater in embryonic microglia (Li et al., 2019).Heritability of common PD-related genes, including SNCA, are known to predispose to microglia and monocyte dysfunction in PD (Andersen et al., 2021;Lee et al., 2023;Navarro et al., 2021), and patients with PD demonstrate a greater percentage of monocyte precursors, which may suggest defective differentiation (Funk et al., 2013).This is consistent with αSyn playing a role in hematopoiesis and lymphopoiesis, with strong SNCA expression in peripheral hematopoietic precursor cells (Pei and Maitta, 2019;Xiao et al., 2014) that appears dysfunctional in PD (Funk et al., 2013).In PD nigral microglia there is some indication of upregulation of SNCA expression from single cell RNAseq analyses (Wang et al., 2024) but downregulation of immune response genes and proteins by multiomics (Lee et al., 2023).Given that microglia originate from primitive macrophages and SNCA expression is greater in embryonic microglia (Li et al., 2019), it is possible that SNCA expression aids microglial differentiation and that PD microglia are immune immature, though this requires further attention.More recently, monomeric αSyn was shown to inhibit the proinflammatory phenotype in cultured microglia (Li et al., 2020).This effect appears to be concentration dependent, with cultured microglia losing their neuroprotective effects when exposed to higher concentrations, at which point oligomer formation becomes likely.Pathologic αSyn from PD patients drives cultured microglia into a highly toxic chronic inflammatory phenotype (Yildirim-Balatan et al., 2024).However, in PD, region-specific microglia activation is confined to the nigrostriatal pathway and frontal cortex (Lavisse et al., 2021) rather than the wide distribution of αSyn pathology.

The toxic effects of αSyn microglia biological processes in PD
Much of the neurodegeneration associated with PD is likely a result of microglial activation.As the resident macrophages in the CNS, microglia are activated by various signals including exogenous αSyn, which acts through a TLR2-dependent pathway (Kim et al., 2013).In mice, αSyn PFFs activate the NLR family pyrin domain-containing 3 (NLRP3) inflammasome through this pathway with subsequent release of cytokines and induced neurodegeneration (Gordon et al., 2018).This has been replicated in cultured mouse and human microglia (Pike et al., 2021).Early and chronic administration of resolvin D1, an inflammation-resolving mediator, to rats overexpressing human αSyn, prevents such microglial activation and PD symptom onset (Krashia et al., 2019).
However, microglia can also play a beneficial role in PD by facilitating αSyn phagocytosis through the Mer tyrosine kinase receptor (Dorion et al., 2024) and TLR4 signaling (Choi et al., 2020).In mice the absence of host microglia in striatal grafts of dopamine neurons facilitates neuronal αSyn accumulation (George et al., 2019) indicating their beneficial role.While microglia are the most efficient cell type involved in synucleinophagy, when hyperactivated they also release exosomes containing αSyn potentially propagating the disease (Guo et al., 2020).Within microglia αSyn triggers a p62-mediated pathway to sequester αSyn into autophagosomes for lysosomal destruction (Choi et al., 2020).
Therefore, depending on the timing, blocking or enhancing microglial function in PD can be harmful.In fact, reducing chaperone-mediated autophagy facilitates microglial activation via inflammasome formation (Wu et al., 2023) and impaired phagocytic capacity of macrophages and monocytes toward αSyn accumulation has been demonstrated in adult mice and elderly human donors, respectively (Bliederhaeuser et al., 2016).Outside of aging, microglia still demonstrate an upper limit to their normal phagocytic potential.A recent mouse model of αSyn accumulation in microglia demonstrated degeneration of dopamine neurons without αSyn aggregation.This was mediated by a strong inflammatory response by the microglia with the production of oxidative species, recruitment of peripheral immune cells, and eventual phagocytic exhaustion (Bido et al., 2021).

Alpha-synuclein in oligodendrocytes
Argyrophilic αSyn accumulations with ultra-structurally filamentous structures in oligodendrocytes are known as glial cytoplasmic inclusions or GCIs, which are diagnostic for multiple system atrophy (Poewe et al., 2022).GCIs have been found in the nigrostriatal tract of long-duration PD patients (Wakabayashi et al., 2000), suggesting that over substantive time αSyn will accumulate in oligodendroglia associated with degenerating neuronal tracts.The numbers of GCIs are low in PD and not seen in many studies, even using new antibody tools assessing different forms and PTMs of αSyn (Sonustun et al., 2022;Wiseman et al., 2024).
Their association with long-term retrograde degeneration of axons has been substantiated in mice injected with a particular αSyn strain (1B fibrils) into the striatum, where this strain of αSyn is retrogradely taken up and transported into axonal segments of cortical neurons that are subsequently pruned by their myelinating oligodendroglia (De Nuccio et al., 2023).Similar αSyn filled axonal segments of cortical neurons associated with changes in their myelinating oligodendroglia have been observed in PD patients, although without GCI formation (Fu et al., 2022).In the nigrostriatal tract, the number of these αSyn containing oligodendroglial inclusions correlated with the severity of the nigral neuronal loss, consistent with an association with PD progression (Wakabayashi et al., 2000).Of interest, single-cell sequencing of the nigra in PD shows a reduction in oligodendroglia with remaining cells characterized by a stress-induced upregulation of S100B (Martirosyan et al., 2024;Smajic et al., 2022) and a general downregulation of many transcripts while increasing their expression of known PD genes and PDassociated genes (Agarwal et al., 2020;Lee et al., 2023).Besides degeneration and αSyn accumulation in some oligodendrocytes in PD, oligodendrocytes also play an active role in maintaining myelination (Fu et al., 2022).In support of increasing this function in PD, increased expression of oligodendrocyte-specific myelin-associated genes was identified in SNCA overexpressing rats and PD patients (Hentrich et al., 2020).
Hypotheses on the origins of αSyn in oligodendrocytes are grounded in their supposed lack of SNCA expression and dogma that any oligodendroglia αSyn pathology is likely due to the transfer of the abundant αSyn in neurons to oligodendroglia.As indicated above, such transfer of αSyn from neurons to oligodendroglia has been shown experimentally (De Nuccio et al., 2023a;Reyes et al., 2014).Mature oligodendrocytes have been found to express little to no SNCA mRNA and do not express high basal levels of αSyn (Asi et al., 2014;De Nuccio et al., 2023a).
However, recent single-cell RNAseq data suggest some upregulation of SNCA in oligodendroglia in PD nigra (Wang et al., 2024), although this has not been found in other similar studies, and αSyn+ oligodendroglia are not common in PD.Increased αSyn expression occurs in oligodendrocyte progenitors (Hsiao et al., 2023), with more evidence of dysfunction and reduced differentiation in this cell type in PD than in mature oligodendroglia (Bryois et al., 2020;Fu et al., 2022).

Alpha-synuclein in peripheral cells
In PD patients, the plasma and serum levels of αSyn were measured as 3.60 ± 2.53 and 0.03 ± 0.04 pg/ml, respectively (Chang et al., 2019).
Although data are largely variable, both values differentiate PD from the healthy controls.The serum level of αSyn also showed a significant correlation with patients' Hoehn and Yahr stages, implying a potential to predict motor symptom severity.Other studies have reported the quantification of total plasma αSyn with varying concentrations on the scale of thousands of pg/ml (Foulds et al., 2013;Koehler et al., 2015), much higher than the Chang et al. report (Chang et al., 2019).The presence of pSer129 αSyn was also found in the human plasma, but again it varies significantly across different studies from several fg/ml to hundreds of pg/ml (Cariulo et al., 2019;Foulds et al., 2011;Lin et al., 2019).While the varying range of total and PTM αSyn reported may be because of different antibodies and methods that have been applied, the multiple protein-hosting cells in the blood could also account for the variance.The blood level of αSyn may impact and reflect the intracellular levels of αSyn in multiple types of cells.αSyn is high in classical and non-classical monocytes, hematopoietic precursor cells, mature erythrocytes, and platelets (Grozdanov and Danzer, 2020;Stefaniuk et al., 2022).For instance, the concentration of αSyn in erythrocytes is around 40 μg/ml (Klatt et al., 2020).In PD, increases in pS129, pY125, and nY39 have been found in their erythrocytes (Abd Elhadi et al., 2019;Vicente Miranda et al., 2017).By assessing the subcellular fractions extracted from PD erythrocytes, pSer129 increased much more in the cytosolic fraction than in the membrane fraction (Tian et al., 2019).It is possible that the changes in the level and species of intracellular αSyn can affect the function of blood cells, which can alternatively impact the pool of αSyn and immune status in the brain.However, studies analyzing associated changes in circulating and CNS αSyn have not been reported except for an emphasis on developing peripheral αSyn biomarkers.

Concluding remarks
The levels and forms of αSyn in different types of neural cells and blood cells impact their cellular functions.Cytopathology developed in one cell type can also potentially introduce pathogenic effects to other cells, inducing or further impacting neuronal dysfunction and degeneration.In PD pathologic αSyn concentrates in neurons and astrocytes while dysfunction of microglia and oligodendrocytes also occurs.The pathologic accumulation of αSyn in neurons and astrocytes is not associated with any significant increase in expression of the protein, with expression levels generally depressed with disease progression.This synergistic, synchronous, and sequential pattern of cellular change that occurs in the brains of PD patients is not currently reflected in αSyn overexpression models of PD or in immune activation models of PD.It will be important to gather more data on the pattern of pathologic αSyn over time in different cell types in populations at risk of and with early PD with new techniques (eg.Nanostring CosMx, 10× Xenium technologies) so that the pathogenic molecular changes can be determined with greater confidence and the initiating cells and processes identified.Then new animal models that mimic the cellular αSyn changes observed in PD will be required.

Declaration of competing interest
The authors claim that there are no competing interests.