"Synucleinopathy:" A Misguided Concept

Elucidating the evolution of Lewy pathology, and the futility of targeting α-synuclein in Parkinson’s disease and other disorders erroneously viewed as “synucleinopathies”

Quoting from the 2020 Nature Genetics paper (1) “Genetic identification of cell types underlying brain complex traits yields insights into the etiology of Parkinson’s disease:”

We “…analyzed gene expression data from post-mortem human brains which had been scored by neuropathologists for their Braak stage. Differential expression was calculated between brains with Braak scores of zero (controls) and brains with Braak scores of 1—2, 3—4, and 5—6. At the latter stages (Braak scores 3—4 and 5—6), downregulated genes were specifically expressed in dopaminergic neurons, while upregulated genes were specifically expressed in oligodendrocytes (Figure 5), as observed in the case-control studies. Moreover, Braak stage 1 and 2 are characterized by little degeneration in the substantia nigra and, consistently, we found that downregulated genes were not enriched in dopaminergic neurons at this stage. Notably, upregulated genes were already strongly enriched in oligodendrocytes at Braak stages 1—2. These results not only support the genetic evidence indicating that oligodendrocytes may play a CAUSAL ROLE in Parkinson’s disease, but indicate that their involvement PRECEDES the emergence of pathological changes in the substantia nigra.” (my emphases)

How might these observations be explained in the context of currently available empirical evidence?

The short answer is that Lewy pathology does not play a causative role in Parkinson’s disease. Rather, α-synuclein-related pathology evolves temporally downstream of, and is a surrogate for, both the deteriorating health of synaptic vesicle (SV) membranes and the perturbation of optimal presynaptic nerve terminal membrane lipid composition (MLC).

The health of these NEURONAL membranes is intimately and strikingly tied to the health of oligodendrocyte lineage cells...underscoring the increasingly obvious physiological reality that oligodendrocytes (OL) are temporally and functionally upstream of CNS projection neurons as drug targets in Parkinson’s disease.

To answer the question “How might these observations be explained in the context of currently available empirical evidence?” in incontrovertible detail, a number of seemingly disparate observations must be brought to bear...particularly with respect to relevant MLCs.

Glycerophospholipids (a.k.a., phosphoglycerides) include the subset of phospholipids known as ether lipids, which have either a 1-O-alkyl (“plasmanyl”) or a 1-O-alk-1′-enyl (“plasmenyl”) linkage at the sn-1 position of the glycerol backbone (2). Plasmenyl phospholipids are known as plasmalogens. The initial steps in ether lipid/plasmalogen biosynthesis occur in peroxisomes, heterogeneous organelles known to be particularly important in affecting cellular lipid metabolism.

Over the last several decades, evidence the plasmalogen content of synaptic vesicles, as well as the plasmalogen content of axolemmal membranes, is notably high has continued to accumulate. Multiple research groups, using varied methods, have highlighted this fact: George H. DeVries and colleagues in the 1970’s and early 1980’s (3), employing “axolemmal enriched fractions” obtained by ultracentrifugation; Lawrence Macala, et al., in the 1980’s (4), using HPTLC (high performance thin-layer chromatography); Xianlin Han, et al. (5) and Reinhard Jahn, et al. (6) in the 2000’s, applying ESI-MS (electrospray ionization mass spectrometry).

More recently, the University of Minnesota’s Jonathan N. Sachs and colleagues have noted that ethanolamine plasmalogens (PlsEtn) “(make) up an astounding 19% of the SV lipid content” (7).

And, building upon these earlier works, a 2020 paper from the University of Maryland’s Jeffery Klauda and colleagues (8) indicates ethanolamine plasmalogens constitute ~60% of the ethanolamine phospholipids in healthy human grey matter…a figure that represents ~14.7% of ALL LIPIDS in such grey matter.

In short, the generally high plasmalogen content of human SVs and of human axolemma, and their respective high ethanolamine plasmalogen content in particular, is remarkably well-established.

To quote Klauda and his co-authors, there is a “ubiquitous presence of ethanolamine plasmalogens in neurological lipid membranes...” (8).

And, as illustrated by Masanori Honsho and Yukio Fujiki in 2017 (9), PlsEtn are preferentially localized to the inner leaflet of plasma membranes, e.g., presynaptic nerve terminal membranes…positioning presynaptic, PlsEtn-containing axolemmal membranes to interact directly with PlsEtn-containing SV membranes.

This is noteworthy in that membranes containing PlsEtn display a markedly increased propensity for bilayer fusion relative to membranes containing phosphatidylethanolamine (PE) lipids (8;10).

Intriguingly, Nancy Braverman and colleagues (11) have noted that levels of phosphatidylethanolamine tightly adapt to changes in ethanolamine plasmalogens, such that the diacyl glycerophospholipid PE “is the main player in the adaptation to plasmalogen insufficiency.”

And, that compensatory shift appears to largely involve the production of PE containing the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) at the expense of PE containing the ω-3 PUFA docosahexaenoic acid (DHA), (11). Such a shift could add to the deleterious effects of diminished plasmalogen levels, given the putative importance of DHA in synaptic integrity and the purported ability of DHA to modulate neurotransmitter release (12;13).

The aforementioned membrane fusion-enhancing properties of PlsEtn likely relate to the vinyl ether bond present in ethanolamine plasmalogens. This double bond ostensibly causes the sn-1 position fatty alcohol moiety and the sn-2 position ester-linked acyl substituent in a given ethanolamine plasmalogen molecule to adopt a parallel alignment. That parallel alignment “streamlines” PlsEtn relative to other phospholipids, e.g., the PE that “is the main player in the adaptation to plasmalogen insufficiency” (11) and that is less efficient at promoting bilayer fusion than PlsEtn (8;10).

Thus, PlsEtn are membrane fusion-enhancing phospholipids and α-synuclein is a putative membrane fusion-enhancing protein. The fusion-competent conformations of SNARE proteins necessary for neurotransmitter release are maintained by chaperone complexes that include α-synuclein (14). And, the PUFA-containing sn-2 chains of PlsEtn appear to regulate the SNARE fusion machinery in their own right (8).

Overexpressing a protein associated with a pathological hallmark of neurodegenerative disease in the presence of (relatively) healthy oligodendrocyte lineage cells—when, in actual clinical disease, it is temporally upstream injury to those very oligodendrocyte lineage cells that triggers the accumulation of that pathological hallmark-associated protein—confounds the effort to discover treatments for that disease.

That being said, well-known Parkinson’s disease researcher Ronit Sharon—in 2017 work (15) employing both the widely used PrP-A53T human α-synuclein Tg mouse line introduced by Virginia M.-Y. Lee, John Trojanowski, and colleagues in 2002 and a Thy-1 wild-type human α-synuclein Tg mouse line—has demonstrated neuronal α-synuclein overexpression perturbs oligodendrocyte phospholipid homeostasis, including PlsEtn homeostasis (“PE-plasmalogen” homeostasis in the Sharon paper’s parlance):

“We performed a systematic study to understand the effect of neuronal-expressed α-Syn on myelin composition. We found that α-Syn expression increased the levels of phospholipids in the absence of evidences for the occurrence of α-Syn or related pathologies. We concluded that α-Syn effect on myelin composition IS AN EARLY EVENT in the sequence of events leading to axonal loss and neurodegeneration" (15); my emphasis.

The distorted chronology of events in these engineered, human α-synuclein-overexpressing mouse models renders them irrelevant in terms of the true etiology of Parkinson’s disease. However, the models do strikingly validate the intimate physiological connection between neuronal α-synuclein and oligodendrocyte-produced phospholipids—including PlsEtn—leveraged in the earlier statement:

“...α-synuclein-related pathology evolves temporally downstream of, and is a surrogate for, both the deteriorating health of synaptic vesicle (SV) membranes and the perturbation of optimal presynaptic nerve terminal membrane lipid composition (MLC).

“The health of these NEURONAL membranes is intimately and strikingly tied to the health of oligodendrocyte lineage cells...underscoring the increasingly obvious physiological reality that oligodendrocytes (OL) are temporally and functionally upstream of CNS projection neurons as drug targets in Parkinson’s disease.”

The intimate connection between α-synuclein and PlsEtn demonstrated in the above Ronit Sharon work is also conspicuously affirmed by more recent work. In a 2021 publication (16)—employing the triple-synuclein-null (TKO) mouse model they had used in 2011 to demonstrate “the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons” (17) in work that appeared in the Journal of Neuroscience—Vladimir Buchman and colleagues demonstrate “a notable reduction in ethanolamine plasmalogens in the midbrain...” (16) of these transgenic animals.

Aside: it should be emphasized that myelinated axons are not restricted to white matter. They are also present in grey matter, although grey matter myelinated axons are not readily resolved with current imaging modalities.

The precise MLC of specific synapses or of particular neuronal subtypes has yet to be elucidated. It is probable, however, the pertinent lipid bilayers resemble those typically seen in animal cells, with a more anionic inner leaflet and a relatively neutral outer leaflet (18).

Interestingly, the UDP-glycosyltransferase UGT8 (a.k.a., ceramide galactosyltransferase (CGT)) is expressed ~exclusively by oligodendrocyte lineage cells in the CNS. UGT8 is the ONLY known gene expressed in the human central nervous system whose protein product catalyzes the conjugation of galactose to the 1-hydroxyl moiety of ceramide, a necessary step in the synthesis of the glycosphingolipid galactosylceramide (GalCer).

And, the ~oligodendrocyte-specific gene gal3st1 (a.k.a., cerebroside sulfotransferase (CST)) produces the ONLY known enzyme expressed in the human CNS capable of catalyzing the sulfation of GalCer at the C3 carbon position of its galactose residue (to form sulfatide species), accomplished in a reaction involving the sulfate donor PAPS.

Relevant RNA-seq data on UGT8 expression and on gal3st1 expression in both human and mouse cortices is contained in a complementary (data) PDF, a Google Drive link to which appears below. The RNA-seq data is culled from a Stanford University-maintained database that displays data generated in the lab of the late Ben Barres; https://brainrnaseq.org/

That database is widely used by the neuroscience community, and the two key papers related to it have nearly 7200 combined citations to date, per Google scholar (19;20).

It is highly likely the sulfatides present in CNS neurons, in general—and in axonal nerve terminals, in particular—are provided by oligodendrocytes. Work reported in the 2010 Xianlin Han, et al., Journal of Neuroscience paper “Neuronal LRP1 Knockout in Adult Mice Leads to Impaired Brain Lipid Metabolism and Progressive, Age-Dependent Synapse Loss and Neurodegeneration” (21) provides mechanistic detail as to how CNS OL may serve as the sole source of neuronal sulfatides:

“ApoE particles (acquire) sulfatides from the myelin sheaths...These sulfatide-containing ApoE particles can be internalized by neurons via lipoprotein receptors and then hydrolyzed in neuronal lysosomes, allowing intracellular release of free cholesterol, sulfatide, and other lipids...all of which are critical for the integrity of neuronal membranes, dendritic spines, and synapses.”

Neuronal reliance on OL for a key component of the outer membrane leaflet of nerve terminals is consistent with the emerging reality that oligodendrocyte injury is a pivotal event in the etiology of Parkinson’s disease. Furthermore, the key role played by neuronal lysosomes in ‘processing’ exogenously-provided sulfatides resonates with the multiple, ongoing drug discovery efforts targeting lysosomes in Parkinson’s disease.

That the lysosome-resident, Parkinson’s disease-implicated enzyme glucosylceramidase ? (a.k.a., “GCase” or as GBA (and the product of the GBA1 gene)) has recently been shown (22) to catalyze galactosylation of cholesterol, forming galactosylated cholesterol (?GalChol)—and the appearance of ?GalChol tracks with the developmental onset of myelination in mice—is particularly striking given that GCase is a focal point in the aforementioned drug discovery efforts targeting lysosomes in Parkinson’s disease.

Also note that, in humans, myelination of CNS axons is a particularly protracted process, extending well into the fifth decade of life (23).

The observation GalCer serves as the source of galactose in the GBA-catalyzed production of ?GalChol (22) adds further intrigue, given both the importance of galactosylated lipids in maintaining myelin integrity and the fact UGT8 is expressed ~exclusively in oligodendrocytes in the CNS, and is the ONLY known gene expressed in the human central nervous system capable of catalyzing GalCer synthesis.

Per the above-referenced RNA-seq database, https://brainrnaseq.org/, mRNA-level GBA expression in human cortical OL is equivalent to, or even slightly exceeds, mRNA-level GBA expression in human cortical neurons...each of which is significantly outstripped by the mRNA-level GBA expression in human cortical astrocytes!

The abundant, mRNA-level GBA expression in human cortical macroglia (oligodendrocytes; astrocytes) relative to that in cortical neurons—given that heterogeneous mutations in GBA are the most common genetic risk factor for Parkinson’s disease identified to date (24)—should provide a red flag for those with a neuroncentric view of the etiology of Parkinson’s disease.

The KEGG ether lipid metabolism reference pathway, promulgated via the widely respected Kyoto Encyclopedia of Genes and Genomes (KEGG), provides an instructive frame of reference here (25); https://www.genome.jp/pathway/map00565 (or see the complementary data PDF here https://drive.google.com/file/d/1MZ_ECeJlmehnaA7AR25UZUGFaV2b-j_8/view?usp=sharing).

UGT8 (“EC 2.4.1.47”) and gal3st1 (“EC 2.8.2.11”) play pivotal roles in CNS sulfatide production. In addition, owing to their roles in the biosynthesis of seminolipid, a glyceroglycolipid abundant in mammalian testes and sperm, UGT8 and gal3st1 are included in the KEGG ether lipid metabolism reference pathway. UGT8 and gal3st1 also participate in the biosynthesis of the glyceroglycolipid SGG (sulfated glyceroglycolipid), which has been demonstrated in the rat brain (26), though not in the human brain to date.

As previously noted, plasmalogens (e.g., PlsEtn) are a subset of ether lipids. Peripheral ether lipids in general, and peripheral plasmalogens in particular, are ostensibly unable to cross the blood brain barrier (27).

In addition to UGT8 and gal3st1, the KEGG ether lipid metabolism pathway includes a number of enzymes pertinent to the elucidation of the genesis of Lewy body pathology: AGPS (“EC 2.5.1.26”); DHRS7B (“EC 1.1.1.101”); DHAPAT, a.k.a., GNPAT (“EC 2.3.1” or “EC 2.3.1.42”); SELENOI, a.k.a., EPT1 (“EC. 2.7.8.1”); CEPT1 (“EC 2.7.8.2”); and TMEM189, a.k.a., PEDS1 (“EC 1.14.19.77”).

The pattern of TMEM189 mRNA expression in the human cortex relative to that in the mouse cortex provides an indispensable clue in understanding the genesis of Lewy body pathology. The TMEM189 data from the Stanford University-maintained RNA-seq database is contained in the above-linked complementary (data) PDF, as is RNA-seq data for other subsequently referenced genes of interest.

At the time the Barres lab human TMEM189 RNA-seq data was generated circa 2016 (20), TMEM189 had not yet been identified as the long-sought human plasmanylethanolamine desaturase, the enzyme responsible for generating ethanolamine plasmalogens (PlsEtn) from plasmanylethanolamine ether lipids by introducing the characteristic vinyl ether double bond at the sn-1 position.

As there is no known plasmanylcholine desaturase enzyme activity, PlsEtn are the “first” plasmalogens generated in the plasmalogen biosynthesis pathway in the mammalian CNS. Plasmenylcholine plasmalogens (PlsCho) cannot be generated in the absence of PlsEtn.

TMEM189 was recently identified as PEDS1 (Plasmanylethanolamine Desaturase 1) via elegant work from two separate groups: Aránzazu Gallego-Garcia, et al. (28) published their findings in SCIENCE in October 2019, just as confirmatory findings from Katrin Watschinger and colleagues (29) were being submitted to PNAS. The Watschinger group’s findings were published online in March 2020.

Complementary validation of the TMEM189/PEDS1-related findings of the Gallego-Garcia and Watschinger groups, employing a completely orthogonal, statistical methodology designed by Stanford University-based authors, also appeared in Nature Genetics in 2021 (30).

The knowledge that TMEM189 has plasmanylethanolamine desaturase activity in mammals sheds new light on a particular aspect of the highly-cited 2012 Nancy Braverman/Ann B. Moser work “Functions of plasmalogen lipids in health and disease”(31).

In that 2012 work, Braverman and Moser cite 2002 work from Thad Rosenberger, et al. (2) addressing the turnover of ether phospholipids in adult rat brain, concluding those 2002 findings are “consistent with a metabolically active role for grey matter plasmalogens, and a relatively inactive, or structural role for myelin plasmalogens ”(31).

Given the order-and-a-half magnitude diminution in the relative expression of TMEM189 in human cortex versus that in mouse cortex—and the narrowing of neural cell type expression of TMEM189 in moving from the lissencephalic rodent brain to the gyrencephalic human brain evident in the Barres lab RNA-seq data—it is highly likely plasmalogens have evolved to play a largely structural role in human neurons relative to the more dynamic role they play in rodent grey matter!

The analogous human cortex/mouse cortex data for the plasmalogen-selective phospholipase A2 encoded by the PLA2G6 gene (a.k.a., PARK14), a Parkinson’s disease risk gene, is strikingly consistent with the view plasmalogens have evolved to play a structural role in human neurons. PLA2G6 displays a nearly identical order-and-a-half magnitude diminution in relative mRNA expression and analogous narrowing of neural cell type expression pattern to that seen for TMEM189 in moving from mouse to man.

Neither TMEM189 nor PLA2G6 show appreciable expression in human cortical neurons at the mRNA level.

The hub-like nature of PLA2G6 in connecting other Parkinson’s disease risk genes (e.g., GBA; PARK2; PARK7; PARK9 (a.k.a., ATP13A2); LRRK2; SNCA) to key plasmalogen-related genes (e.g., TMEM189; EPT1; CEPT1; PLA2G4B) in specified STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) functional interaction networks (32) is also visually and conceptually stunning. For example, see the data for the above-specified network, culled from STRING v11.5 https://version-11-5.string-db.org/ and included in the above-linked complementary data PDF.

Comparing that STRING network in man to the analogous STRING network (32) in mouse (mus musculus) also supports the conclusion the role of plasmalogens in CNS physiology has evolved in moving from mouse to man: see the mus musculus STRING data for the above-specified network, culled from STRING v11.5 and contained in the complementary data PDF.

The respective STRING networks displayed in the complementary data PDF are generated by applying the default confidence score setting (0.400), which is the ‘floor’ for functional interactions of (at least) medium confidence.

That astrocytes and oligodendrocytes appear to be the sole sources of PlsEtn in the human cortex—and that astrocytes appear more likely to catabolize the PlsEtn they (astrocytes) produce than are oligodendrocytes to catabolize the PlsEtn they (OL) produce vis-à-vis the mRNA expression levels of the plasmalogen-selective phospholipase PLA2G6 in astrocytes versus in oligodendrocytes—is consistent with OL serving as the PRIMARY source of the exogenous PlsEtn human neurons ostensibly require.

And, the potential for temporally upstream oligodendrocyte injury—manifesting, in part, as diminished presynaptic nerve terminal inner membrane leaflet levels of PlsEtn—to affect the genesis of Lewy pathology is intriguingly underscored by findings from Giuliana Fusco and colleagues published in Nature Communications in 2021 (33).

Fusco, et al., demonstrate (in vitro) that α-synuclein preferentially binds the inner presynaptic plasma membrane leaflet. They then go on to show that the interaction of α-synuclein with that inner leaflet, in the context of α-synuclein-mediated docking of synaptic vesicles to the inner leaflet, is altered by changes in the inner leaflet’s lipid composition.

The findings of Fusco, et al., also elegantly echo work published slightly earlier, in PNAS (34), by Upneet Kaur and Jennifer C. Lee of NHLBI (NIH). Using a technique known as cellular unroofing, Kaur and Lee demonstrate that α-synuclein bound to human cytoplasmic membranes “is conformationally dynamic (and) exquisitely sensitive to lipid/protein composition” of the membranes with which it interacts.

Kaur and Lee’s findings are particularly striking as the unroofed (human) cell platform they employ to investigate α-synuclein/membrane interactions has been shown to “(retain) native-like properties of the inner leaflet of the plasma membrane..." (34).

Aside: Having Kaur and Lee recapitulate their 2020 work, employing HAP1 cells and HAP1 TMEM189 (-) 1 cells in addition to the originally-employed cells, would likely provide striking empirical evidence of the importance of PlsEtn (and their loss) in α-synuclein/inner plasma membrane interactions, and in the SUBSEQUENT development of Lewy pathology.

As Reinhard Jahn, et al. (35) recently noted, “A key open question is how neurons ensure (presynaptic) membrane homeostasis and, as a result, maintain the SV lipid composition.”

Counterintuitively, oligodendrocytes are the key player in that dynamic.

Although human cortical neurons do not appear to express PEMT (phosphatidylethanolamine N-methyltransferase), an enzyme which converts phosphatidylethanolamine to phosphatidylcholine, they do appear to express sufficient CEPT1 (choline/ethanolamine phosphotransferase 1) to produce their own PlsCho from exogenously supplied PlsEtn.

In concert with phospholipase C, CEPT1 can convert PlsEtn to PlsCho. Again, as there is no known plasmanylcholine desaturase enzyme activity, PlsCho cannot be generated in the absence of PlsEtn.

The diminution in the relative expression of the PlsCho-preferring phospholipase PLA2G4B and the narrowing of its neural cell type expression to largely exclude cortical neurons in moving from mouse to human, per the Barres lab RNA-seq database, is also consistent with plasmalogens having evolved to play a structural (rather than dynamic) role in the human cortex...and likely beyond.

Furthermore, UCSD’s Edward A. Dennis and co-authors have recently demonstrated that the human calcium-dependent, cytosolic phospholipase A2 (cPLA2) encoded by the PLA2G4A gene is plasmalogen-preferring in vitro (36). Consistent with plasmalogens having evolved to play a structural (rather than dynamic) role in human grey matter, human cortical neurons show no appreciable PLA2G4A mRNA-level expression, per the Barres lab RNA-seq database.

That plasmalogens are key components of CNS paranodal junctions (PNJs), and contribute to the stability of those cognition critical structures, may also play into the (apparently) limited capacity of human cortical OL to catabolize the plasmalogens they produce, reflected in the very limited PLA2G6 mRNA, PLA2G4A mRNA, and PLA2G4B mRNA expression levels in such OL.

There are multiple avenues via which oligodendrocytes might supply neurons with plasmalogens, including exosomes; shed microvesicles; tunneling nanotubes; unapposed hemichannels; myelinic channels (37); and double-walled coated invaginations (DWCI).

DWCI represent incursions of oligodendroglial cytoplasm-containing structures into the axoplasm in myelinated axons, and have been observed in the CNS of both primates and rodents (38;39).

DWCI may give rise to freestanding, oligodendroglial cytoplasm-containing double-walled coated vesicles within the axoplasm.

Just as the aforementioned 2010 Journal of Neuroscience article from Xianlin Han and colleagues elucidates how OL may serve as the source of neuronal sulfatides, a series of works from esteemed neuroscientist Klaus-Armin Nave and colleagues supports the conclusion herein that OL-derived exosomes are likely the primary vehicle via which OL provide PlsEtn to neurons.

The three known, major metabolic pathways wholly or partially affected in peroxisomes are α-oxidation of certain fatty acids (e.g., phytanic acid); ?-oxidation of very long chain fatty acids (VLCFA) and other substrates not degraded in mitochondria; and ether lipid biosynthesis (40).

In a 2007 Nature Genetics paper (41), Nave, et al., demonstrate that selectively inactivating the peroxisomal import factor peroxin-5 (PEX5) in oligodendrocytes—thus disrupting the formation of functional oligodendroglial peroxisomes—leads to widespread axonal degeneration. As KU Leuven’s Myriam Baes and INSERM’s Patrick Aubourg would later note of this work, the phenotype of the OL-specific PEX5 knockout mouse “unequivocally demonstrates that a primary peroxisomal dysfunction in oligodendrocytes affects mostly and primarily the integrity of axons” (40).

One consequence of OL-specific PEX5 knockout in mice is to lower myelin plasmalogen content, and concomitantly increase myelin phosphatidylethanolamine content, in a statistically significant manner (41). This empirical observation is consistent with the conclusion of Braverman, et al., that PE “is the main player in the adaptation to plasmalogen insufficiency” (11).

A follow-up paper from Celia Kassmann, Klaus-Armin Nave, et al. (42) published in FEBS Letters in 2011 demonstrates both CNS peroxisomes and PNS peroxisomes in mice “...are highly abundant in myelinated fiber tracts, but largely contained in the glial compartment and rarely (seen) within axons.”

This peroxisomal dynamic in the mammalian brain may well extend to the myriad myelinated axons in human grey matter, e.g., to thinly myelinated axons in the substantia nigra (SN), where OL outnumber neurons (at least) 3:2, and outnumber dopaminergic projection neurons, in particular, to a much greater extent.

A 3:2 outnumbering of SN neurons by oligodendrocytes is, in fact, likely a very conservative estimate. Recent work in human postmortem midbrain tissue sections demonstrates “(a) massive oligodendrocyte population...”(43) therein. And, a 30 March 2022-posted bioRxiv preprint (44) from Icahn School of Medicine-based authors, which describes a single-cell transcriptomic atlas of the human substantia nigra in Parkinson’s disease, states “the cell populations of the aged human SN are composed predominantly by oligodendrocytes, followed in descending order by neurons, microglia, astrocytes, endothelia, OPC, pericytes, fibroblast (sic), and T cells.”

In work from Nave and colleagues published the year before the FEBS Letters paper appeared, it is also strikingly shown that neither neuron-specific ablation of functional peroxisomes in mice nor astrocyte-specific ablation of functional peroxisomes in mice leads to the axonopathy seen when oligodendroglial peroxisomes are functionally ablated (45).

A 2014 mini-review by Celia Kassmann in the journal Biochimie (46) elegantly summarizes the peroxisome-related findings of Nave and colleagues produced over the preceding several years. Figure 3 in that 2014 work is particularly noteworthy. The speculative metabolic pathways contained in the figure are less important in the current context than is the eye-opening, empirically-based proximity of ether lipid synthesis-initiating oligodendrocyte peroxisomes to the axonal compartment depicted therein.

The physical proximity of the peroxisomes of TMEM189-expressing, plasmalogen-producing oligodendrocytes to axons in the mammalian brain aligns with the observation oligodendrocytes are the likely primary source of exogenous PlsEtn human neurons require to maintain optimal synaptic vesicle and nerve terminal MLCs.

Tellingly, in terms of the sequence of events triggering the formation of Lewy pathology, mouse models of peroxisomal biogenesis disorders (e.g., Pex2-/-; Pex5-/-; Pex13-/-) display increased α-synuclein oligomerization, phosphorylation, and deposition in cytoplasmic inclusions (47).

The empirical evidence supplied in the very highly-cited Eva-Maria Kr?mer-Albers, Klaus-Armin Nave, et al., PLoS Biology paper “Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte-Neuron Communication” (48) published in 2013 supports the interpretation OL-derived exosomes are the primary vehicle via which OL provide PlsEtn to neurons. This work provides definitive evidence that myelinating oligodendrocytes in the mammalian CNS release exosomes along myelin internodes and at non-compact myelin-containing paranodal junctions. And, furthermore, that axons and neuronal somata take up those exosomes and make use of their cargo.

In follow-up work entitled “Oligodendrocytes support axonal transport and maintenance via exosome secretion” (49) published in PLoS Biology in 2020, the authors elegantly extend their 2013 findings.

It should also be noted that, given the “massive oligodendrocyte population” (43) in the human midbrain, there is a high probability exosomes released by myelinating oligodendrocytes—and potentially by non-myelinating, perineuronal OL—are accessible to midbrain dopaminergic projection neurons with unmyelinated axons, e.g., those projecting to the striatum via the medial forebrain bundle (MFB).

The physiological significance of oligodendrocyte-dopaminergic neuron (DaN) exosomal communication is driven home in a just-published, bioinformatics-based paper (50) in Frontiers in Aging Neuroscience: “Despite bulk studies focusing on intracellular mechanisms of PD inside DaNs, few studies have explored the pathogeneses outside DaNs, or between DaNs and other cells...Our bioinformatics analysis showed that the alteration in intercellular communications involving DaNs might be a previously underestimated aspect of PD pathogeneses with novel translational potential.”

The fact exosomes are enriched in phosphatidylethanolamine- and phosphatidylcholine-containing ether lipids, including PlsEtn, has been extensively documented (51;52), providing additional empirical evidence OL-derived exosomes are the probable primary source of the PlsEtn human neurons require.

And, in addition to carrying lipids in their lumenal cargo, the lipid bilayers of the exosomal vesicles themselves may contain phospholipids, including PlsEtn.

Exosomes isolated from primary mouse oligodendrocyte cultures have also been shown to contain sulfatides (53). That suggests exosomes secreted by human oligodendrocyte lineage cells may serve as an additional source of sulfatides for neurons, complementing the myelin sheath-derived, neuronal lysosome-liberated sulfatides described earlier (21).

The critical role oligodendrocyte-produced sulfatides play in sustaining normal CNS function is also underscored by the finding sulfatides are essential for the maintenance of ion channels in the axolemma of myelinated axons (54).

And, the ramifications of temporally upstream oligodendrocyte injury leading to neuronal PlsEtn deficiency are further underscored by recent work from Pedro Brites and colleagues (55). This group’s findings indicate that, at least in mouse, plasmalogen deficiency is associated with a distal repositioning of the action potential initiation-critical axon initial segment (AIS), as well as with diminished neuronal excitability.

Each of the key neuronal membrane components supplied to neurons by oligodendrocytes—PlsEtn and sulfatides—is also a player in modulating membrane composition, membrane rigidity/fluidity, and membrane curvature. This is particularly noteworthy in that membrane composition, fluidity, and curvature are thought to play critical roles with respect to α-synuclein’s interactions with biological membranes.

Thus, the Braverman, et al.-identified shift toward phosphatidylethanolamine (PE) phospholipids in response to plasmalogen insufficiency noted above (11)—a shift that would occur temporally downstream of PD-triggering OL injury, as OL PlsEtn production and/or the capacity to exocytose falls—would clearly alter the lipid composition (and fluidity, and curvature) of the key biological membranes with which α-synuclein interacts.

Obviously, the physiological mechanisms via which the health of neuronal synaptic vesicle and nerve terminal membranes is intimately and strikingly tied to the health of oligodendrocyte lineage cells have already been elucidated, empirically speaking. It simply requires the ability to step away from a neuroncentric view of CNS function (and of the pathophysiology of Parkinson’s disease and other neurodegenerative conditions) to appreciate that reality!

Klaus-Armin Nave’s highly-cited November 2010 Nature review article “Myelination and support of axonal integrity by glia” (56) provides additional support for the conclusion herein that oligodendrocytes are functionally and temporally upstream of CNS projection neurons as drug targets in Parkinson’s disease and other so-called synucleinopathies:

“Degeneration of (axons) in the presence of apparently normal myelin—for example, that assembled by oligodendrocytes lacking PLP or CNP—...is preceded by significant perturbation of fast axonal transport. This raises the intriguing possibility that oligodendroglial support is required for the timely delivery of presynaptic components such as...proteins that control the fast calcium-regulated neurotransmitter release.”

Klaus continues: “Little is known about how the steady state of...presynaptic proteins is controlled in the terminals of long axons, but the effect of reduced transport rates (as a consequence of oligodendroglial injury) is probably equivalent to that of reduced expression rates (of synaptic proteins)” (56).

Klaus’ last point is particularly noteworthy in light of a bioRxiv preprint posted 8 February 2022 from the lab of the University of Wisconsin-Madison’s Edwin R. Chapman (57). Chapman and co-authors present compelling evidence synaptic vesicle proteins in mammalian neurons are selectively delivered to axons, without entering dendrites. And, furthermore, that neurons subsequently axonally transport such SV proteins to the presynaptic plasma membrane, creating a depot which allows SV biogenesis to occur directly from the presynaptic plasma membrane.

Thus, reduced axonal transport rates in long axons occurring as the result of oligodendrocyte injury may directly and deleteriously affect synaptic vesicle biogenesis, the synaptic vesicle cycle, and the integrity of neurotransmission...in part owing to the altered capacity of the presynaptic plasma membrane to serve as a depot for synaptic vesicle proteins temporally downstream of the compensatory shift from PlsEtn-laden presynaptic plasma membranes to phosphatidylethanolamine-laden presynaptic plasma membranes which accompanies Parkinson’s disease-triggering OL lineage cell injury.

The fact oligodendrocytes are temporally and functionally upstream of CNS projection neurons as drug targets in Parkinson’s disease and other so-called synucleinopathies is increasingly difficult to deny. And, the findings of the Nature Genetics paper (1) quoted off the top are not one-off. In a 2020 Nature Communications paper (58) published by a separate group of authors several months after the 2020 Nature Genetics paper appeared it is noted “A single-cell atlas of the human substantia nigra reveals...a distinct cell type association between PD risk and oligodendrocyte-specific gene expression."

From a clinical perspective, a 2021 paper published in Human Brain Mapping (59) employs a combination of diffusion tensor imaging (DTI) and fixel-based analysis (FBA) to demonstrate “...strengthened and weakened white matter integrity that is subject to symptom laterality in a large drug-na?ve de novo PD cohort...” The authors go on to note their findings “...suggest that (Parkinson’s) disease gives rise to tissue degeneration and potential re-organization in the early stage.”

Such clinical findings are consistent with compensatory, myelin-related changes affected to circumvent the initially subclinical effects of Parkinson’s disease-triggering oligodendrocyte injury.

Also of note, Rutgers-Robert Wood Johnson Medical School’s Yoon-Seong Kim and colleagues posted a medRxiv preprint in January 2022 presenting “evidence for a novel age-associated oligodendrocyte subtype that appears during normal aging, characterized by elevated protein folding and chaperone-mediated autophagy pathways” (60). UPDATE: this Yoon-Seong Kim, et al., work has subsequently been published in peer-reviewed form in NATURE Aging; https://pubmed.ncbi.nlm.nih.gov/38491288/

In their work, Kim, et al., go on to conclude that “(their) study suggests a previously undescribed role for oligodendrocytes in aging and PD pathogenesis” (60).

Interestingly, in a 2020 SCIENCE Advances article (61) entitled “Incomplete annotation has a disproportionate impact on our understanding of Mendelian and complex neurogenetic disorders" (e.g., Parkinson’s disease), John Hardy and colleagues observe that “Incomplete annotation of genes disproportionately affects oligodendrocytes...suggesting that incomplete annotation (is) disproportionately limiting our understanding of this cell type."

That observation resonates with November 2019 work from UTSWMC’s Genevieve Konopka, et al. (62) published in PNAS, which notes “...oligodendrocytes have undergone more pronounced accelerated gene expression evolution in the human lineage (than have) neurons” and the findings of Erasmus University Medical Center’s Menno P. Creyghton and colleagues in a January 2020 Nature Communications paper (63) that “Hominin-specific (gene) regulatory elements selectively emerged in oligodendrocytes...”

The urgency of correcting the collective ignorance suggested by Hardy, et al.’s 2020 findings is reinforced by 8 April 2022-published findings in IET Systems Biology (64). In that work, the authors, in response to accumulating evidence implicating the cingulate cortex in PD-related cognitive impairment, carry out transcriptomic and regulatory network analyses of publicly available RNAseq data from postmortem PD and healthy control cingulate cortices.

They find, in part, “Importantly, myelin genes and the oligodendrocyte development pathways were markedly downregulated, indicating disrupted myelination in PD cingulate cortex. Cell-type-specific signatures revealed that myelinating oligodendrocytes were the major cell type damaged in the PD cingulate cortex. Furthermore, downregulation of myelination pathways in the cingulate cortex were shared and validated in another independent RNAseq cohort of dementia with Lewy bodies (DLB)...indicating the similarity of underlying causes for cognitive deficits in” (64) diseases exhibiting Lewy pathology.

The red flag for those with a neuroncentric view of Parkinson’s disease etiology mentioned above in connection with the macroglial expression of GBA in the human cortex should become a flashing red light when such neuroncentrists view the evolution of the mRNA-level expression of CDNF (Cerebral Dopamine Neurotrophic Factor) in moving from mouse to man: see the Barres lab RNA-seq database CDNF data in the above-linked complementary data PDF.

The evolution of the mRNA-level expression of GDNF (Glial Cell Derived Neurotrophic Factor)—which UniProtKB/Swiss-Prot describes as “(enhancing) survival and morphological differentiation of dopaminergic neurons and (increasing) their high-affinity dopamine uptake”—is equally instructive in moving from mouse to man. The expression of GDNF is apparently oligodendrocyte-specific in the human cortex; see the Barres lab RNA-seq database GDNF data in the complementary (data) PDF.

Lewy pathology is an epiphenomenon. It develops temporally downstream of perturbations of the fragile biophysics of neurotransmitter release from vulnerable CNS projection neurons, i.e., those projection neurons with axons that are disproportionately long and of thin caliber relative to the size of their cell somata (65). Mutations in the α-synuclein gene, SNCA, simply** confer heightened sensitivity to deviations from optimal synaptic vesicle health/nerve terminal membrane lipid composition in such neurons.

And, synaptic vesicle health and nerve terminal membrane lipid composition are intimately and strikingly tied to the health of oligodendrocyte lineage cells.

**In relatively rare instances, SNCA mutations—owing to loss-of-function effects which do NOT warrant targeting Lewy pathology as a treatment strategy—may themselves trigger such deviations from optimal synaptic vesicle health/nerve terminal membrane lipid composition in vulnerable CNS projection neurons.

The work of UCSF’s Robert H. Edwards and colleagues in the 2017 Nature Neuroscience paper “α-Synuclein promotes dilation of the exocytotic fusion pore” (66) dovetails with the assessment Lewy pathology is an epiphenomenon that occurs temporally downstream of perturbations of the fragile biophysics of neurotransmitter release. As Edwards, et al., conclude: “Thus, synuclein exerts dose-dependent effects on dilation of the exocytotic fusion pore. Remarkably, mutations that cause Parkinson’s disease abrogate this property of α-synuclein...”

In addition to membrane deformation/membrane curvature-promoting proteins such as α-synuclein playing a role in fusion pore dynamics, membrane curvature-promoting phospholipids are also key players in fusion pore dilation (67).

Plasmalogen phospholipids are, in fact, enriched in regions of high membrane curvature. And, modification of biological membrane properties owing to reduced plasmalogen content is likely to affect key cellular exocytotic and endocytotic processes, which typically involve highly curved membrane bilayers (8).

The adsorption of α-synuclein to synaptic vesicles, in particular, is a function of biological membrane properties such as membrane curvature and the density of ‘lipid packing defects,’ each of which PlsEtn have been shown to directly affect (8;68;69).

Tellingly, Jennifer L. Gallop of the University of Cambridge and her colleagues have noted that “The molecular mechanisms of membrane curvature rarely act alone, but instead cooperate in diverse ways...” (70). That observation strongly implies ethanolamine plasmalogen deficiency in neuronal membranes will disrupt α-synuclein’s role in key membrane curvature-dependent exocytotic and endocytotic events.

It is also of note that, among ethanolamine glycerophospholipids, PlsEtn show the greatest propensity to stabilize membrane curvature (71):

plasmenylethanolamines ? plasmanylethanolamines > phosphatidylethanolamines (PE)

In fact, it is the obligatory “compensatory shift” from optimally constituted, high PlsEtn content synaptic vesicle (SV) membranes and high PlsEtn content nerve terminal plasma membrane (PM) inner leaflets to PE-laden SV membranes and PE-laden nerve terminal PM inner leaflets occurring under the conditions of neuronal PlsEtn insufficiency which develop temporally downstream of Parkinson’s disease-triggering oligodendrocyte lineage cell injury that directly leads to the appearance of Lewy pathology—and, ultimately, to the demise of the various projection neurons known to be vulnerable (65) in Parkinson’s disease.

There is a clear-cut, “physiological straight line” between temporally upstream, neuronal lipid homeostasis-altering/Parkinson’s disease-triggering oligodendrocyte lineage cell injury and the advent of Lewy Pathology.

The recently revealed ATG9-mediated coupling of presynaptic macroautophagy (“autophagy”) to the synaptic vesicle cycle (72;73) highlights a particularly intriguing point on that line. To wit, ATG9-mediated coupling of autophagy to the synaptic vesicle cycle relies heavily upon exo-endocytic cycling of ATG9 through the plasma membrane compartment in presynaptic nerve terminals...cycling which is ostensibly deleteriously impacted by the compensatory shift from optimally PlsEtn-supplied presynaptic nerve terminal membranes to PE-laden presynaptic nerve terminal membranes that occurs as the pathophysiology of Parkinson’s disease unfolds.

Continuing along that physiological straight line from temporally upstream oligodendrocyte lineage cell injury to the advent of Lewy pathology, Nicholas T. Ktistakis’ description of the cellular structures that arise upon disrupting autophagosome biogenesis is noteworthy: “...mutations that block late steps in autophagosome biogenesis often produce preautophagosomal structures with multiple bilayers that are devoid of proteins and are stacked together in a multilamellar morphology” (74).

That description bears a striking resemblance to the unexpectedly lipid-rich ultrastructural core of Lewy bodies reported in the now very highly-cited 2019 Sarah Shahmoradian, et al., Nature Neuroscience paper (75) “Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes:”

“...dense ‘dark’ L-shaped structures resembling stacks of compacted membrane sheets with a membrane layer spacing of ~6 nm were distinguished...They cannot be attributed to myelin, since the average membrane spacing of myelin sheaths is 10.7 nm in the central nervous system.”

Interestingly, defective ATG9 trafficking—as may occur temporally downstream of PD-triggering oligodendrocyte lineage cell injury and the compensatory shift to higher phosphatidylethanolamine (PE) content nerve terminal plasma membranes—has been associated with impaired clearance of protein aggregates (76).

And, cells under autophagic stress, e.g., substantia nigra dopaminergic projection neurons in sufferers of Parkinson’s disease, can continuously (attempt to) produce autophagosomes in large numbers (77), suggesting how the Shahmoradian, et al.-described “...dense ‘dark’ L-shaped structures resembling stacks of compacted membrane sheets...” (75) in Lewy body ultrastructure may arise.

The observations in the latter two paragraphs make sense of the chronology of events-related statement in the 2020 Nature Genetics paper quoted off the top (1): “These results not only support the genetic evidence indicating that oligodendrocytes may play a causal role in Parkinson’s disease, but indicate that their involvement PRECEDES the emergence of pathological changes in the substantia nigra.” (my emphasis)

The aforementioned 2019 work of Sarah Shahmoradian and colleagues on the composition of Lewy bodies/Lewy neurites also resonates with Lewy pathology being intimately related to disrupted presynaptic autophagy. In the 13 February 2019 bioRxiv preprint version of their work (78), the authors note “...subsequent analysis confirmed the high cell membrane related lipid content (of Lewy bodies) indicated by other methods, with the mass spectra showing strong peaks corresponding to sphingomyelin (SM) and phosphatidylcholine (PC) for aSyn-immunopositive inclusions isolated from both the (substantia nigra) and hippocampal CA2 sector...”

The observation Lewy bodies contain high levels of sphingomyelin dovetails with the idea Lewy pathology evolves as the result of disrupted presynaptic autophagy—and the disrupted coupling of that presynaptic autophagy to the synaptic vesicle cycle—as sphingomyelin appears to be an important regulator of ATG9 trafficking and of the maturation of autophagic membranes (79).

The high phosphatidylcholine content of Lewy bodies noted by Shahmoradian, et al. (78) is also consistent with the likelihood Lewy pathology evolves as the result of disrupted presynaptic autophagy, given that autophagosomes are phospholipid-enriched structures whose biogenesis appears to require de novo phosphatidylcholine synthesis (74;80).

To reiterate, the compensatory shift from optimally constituted, high PlsEtn content synaptic vesicle (SV) membranes and high PlsEtn content nerve terminal plasma membrane (PM) inner leaflets to PE-laden SV membranes and PE-laden nerve terminal PM inner leaflets occurring under the conditions of neuronal PlsEtn insufficiency that develop downstream of PD-triggering OL lineage cell injury may inhibit or dysregulate ATG9 cycling through the nerve terminal plasma membrane.

Even should such ATG9 (i.e., ATG9A in man) cycling continue unabated, however, that compensatory PlsEtn-to-PE shift will have additional, physiological consequences for ATG9-mediated phagophore development/autophagosome biogenesis.

ATG9’s scramblase activity is thought to allow it to act in concert with ATG2 to pull lipids from various sources, including the plasma membrane, and distribute those lipids to nascent phagophores (73). The autophagosomes which emerge from this process are phospholipid-enriched structures and are largely devoid of proteins and cholesterol (74). And, at least in a human cancer cell line (HCT116), de novo choline phospholipid synthesis is required for autophagosome formation/maturation, with such synthesis including the production of plasmenylcholine (PlsCho) (80).

Multiple investigators have also demonstrated CEPT1 (choline/ethanolamine phosphotransferase 1), the enzyme mentioned earlier as allowing human neurons to produce their own PlsCho from exogenously supplied PlsEtn, is present at sites of autophagosome biogenesis (81;82).

However, temporally downstream of PD-triggering OL lineage cell injury and the resulting compensatory shift from PlsEtn-laden to PE-laden SV membranes and nerve terminal plasma membranes ATG9 will lose its primary source of PlsEtn for delivery to nascent phagophores.

Again, as there is no known plasmanylcholine desaturase enzyme activity, PlsCho cannot be generated in the absence of PlsEtn. In much the same way PE substitutes for PlsEtn under conditions of plasmalogen insufficiency11 phosphatidylcholine (PC) is thought to substitute for PlsCho under conditions of plasmalogen insufficiency (71).

Thus, temporally downstream of PD-triggering OL lineage cell injury, presynaptic phagophore biogenesis, to the extent it is possible, will produce autophagosomes with suboptimal phospholipid content. In an analogous manner to that seen in the compensatory shift from PlsEtn to PE, the shift from PlsCho to PC (in autophagosome double bilayer membranes) will have a deleterious impact on the capacity to generate and sustain membrane curvature and to accommodate membrane fusion.

Sarah E. Hill and Daniel A. Colón-Ramos have recently pointed out that “...one of the most striking aspects of neuronal autophagy is its spatial organization. Autophagosomes form in distal axonal compartments near synapses and undergo retrograde transport” (83).

As the synaptic activity-coupled neuronal presynaptic (macro)autophagy ‘cycle’ includes “multiple sequential fusions (of autophagosomes) with late endosomes and lysosomes during transport to the cell body...” (83), the compensatory shift from PlsCho to PC in neuronal autophagosomes will have physiological consequences...perhaps including the axonal and/or somal accumulation of ‘spent,’ incompletely-processed autophagic double-membrane vesicles which ultimately end up at the core of Lewy bodies.

A compensatory shift to phosphatidylethanolamine-laden synaptic vesicle membranes and phosphatidylethanolamine-laden nerve terminal plasma membrane inner leaflets may also dysregulate lipidation (with phosphatidylethanolamine) of the key autophagosome biogenesis protein LC3.

LC3, encoded by the MAP1LC3B gene, is a mammalian homolog of the yeast autophagy protein ATG8. Empirically, PE-lipidated LC3 (LC3-PE, a.k.a., LC3-II)—and the ratio of LC3-II to non-lipidated, cytosolic LC3 (i.e., LC3-I)—are widely used to monitor autophagy (84).

And, in primary mouse hippocampal neurons, induction of autophagy leads to the accumulation of LC3 in synapses (85)...suggesting abnormally high synaptic vesicle PE content and abnormally high plasma membrane inner leaflet PE content may be problematic, e.g., provoking ectopic LC3 lipidation (86) and association with synaptic vesicles and/or the inner leaflet of nerve terminal plasma membranes (rather than association with nascent, perisynaptic phagophores).

In keeping with that possibility, model systems such as C. elegans and Drosophila display hotspots of autophagosome formation “in close proximity to or directly within presynaptic terminals” (87).

The fact many phagophore formation-critical proteins have been shown to associate with the scaffolding proteins of the active zone, synaptic vesicles themselves, or with the perisynaptic endocytic zone (87) further underscores the potential for such LC3-related dysregulation temporally downstream of PD-triggering OL injury and the resultant compensatory shift to PE-laden SV/nerve terminal plasma membranes.

Plasmalogen insufficiency also has implications for ATP production in neuronal mitochondria.

The source(s) of the “plasmalogenase” enzymatic activity that cleaves the vinyl ether linkage in plasmalogens was still a mystery when, in a JBC paper published in 2018, Richard W. Gross and co-authors demonstrated cytochrome c exhibits such activity under conditions of oxidative stress (88). Thus, dopaminergic and other fragile projection neurons under stress may actually catabolize their own (dwindling) PlsEtn reserves—and, by extension, their capacity to endogenously produce PlsCho.

Although phosphatidylethanolamine (PE) itself is known to play important roles in mammalian mitochondria, “...notable loss of plasmenylethanolamine in the brain, despite the presence of some counterbalancing gain of diacyl PE, is expected to have a negative impact on physical stability of the (mitochondrial) cristae membrane, biological maintenance of the cristae ultrastructure, integrity of the structure and function of the individual respiratory (chain) complexes and their supercomplexes, and production of ATP...” (71).

Clearly, temporally upstream oligodendrocyte injury and the resulting neuronal PlsEtn/sulfatide insufficiencies precipitate a truly (neuro)degenerative cascade of events!

Although outside the scope of this communication, that temporally upstream OL injury and resulting ‘cascade of events’ obviously have implications for the pathobiology of Alzheimer’s disease and beyond...implications which have been worked out in great detail by the author.

In their plasmalogenase enzymatic activity-assigning 2018 work, Gross, et al., also highlight the lysoplasmalogenase TMEM86B: “...despite intense efforts to identify plasmalogen hydrolysis by (TMEM86B), the results demonstrated that this enzyme does not possess plasmalogenase activity” (88).

The lysoplasmalogenase activity of TMEM86B requires plasmalogens to first have had their sn-2 acyl substituents removed, e.g., by one of the key plasmalogen-preferring phospholipase A2’s already discussed as being significantly downregulated in the human cortex relative to their mRNA-level expression in the mouse cortex—those key plasmalogen-preferring phospholipase A2’s showing no appreciable expression in human cortical neurons, in particular: PLA2G6; PLA2G4A; PLA2G4B.

TMEM86B follows this same pattern, displaying an order-and-a-half magnitude downregulation in mRNA-level expression in the human cortex relative to the mouse cortex, and no appreciable expression in any of the human neural cell types represented in the Barres lab RNA-seq database...such a diminution of lysoplasmalogenase activity being consistent with plasmalogens having evolved to play a structural (rather than dynamic) role in the human cortex, and likely beyond.

There is a highly plausible explanation of the observation in the 2020 Nature Genetics paper (1) cited off the top that oligodendrocyte perturbation precedes the appearance of substantia nigra pathology in Parkinson’s disease. That likelihood involves (at least) three key proteins: kallikrein-related peptidase 6 (KLK6), transferrin (TF), and fatty acid 2-hydroxylase (FA2H).

At the level of mRNA in the human cortex, each of these three proteins is overwhelmingly oligodendrocyte-expressed relative to other neural cell types, each protein arguably on the verge of being an oligodendrocyte-specific protein; https://brainrnaseq.org/ (e.g., search each protein in the Stanford University-maintained database reflecting work in the lab of the late Ben Barres).

In the context of the “massive oligodendrocyte population” (43) in the human midbrain and the observation “the cell populations of the aged human SN are composed predominantly by oligodendrocytes...” (44) noted previously, it is likely nigral KLK6, TF, and FA2H levels are intimately tied to oligodendrocyte lineage cells.

Remarkably, of the ten brain regions and seventeen non-nervous system tissues represented in the Genotype-Tissue Expression (GTEx) Consortium RNA-seq data displayed in each of the respective GeneCards entries (KLK6; TF; FA2H), substantia nigra shows the highest mRNA-level expression of any human tissue surveyed in each instance.

The serine protease KLK6 has been shown to cleave α-synuclein, both in vitro and in vivo. KLK6, ostensibly the most abundantly-secreted serine protease in the CNS (89), also inhibits in vitro polymerization of α-synuclein and has been postulated to prevent α-synuclein aggregation in vivo (90-93).

On the order of 70% of brain iron is associated with oligodendrocytes (23;94), and, transferrin has been convincingly implicated in the regulation of ferroptosis (95). The oligodendrocyte-centric nature of TF expression and recent evidence (in mouse) that oligodendrocytes secrete ferritin heavy chain in protection of neurons (96) strongly suggests oligodendrocytes play a direct role in intraneuronal iron homeostasis (39).

Blocking either oligodendroglial release of extracellular vesicles or production of ferritin heavy chain in (mouse) oligodendrocytes leads to axonal damage in aging mice (96).

In the context of such a dynamic, the presence of a probable iron responsive element (IRE) in SNCA mRNA (97-99) is noteworthy and highly suggestive of dysregulated neuronal iron homeostasis temporally downstream of PD-triggering OL injury leading to increased neuronal α-synuclein expression.

Per the Genecards entry for the FA2H gene, FA2H is six-fold overexpressed in the human substantia nigra. And, in 2010, John Hardy and colleagues identified mutations in the FA2H gene (100) that lead to a novel form of NBIA (Neurodegeneration with Brain Iron Accumulation).

In addition, 2-hydroxylated forms of sulfatide, which require FA2H enzyme activity to produce, are seen to display dichotomous expression in the human brain: “The distribution analysis reveals that the composition ratios of non-hydroxylated/hydroxylated ST (sulfatide) are clearly reversed at the border between white and gray matter; the hydroxylated group is the dominant ST species in the gray matter”(101).

Instructively, paraphrasing from a 24 September 2008 J. Neuroscience paper from Matthias Eckhardt and colleagues (102), because axon degeneration appears to start rather early with respect to myelin degeneration in FA2H-/- mice, 2-hydroxylated sphingolipids—including 2-hydroxylated sulfatides—may be required for long-term glial support of axon function.

Collectively, diminished nigral KLK6, TF, and FA2H levels likely trigger α-synuclein-related pathology as an epiphenomenological correlate of Parkinson’s disease temporally downstream of PD-initiating OL injury.

A brief aside for those who may question how oligodendrocyte injury serving as the trigger for the development of CNS Lewy pathology explains Lewy pathology found in the peripheral (PNS) and enteric (ENS) nervous systems:

The major CNS myelin protein PLP1 (proteolipid protein 1), encoded by the PLP1 gene, fits the same paradigm as KLK6, TF, and FA2H. That is, at the level of mRNA expression in the human cortex, PLP1 arguably borders on being expressed oligodendrocyte-specifically. And, of the ten brain regions and seventeen non-nervous system tissues represented in the GTEx RNA-seq data in the PLP1 GeneCards entry, the substantia nigra shows the highest mRNA-level PLP1 expression.

PLP1 has also been implicated in ferroptosis (103).

The advent of the vertebrate-brain-enabling neuroprotective function of CNS myelin sheaths—which occurred ~400 million years ago, and involved a transition from (type I integral membrane protein) Po-containing CNS myelin to tetraspan membrane protein PLP (proteolipid protein)-containing CNS myelin—was elegantly pinpointed in work from the Cleveland Clinic’s Bruce Trapp and colleagues published in the Journal of Cell Biology in 2006 (104).

Outside the CNS, PNS Schwann cells (SCs) and satellite glia, the latter known to completely surround individual neuronal cell bodies and synapses in peripheral ganglia, each have PLP1 as a canonical marker (105). And, mammalian ENS glia have been shown to “unexpectedly” express both PLP1 mRNA and PLP1 protein, as well as to “express many genes characteristic of the myelinating glia, Schwann cells and oligodendrocytes...” (106).

As Heiko Braak, Kelly Del Tredici, and colleagues have observed (107), “Only projection neurons with a long and sparsely myelinated or unmyelinated axon become involved in PD, while short-axoned cells or neurons with a thickly myelinated axon resist (Lewy pathology). Vulnerable nerve cell types occur in peripheral, enteric, and central portions of the nervous system (PNS/ENS/CNS).”

Auerbach’s (“myenteric”) plexus—a network of peripheral (“enteric”), interconnected neurons/nerve fibers found in the wall of the gut, and which runs the entire length of the human alimentary canal—contains myelinated axons in man (108). Auerbach’s plexus is also known to develop Lewy pathology in Parkinson’s disease (109).

Although expressed at much lower levels in the PNS than in the CNS, PLP1 has been shown to contribute to peripheral myelination, and, most importantly, to be directly involved in the preservation of PNS axons (110)...axonal preservation being a well-known function of PLP1 with respect to CNS axons.

It is thus reasonable to assert the initial insults in the PNS/ENS which lead to the development of extra-CNS Lewy pathology are glial in nature, not neuronal—particularly in light of the fact the ENS glia:ENS neuron ratio in the human gut is estimated to be approximately 4:1 (106), and may reach 7:1 in Auerbach’s plexus (111).

As Brian Gulbransen observed in a 2014 review of enteric glia (112), a “general rule seems to be that the ratio of (enteric) glial cells to (enteric) neurons increases with species size.” Gulbransen goes on to note that the need for additional enteric glia in larger animals “may reflect the high metabolic needs of the much larger (enteric) neurons. Enteric glial cell body size remains relatively constant across species analyzed to date despite wide variability of (enteric) neuron sizes. Thus, the amplified size mismatch in larger species may require additional glia” (112).

That temporally upstream injury to PNS glia (SCs; satellite glia) and/or ENS glia, and the resulting perturbation of the neuroprotective function(s) those glia provide, would serve as the trigger for the development of Lewy pathology in vulnerable PNS/ENS projection neurons resonates with the overwhelming weight of evidence presented herein that oligodendroglial injury serves as the trigger for the development of Lewy pathology in vulnerable CNS projection neurons.

And, the conclusion Lewy pathology in gut neurons develops temporally downstream of ENS glial cell injury is bolstered by a more recent Brian Gulbransen publication (113), the 2021 PNAS article “Circuit-specific enteric glia regulate intestinal motor neurocircuits.”

The title of an 18 July 2021 article by Celia Henry Arnaud in Chemical & Engineering News (C&EN) poses the question “Is Parkinson’s disease caused by dysfunctional lipids?” (114).

Parkinson’s disease is not caused by “dysfunctional” lipids, per se. It is, however, largely caused by a compensatory shift in neuronal membrane lipid composition (MLC) which produces PE-laden synaptic vesicle membranes and PE-laden presynaptic nerve terminal plasma membranes incapable of supporting the fragile biophysics of neurotransmitter release from vulnerable CNS projection neurons over time.

And that compensatory shift in neuronal MLC is triggered by temporally upstream injury to oligodendrocyte lineage cells.

Lewy pathology is clearly an ill-advised and ill-fated drug target in Parkinson’s disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Furthermore, the concept of “synucleinopathies” is misguided and represents a major impediment to progress in developing efficacious, disease-modifying treatments for sufferers of those devastating illnesses.

Oligodendrocytes are clearly temporally and functionally upstream of CNS projection neurons as drug targets in Parkinson’s disease and other so-called “synucleinopathies.”

May the research community at large grasp those profoundly important truths sooner, rather than later.

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