The catecholaldehyde hypothesis for the pathogenesis of catecholaminergic neurodegeneration: What we know and what we don’t know

3,4-dihydroxyphenylacetaldehyde (DOPAL) is the focus of the catecholaldehyde hypothesis for the pathogenesis of Parkinson disease and other Lewy body diseases. The catecholaldehyde is produced via oxidative deamination catalyzed by monoamine oxidase (MAO) acting on cytoplasmic dopamine. DOPAL is autotoxic, in that it can harm the same cells in which it is produced. Normally DOPAL is detoxified by aldehyde dehydrogenase (ALDH)-mediated conversion to 3,4-dihydroxyphenylacetic acid (DOPAC), which rapidly exits the neurons. Genetic, environmental, or drug-induced manipulations of ALDH that build up DOPAL promote catecholaminergic neurodegeneration. A concept derived from the catecholaldehyde hypothesis imputes deleterious interactions between DOPAL and the protein alpha-synuclein (  S), a major component of Lewy bodies. DOPAL potently oligomerizes  S, and  S oligomers impede vesicular and mitochondrial functions, shifting the fate of cytoplasmic dopamine toward MAO-catalyzed formation of DOPAL — destabilizing vicious cycles. Direct and indirect effects of DOPAL and of DOPAL-induced misfolded proteins could “freeze” intra -neuronal reactions, plasticity of which is required for neuronal homeostasis. The extent to which DOPAL toxicity is mediated by interactions with  S, and vice versa, are poorly understood. Because of numerous secondary effects such as augmented spontaneous oxidation of dopamine by MAO inhibition, there has been insufficient testing of the catecholaldehyde hypothesis in animal models. The clinical pathophysiological significance of genetics, emotional stress, environmental agents, and interactions with numerous proteins relevant to the catecholaldehyde hypothesis are matters for future research. The imposing complexity of intra-neuronal catecholamine metabolism seems to require a computational modeling approach to elucidate clinical pathogenetic mechanisms and devise pathophysiology-based, individualized treatments. formation of spontaneous oxidation products.


MAO and intra-neuronal metabolism of catecholamines
In 1928 Mary L.C. Hare described an enzyme in the liver that oxidized and deaminated the dietary amine tyramine and resulted in the formation of ammonia (Hare 1928). She noted that the amount of ammonia generated by this amine oxidase was one-half that of the oxygen consumed.
Subsequently, Kohn explained this in terms of formation of hydrogen peroxide (which is then metabolized by catalase) and an aldehyde, 4-hydroxyphenylacetaldehyde (Kohn 1937).
Demonstration that the catecholamine adrenaline and tyramine competed for the same enzyme led to abandonment of the notion of a separate "adrenaline oxidase." After Julius Axelrod showed that the major route of exogenously administered catecholamine is O-methylation catalyzed by catechol-O-methyltransferase (COMT) (Axelrod 1966), routes of inactivation of endogenous catecholamines were unclear. Eventually it was recognized that in contrast with exogenously administered catecholamines, catecholamines Although most of MAO activity in the brain is of the B type, MAO-A figures prominently in the oxidative deamination of striatal dopamine (Demarest & Moore 1981;Wachtel & Abercrombie 1994;Dyck et al. 1993;Kumagae et al. 1991;Colzi et al. 1990). Based on mRNA contents, MAO-A is expressed in noradrenergic and dopaminergic cell groups, with relatively little MAO-B expression (Jahng et al. 1997). Substantia nigra dopaminergic neurons of mice express both MAO-A and MAO-B, with a predominance of MAO-A (Graves et al. 2020).
Administration of drugs that are selective MAO-B inhibitors in vitro can inhibit MAO-A in vivo (Eisenhofer et al. 1986;Fowler et al. 2015;Naoi et al. 2016). Bases for this phenomenon are unclear but seem to be related to the chronicity of dosing (Bartl et al. 2014). In humans, treatment with the MAO-B inhibitors deprenyl or rasagiline decreases plasma levels of 3,4dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylglycol (DHPG), the respective main intra-neuronal metabolites of dopamine and norepinephrine (Eisenhofer et al. 1986;Goldstein et al. 2016a) (Figures 1-3).

DOPAL toxicity
After Blaschko's expression of concern in 1952 that aldehydes produced from MAOcatalyzed oxidative deamination of catecholamines might be toxic (Blaschko 1952), about 40 years elapsed before the first evidence of catecholaldehyde toxicity was reported by Mattammal et al. Applying a gas chromatography-mass spectrometry method they noted the presence of DOPAL in post-mortem substantia nigra tissue from patients with PD but not in control subjects (Mattammal et al. 1993). In the same study they reported that incubation of dopamine with MAO-B resulted in 4-5-fold increases in covalent binding to DNA, bovine serum albumin, and microtubular protein. This finding presaged the relatively recent discovery of DOPAL-mediated oligomerization and quinone adduct formation ("quinonization") of numerous proteins in catecholaminergic cells (Jinsmaa et al. 2018).
Mattammal et al. also reported that (1) DOPAL is taken up into striatal synaptosomes via the cell membrane dopamine transporter (DAT); (2) DOPAL concentration-and time-dependently releases DA from synaptosomes, due to damaging the synaptosomal membranes; (3) in rat pheochromocytoma PC12 cells differentiated with nerve growth factor, DOPAL concentration-dependently depletes cell contents of dopamine, DOPAC, and homovanillic acid (HVA, the end-product of dopamine metabolism) and kills cells based on both cell counts and release of lactate dehydrogenase into the medium; (4) in primary cultures of ventral midbrain from rat embryos, at DOPAL concentrations of 7.5-20 µM there are reductions in dopamine uptake without a reduction in the number of tyrosine hydroxylase (TH)-positive cells, and at higher concentrations there is cellular loss; and (5) (Li et al. 2001). The relevance of this finding to pathophysiology is unclear, because hydrogen peroxide is broken down by catalase and because within cells MAO acting on dopamine may not actually increase cytoplasmic hydrogen peroxide levels (Graves et al. 2020).
Burke et al. examined the in vivo toxicity of exogenously administered DOPAL in rat substantia nigra and ventral tegmental area. At 5 days post-surgery, DOPAL at concentrations as low as 100 ng was found to be toxic selectively to nigral dopaminergic neurons (Burke et al. 2003). Several years later, another in vivo animal study of exogenously administered DOPAL in Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 3 May 2021 doi:10.20944/preprints202105.0002.v1 Goldstein,p. 8 rats reported that DOPAL evokes neuronal loss in the substantia nigra pars compacta and loss of striatal dopaminergic terminals and produces rotational asymmetry as is seen in other PD animal models (Panneton et al. 2010).

DOPAL detoxification by aldehyde dehydrogenase (ALDH)
Blaschko theorized that enzymatic metabolism via mitochondria detoxifies the aldehydes produced by amine oxidases acting on monoamines. He wrote, "…the amine oxidase reaction results in the formation of two toxic metabolites, both of which are removed by reactions which take place in the mitochondria. One of these metabolites is ammonia which is removed in the mitochondria by urea synthesis, the other is an aldehyde which is further metabolised in reactions known to be linked with the cytochrome-cytochrome oxidase system. The location of amine oxidase in the mitochondria thus ensures that these two end products of the enzymatic reaction are rendered harmless not far from the site where they are formed" (Blaschko 1952). In Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 3 May 2021 doi:10.20944/preprints202105.0002.v1 Goldstein,p. 9 1957 he wrote, "Oxidative deamination by amine oxidases leads to the formation of an aldehyde, but the aldehyde is usually further oxidized to the corresponding carboxylic acid." (Blaschko 1957).
DOPAL is converted to the acid DOPAC via aldehyde dehydrogenase (ALDH), and the DOPAC produced rapidly exits the cells. Thus, in PC12 cells DOPAC is extruded actively via a sulfonylurea-sensitive transporter (Lamensdorf et al. 2000b). One may conceptualize that MAO keeps cytoplasmic dopamine levels low, and ALDH in series keeps DOPAL levels low.
In the 1960s Youdim and Sandler published that reserpine inhibits ALDH (Youdim & Sandler 1968). To explain this phenomenon they wrote, "A facilitated metabolism of catecholamine, giving rise in its turn to an increased production of aldehyde, may cause substrate inhibition of aldehyde dehydrogenase." Subsequent experimental evidence has supported the concept that DOPAL can inhibit its own metabolism by ALDH ( Figure 4). There is a linear negative relationship between the percent of control ALDH activity and DOPAL concentrations in the range of 0-60 µM (MacKerell & Pietruszko 1987).
ALDH is an important determinant of intracellular DOPAL levels. Mice with double knockout of the genes encoding the mitochondrial and cytosolic forms of ALDH have DOPAL buildup (Wey et al. 2012). The fungicide benomyl inhibits ALDH and also results in DOPAL accumulation (Casida et al. 2014). Farm chemicals inhibiting ALDH may contribute to the incidence of PD (Fitzmaurice et al. 2013;Fitzmaurice et al. 2014;Ritz et al. 2016). In humans it has been reported that decreased ALDHA1A gene expression in blood is part of a "molecular signature" that can identify early PD (Molochnikov et al. 2012). ALDH1A1 gene expression and protein are decreased in substantia nigra specimens in patients with PD (Grunblatt et al. 2018;Mandel et al. 2005).
The mitochondrial complex 1 inhibitor rotenone indirectly decreases ALDH activity (Goldstein et al. 2015) by interfering with its co-factor NAD + , and rotenone therefore increases endogenous DOPAL production in PC12 cells (Lamensdorf et al. 2000a). Predictably, ALDH2 activation is protective in cellular and animal models of rotenone-induced neurotoxicity (Chiu et al. 2015).
Graves et al. (Graves et al. 2020) recently advanced the concept that rather than MAO resulting in hydrogen peroxide production in the cytoplasm, because of the positioning of MAO in the outer mitochondrial membrane electrons are shuttled through the intermembrane space to increase activity of the electron transport cascade leading to ATP production. The model does not mention the consequences of DOPAL production or that generation of NAD + is required for ALDH to detoxify DOPAL.

Vesicular uptake as a detoxification mechanism
Active uptake of cytoplasmic dopamine into vesicles via the vesicular monoamine transporter (VMAT) is not only required for dopaminergic neurotransmission but also serves as a detoxification mechanism (Gainetdinov et al. 1998;Fumagalli et al. 1999;Staal & Sonsalla 2000;Guillot & Miller 2009) (Figure 2). This includes autotoxicity exerted by dopamine itself (Weingarten & Zhou 2001). Animals with reduced activity of the type 2 VMAT have evidence of progressive nigrostriatal and locus ceruleus neurodegeneration (Caudle et al. 2007;Taylor et al. 2014), while increased VMAT activity is neuroprotective (Lohr et al. 2014;Munoz et al. 2012). Unexpectedly, mice with genetically determined very low VMAT2 activity do not have evidence of DOPAL buildup (Goldstein et al. 2014b). These animals have elevated tissue DOPAC/DOPAL ratios, suggesting differential survival based on increased DOPAL detoxification by ALDH.

The double hit concept
For a given rate of cytoplasmic dopamine synthesis, DOPAL levels are determined by dopamine uptake into vesicles and DOPAL metabolism (Figure 2). Although a small amount of DOPAL is metabolized to 3,4-dihydroxyphenylethanol (DOPET) by aldehyde/aldose reductase (AR), the main enzymatic fate of cytoplasmic DOPAL is metabolism to DOPAC via ALDH.
Accordingly, blockade by reserpine of vesicular uptake increases endogenous DOPAL levels in PC12 cells, and concurrent inhibition of ALDH increases DOPAL levels further (Goldstein et al.  Goldstein,p. 11 2012). Post-mortem neurochemical evidence for a "double hit" of a vesicular storage defect with decreased ALDH activity has been reported in putamen tissue from patients with PD ).

DOPAL-protein interactions, with emphasis on alpha-synuclein
DOPAL interacts with numerous intra-cellular proteins related to catecholamine production, storage, recycling, and metabolism ( Figure 4). As noted above, DOPAL interferes with synaptosomal uptake and retention of tracer-labelled dopamine (Mattammal et al. 1995).
DOPAL covalently binds to and inhibits the activity of TH (Mexas et al. 2011), the rate-limiting enzyme in catecholamine biosynthesis (Nagatsu et al. 1964). The ability of DOPAL to do so depends on both the catechol and aldehyde moieties (Vermeer et al. 2012). DOPAL also forms quinoprotein adducts with and inhibits the activity of LAAAD (Jinsmaa et al. 2018) and may decrease its own metabolism by ALDH (MacKerell & Pietruszko 1987).
In 1997, first genotypic abnormality producing familial PD was identified-A53T mutation of the gene encoding the protein alpha-synuclein (S) (Polymeropoulos et al. 1997). In the same year, Lewy bodies, a histopathologic hallmark of idiopathic PD, were found to contain abundant S (Spillantini et al. 1997). Since then the view has evolved that PD as typically encountered clinically is a form of synucleinopathy. Other synucleinopathies include multiple system atrophy (MSA), in which -syn is deposited in glial cytoplasmic inclusions in the brain (Wakabayashi et al. 1998); dementia with Lewy bodies (Baba et al. 1998); and pure autonomic failure (PAF) (Arai et al. 2000;Kaufmann et al. 2001).
Since then it has been suggested that dopamine oxidation products contribute to the oligomerize -syn (Huenchuguala et al. 2019;Munoz et al. 2015;Pham et al. 2009).
The literature on DA oxidation and synucleinopathy has generally overlooked DOPAL (Asanuma et al. 2003;Segura-Aguilar 2017;Mazzulli et al. 2006;Mor et al. 2017;Burbulla et al. 2017;Leong et al. 2009;Mor et al. 2019), and the literature on DOPAL and synucleinopathy has generally overlooked DA-Q (Burke et al. 2008;Follmer et al. 2015;Jinsmaa et al. 2018;Cagle et al. 2019). In the few studies where DOPAL and dopamine have been compared directly in terms of oligomerizing -syn, DOPAL has been found to be far more potent (Burke et al. 2008;Jinsmaa et al. 2014;Jinsmaa et al. 2018;Jinsmaa et al. 2020). A recent study found that DOPAL is not only more potent than dopamine in oligomerizing S but also that DOPAL quinonizes S (Jinsmaa et al. 2020). Even in the setting of dopamine oxidation evoked by Cu (II) or tyrosinase, dopamine does not quinonize S ( Figure 5).
The differences in potencies of DOPAL and dopamine in oligomerizing and quinonizing Goldstein, p. 14 2020). So far there has been no demonstration that DOPAL-induced cytotoxicity is mediated by protein quinonization.
Braak's "gut first" concept states that "a putative environmental pathogen capable of passing the gastric epithelial lining might induce S misfolding and aggregation in specific cell types of the submucosal plexus and reach the brain via a consecutive series of projection neurons" (Braak et al. 2006). Almost half of the synthesis and metabolism of dopamine in the body takes place in non-neuronal cells of the gut (Eisenhofer et al. 1997). One may speculate that DOPAL produced locally from abundant non-neuronal dopamine reacts with S to induce a pathogenic cascade.
A recent study involved DOPAL injection into the vagus nerve, to help understand the frequent association of autonomic failure with PD (Sun et al. 2021). DOPAL-treated rats had evidence of baroreflex dysfunction, orthostatic hypotension, and time-dependent associated changes in S monomers/trimers. The data suggest the plausibility of "body-first" subtype of PD that features autonomic abnormalities (Berg et al. 2021).   Goldstein,p. 15 As scanty as the literature is about DOPAL-related neurotoxicity, there is even less literature about DOPEGAL, which is the catecholaldehyde produced by the action of MAO on norepinephrine ( Figure 3). An early neuropathological feature of Alzheimer's disease is deposition of the protein Tau in neurons of the pontine locus ceruleus, which is the main source of norepinephrine in the brain. Kang et al. recently reported that DOPEGAL activates asparagine endopeptidase, which cleaves Tau and results in aggregation-and propagation-prone forms of the protein, leading to both locus ceruleus degeneration and spread of Tau pathology (Kang et al. 2020). The same groups has reported that DOPAL induces activation of asparagine endopeptidase (Kang et al. 2018). Intra-dermal DOPEGAL injection has also been reported to produce mechanical hyperalgesia in an animal model of alcoholic peripheral neuropathy (Dina et al. 2008).

Stress and the catecholaldehyde hypothesis
Catecholaminergic systems operate differently from other neurotransmitter systems in that the neurotransmission is relatively slow (Greengard 2001), and the sites of neurotransmitter release are not necessarily synaptic and can occur at varicosities along widely arborizing axons (Vizi 1982;van der Velden et al. 2017). Catecholamine neurons behave as if continuously in "idle," analogous to a bank robber's getaway car (Goldstein 2013). When needed, on top of this idling, there is augmented release in response to and even in anticipation of emergencies and activities of daily life.
One may ask what the value is of "leaky" vesicles that cause a high rate of turnover of catecholamines even without exocytotic release. Extending on the automotive analogy, an explanation offered by Eisenhofer is based on "gearing down" (Eisenhofer et al. 2004). In the human heart about 3/4 of norepinephrine turnover is due to intraneuronal metabolism of norepinephrine leaking from storage vesicles. Norepinephrine stores are in a highly dynamic equilibrium with the cytoplasm, and passive leakage is balanced by active uptake via the VMAT.
Having leaky stores may gear down the requirement for increases in catecholamine synthesis to match increases in catecholamine release and neurons with a capacity for more extended range of sustainable release rates in response to stress than would otherwise be possible.
Activation of the "central stress system," conceptualized to be embedded in the central autonomic network (Benarroch 1993;Goldstein 2020b), increases exocytotic release of catecholamines in the brain and periphery (Valentino et al. 2017;Carlson et al. 1968;Shanks et al. 1994). Most of neurotransmitter catecholamines are recycled by neuronal reuptake (Axelrod and Kopin 1969;Hertting and Axelrod 1961)  Although direct evidence is lacking, the results of several studies fit with this notion. In rats, repeated immobilization, which activates catecholaminergic neurons inside and outside the brain (Pacak et al. 1992(Pacak et al. , 1993bKvetnansky et al. 1992a, b), reduces the numbers of substantia nigra dopaminergic and locus ceruleus noradrenergic neurons (Sugama et al. 2016), as in PD (Zarow et al. 2003). Exposure to multiple, random unpredictable stressors exacerbates the loss of TH-positive neurons in the substantia nigra that is evoked by the neurotoxin 6hydroxydopamine (Hemmerle et al. 2014). In mice, chronic exposure to a mild stress paradigm augments neurotoxin-related neurodegeneration (Janakiraman et al. 2017). Chronic restraint (a severe stressor) promotes rotenone-induced neurotoxic effects in the brain, including loss of substantia nigra TH-positive cells and reduced striatal concentrations of dopamine (Dodiya et al. 2020). Rotenone alone did not cause overt nigral neuronal loss in this study.

MAO inhibitor trials and the catecholaldehyde hypothesis
Since MAO is required for DOPAL formation, a seemingly straightforward test of the catecholaldehyde hypothesis would be to assess whether MAO inhibitors slow the progression of PD; however, results of large multi-center trials of the MAO-B inhibitors deprenyl and rasagiline failed to demonstrate efficacy convincingly (Group 1996;Ward 1994;Fabbrini et al. 2012;Olanow et al. 2009;de la Fuente-Fernandez et al. 2010).
There are two potential explanations for this failure. One is that the subjects in these clinical trials already had symptomatic PD, and the neurodegenerative process might already have been advanced by the time their symptoms manifested clinically. There seems to be a long preclinical period during which catecholaminergic neurons are dysfunctional-we call this the "sick-before-dead" phenomenon (Lamotte et al. 2019). In at-risk individuals, cerebrospinal fluid indices of central dopamine deficiency predict the later development of PD (Goldstein et al. 2018b).
Second, as inspection of the steps in Figure 1 would predict, MAO inhibitors increase the spontaneous oxidation of cytoplasmic dopamine, as indicated by increased levels of Cys-DA Goldstein et al. 2016a). We call this the "MAOI tradeoff." The catecholaldehyde hypothesis has not yet been put to a correct test in humans.
The  Goldstein,p. 18 S in sympathetic noradrenergic nerves (Isonaka et al. 2019), an enriched enough population might be identified for efficient testing of the catecholaldehyde hypothesis.

Gaps in knowledge and goals for the future
Even cursory inspection of the concept diagrams in Figures 2-4 and 6 bring to mind gaps in knowledge and challenges that future research can address. This section frames the issues in terms of as yet unanswered-but answerable-questions.
( Although such modeling has identified specific sites of abnormal catecholamine synthesis, storage, and metabolism in Lewy body diseases , efforts to apply computational modeling so far have not incorporated homeostasis, allostatic load, genetics, aging, stress, and autotoxicity.

Conclusions
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 3 May 2021 doi:10.20944/preprints202105.0002.v1 Goldstein,p. 19 According to the catecholaldehyde hypothesis, the answers to the four questions that began this review are as follows. Catecholamine autotoxicity has long been considered a possible pathogenetic mechanism of catecholaminergic neurodegeneration in PD, based on the well known tendency of dopamine to oxidize spontaneously, resulting in the formation of several potentially toxic oxidation products. The catecholaldehyde hypothesis, which is based on enzymatic rather than spontaneous oxidation of cytoplasmic dopamine, is relatively new. It is hoped that this review will spur interest in further research on DOPAL-induced catecholamine autotoxicity for understanding mechanisms, identifying biomarkers, and devising novel treatment and prevention strategies for diseases that are posing an increasing public health burden as populations age.