The “Sick-but-not-Dead” Phenomenon Applied to Catecholamine Deficiency in Neurodegenerative Diseases

Abstract

The catecholamines dopamine and norepinephrine are key central neurotransmitters that participate in many neurobehavioral processes and disease states. Norepinephrine is also the main neurotransmitter mediating regulation of the circulation by the sympathetic nervous system. Several neurodegenerative disorders feature catecholamine deficiency. The most common is Parkinson’s disease (PD), in which putamen dopamine content is drastically reduced. PD also entails severely decreased myocardial norepinephrine content, a feature that characterizes two other Lewy body diseases—pure autonomic failure and dementia with Lewy bodies. It is widely presumed that tissue catecholamine depletion in these conditions results directly from loss of catecholaminergic neurons; however, as highlighted in this review, there are also important functional abnormalities in extant residual catecholaminergic neurons. We refer to this as the “sick-but-not-dead” phenomenon. The malfunctions include diminished dopamine biosynthesis via tyrosine hydroxylase (TH) and L-aromatic-amino-acid decarboxylase (LAAAD), inefficient vesicular sequestration of cytoplasmic catecholamines, and attenuated neuronal reuptake via cell membrane catecholamine transporters. A unifying explanation for catecholaminergic neurodegeneration is autotoxicity exerted by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate in cytoplasmic dopamine metabolism. In PD, putamen DOPAL is built up with respect to dopamine, associated with a vesicular storage defect and decreased aldehyde dehydrogenase activity. Probably via spontaneous oxidation, DOPAL potently oligomerizes and forms quinone-protein adducts with (“quinonizes”) α-synuclein (AS), a major constituent in Lewy bodies, and DOPAL-induced AS oligomers impede vesicular storage. DOPAL also quinonizes numerous intracellular proteins and inhibits enzymatic activities of TH and LAAAD. Treatments targeting DOPAL formation and oxidation therefore might rescue sick-but-not-dead catecholaminergic neurons in Lewy body diseases.

Dopamine and norepinephrine (NE) are catecholamines that play important roles as central neurotransmitters in movement, learning, memory, reward, attention, and distress.^1–3 Outside the brain, NE is the major neurotransmitter of the sympathetic nervous system in circulatory regulation.^4,5

Catecholamine Depletion in Neurodegenerative Diseases

Catecholamine depletion characterizes a variety of neurodegenerative diseases, the most common of which is Parkinson’s disease (PD). Other such conditions involving catecholamine deficiency in affected regions include pure autonomic failure (PAF),^6,7 multiple system atrophy (MSA),^8–10 and dementia with Lewy bodies (DLB).¹¹

PD features profoundly decreased dopamine content in the striatum^12,13—especially in the putamen.¹⁴ Frontal cortex normally contains relatively low dopamine concentrations that do not seem to be decreased in PD.¹⁵ Contents of NE in most brain areas are also decreased,^16–18 including in frontal cortex.¹⁵ In the locus ceruleus, the main source of NE in the brain, NE deficiency has been reported specifically in PD with dementia.¹⁹ Since frontal cortical noradrenergic innervation is derived from the locus ceruleus, there seems to be greater loss of NE at the level of the terminals than at the level of the neuronal cell bodies. It has been reported that in PD there is even greater loss of neurons in the locus ceruleus than in the substantia nigra.²⁰

Outside the brain, in PD there is very prominent loss of NE in the left ventricular myocardium.^21–23 The extent of decrease in PD—90 to 99%—is similar to that of putamen dopamine. Several in vivo neuroimaging studies have indicated that in PD noradrenergic deficiency is cardioselective^24–27; however, postmortem NE tissue contents in extracardiac regions in PD have not yet been reported.

PD is characterized pathologically by Lewy bodies, which are intraneuronal cytoplasmic inclusions that have particular light microscopic characteristics upon hematoxylin/eosin staining (►Fig. 1, left panel). Lewy bodies contain deposits of the protein α-synuclein (AS)²⁸ as well as of two proteins intimately involved with catecholaminergic functions—tyro-sine hydroxylase (TH)^29,30 (►Fig. 1, right panel), which is the rate-limiting enzyme in catecholamine biosynthesis, and the type 2 vesicular monoamine transporter (VMAT2),³¹ which is required for vesicular uptake of cytoplasmic catecholamines.

Fig. 1.

Lewy bodies. The left panel shows an intracellular Lewy body stained with hematoxylin/eosin from a patient with pure autonomic failure (PAF). The right panel shows an immunofluorescence microscopic image of an extracellular Lewy body and nearby Lewy neurite from sympathetic ganglion tissue of a patient with Parkinson’s disease and orthostatic hypotension. In the right panel, red corresponds to immunoreactive tyrosine hydroxylase (TH), green α-synuclein (AS), blue DAPI to stain nuclei, and yellow TH-AS colocalization. Note TH in the center of the Lewy body and colocalized TH and AS in the halo. The Lewy neurite contains AS. Scale bar 10 μm. (Image courtesy of R. Isonaka.)

Two other Lewy body diseases related to PD are PAF and DLB. All PAF patients have neurogenic orthostatic hypotension (nOH) without clinical signs or symptoms of central neurodegeneration.^32,33 PAF is a rare disease, and so the literature on Lewy bodies in PAF is sparse; however, all postmortem studies of PAF have noted Lewy bodies in the brain or sympathetic ganglion tissue.^34–38 PAF patients have AS deposition in sympathetic ganglion tissue^29,36,39 and sympathetic nerves.^40–43 Moreover, PAF can evolve into PD or DLB.⁴⁴ It is therefore reasonable to conceptualize that PAF is by definition a form of Lewy body disease. If so, then a patient with nOH and noradrenergic deficiency but lacking demonstrable Lewy bodies or AS in sympathetic nerves would not be considered to have PAF.^45,46

DLB patients also have AS deposition in Lewy bodies,⁴⁷ including in sympathetic ganglion tissue^29,48 and cutaneous sympathetic nerves.^43,48 The frequency of nOH in DLB seems to be intermediate between PAF and PD.⁴⁹

As in PD, in MSA there is drastic putamen dopamine deficiency.⁹ Catecholamine levels in other brain regions have not been reported; however, counts of catecholaminergic neurons are decreased in the A1 and C1 regions of the ventrolateral medulla.⁵⁰ Cerebrospinal fluid levels of homovanillic acid, the end-product of dopamine metabolism, are decreased in MSA,⁸ as are levels of 3,4-dihydroxyphenylglycol, the main neuronal metabolite of NE.⁵¹ Outside the brain, there is no evidence for generalized catecholamine deficiency in MSA,^6,7,52 although a minority of MSA patients do have neuroimaging or postmortem neuropathologic evidence of loss of cardiac noradrenergic nerves.^53–55

Functional Abnormalities in Residual Catecholaminergic Neurons: The “Sick-but-not-Dead” Phenomenon

In PD, the extent of loss of nigral dopaminergic neurons⁵⁶ or striatal dopaminergic innervation as indicated by immunoreactive TH⁵⁷ is far less than the extent of decrease in putamen dopamine content.¹⁴ How can there be a greater loss of a neurotransmitter than of the nerves that contain the neurotransmitter?

A potential resolution of this apparent paradox is decreased neurotransmitter synthesis, vesicular storage, or recycling in the residual terminals. Let us presume that the innervation is 20% of control, whereas the dopamine content is 4% of control. If the ability to sequester dopamine in vesicles in the residual terminals were 20% of control, then the tissue content of the neurotransmitter would be 20%× 20%=4% of control.

We have introduced the term “sick-but-not-dead” to refer to the occurrence of functional abnormalities in extant residual catecholaminergic neurons in diseases involving catecholaminergic neurodegeneration.²³ The following discussion describes examples of the sick-but-not-dead phenomenon in catecholaminergic neurons in the brain or heart in Lewy body diseases.

There is growing evidence for a vesicular storage defect in dopaminergic neurons in PD.^58–62 Decreased VMAT2 activity can precede loss of nigrostriatal dopaminergic neurons,⁶³ and mice with genetically determined low VMAT2 activity have aging-related denervation of central dopaminergic and noradrenergic neurons.^64,65 Meanwhile, animals with increased VMAT2 activity are relatively resistant to manipulations that produce catecholaminergic neurodegeneration.^64–69

Several in vivo and postmortem studies have indicated an analogous vesicular storage defect in residual myocardial sympathetic noradrenergic neurons in Lewy body diseases.^{21–23,59,61}

Another functional abnormality contributing to putamen dopamine deficiency in PD is decreased dopamine biosynthesis from 3,4-dihydroxyphenylalanine (DOPA) via L-aromatic-amino-acid decarboxylase (LAAAD).^9,70 Decreased LAAAD activity has also recently been reported in residual cardiac sympathetic nerves in PD.²³

A third functional abnormality in putamen dopaminergic terminals is decreased activity of aldehyde dehydrogenase (ALDH). ALDH is gaining increasing attention as a factor relevant to PD pathogenesis.^71–74 Postmortem neurochemical studies have reported decreased ALDH activity, based on low 3,4-dihydroxyphenylacetic acid (DOPAC)/3,4-dihydroxyphenylacetaldehyde (DOPAL) concentration ratios in putamen tissue from patients with PD or MSA.^75,76

ALDH deficiency promotes DOPAL accumulation,^77,78 because ALDH inhibition decreases the metabolism of cytoplasmic DOPAL. Animals with genetically determined low ALDH activity and DOPAL buildup have aging-related abnormalities resembling those in PD.⁷⁷ Meanwhile, animals with increased ALDH activity are relatively resistant to manipulations that produce dopaminergic neurodegeneration.⁷⁹ ALDH1A1, a marker of nigral dopaminergic neurons, seems to be protective.⁸⁰ Overexpression of ALDH1A1 reduces oxidation-induced toxicity in SH-SY5Y neuroblastoma cells.⁸¹ The metabolic stressor and pesticide rotenone, which produces an adult rat model of PD,^82–84 decreases ALDH activity indirectly by blocking mitochondrial complex 1; this decreases generation of NAD+, a required cofactor for ALDH. As one would predict from this effect, rotenone evokes DOPAL accumulation.^85,86 DOPAL is not normally detected in myocardial tissue (unpublished observations).

Several clinical pathophysiologic states are associated with myocardial NE depletion. These include heart failure,^87–92 myocardial infarction,^93,94 long-term diabetes,⁹⁵ and Lewy body diseases.^21,22,96

As for putamen dopamine, one might presume that myocardial NE depletion in Lewy body diseases reflects denervation; however, studies using immunoreactive TH as a marker of myocardial noradrenergic innervation have noted about a 75% average decrease in PD,^97–102 whereas the extent of decrease in tissue NE content is in the range of 90 to 99%.^21–23 The greater magnitude of NE depletion than of loss of sympathetic noradrenergic innervation indicates the sick-but-not-dead phenomenon in residual myocardial noradrenergic nerves in Lewy body diseases.

Numerous mechanisms might determine denervation-independent decreased NE stores in sympathetic nerves (►Fig. 2). These include decreased vesicular uptake of cytoplasmic catecholamines via the VMAT2⁶¹; increased vesicular permeability¹⁰³; decreased axonal transport of vesicles or vesicle-associated proteins¹⁰⁴; decreased enzymatic activities of TH,¹⁰⁵ LAAAD,¹⁰⁶ or vesicular dopamine-β-hydroxylase (DBH)¹⁰⁷; increased exocytotic release of vesicular NE¹⁰⁸; and decreased neuronal NE recycling via the uptake-1 process mediated by the cell membrane NE transporter (NET).¹⁰⁹

Fig. 2.

Sites of functional abnormalities of catecholamine synthesis, storage, release, recycling, and metabolism in myocardial sympathetic nerves in Lewy body diseases. Reactions are in italics and amounts of reactants in plain text. Font sizes correspond roughly to amounts of reactants. Green arrows indicate dopamine (DA) synthesis and blue arrows norepinephrine (NE) vesicular uptake and leakage. Red X marks placed to indicate sites of abnormalities in residual sympathetic nerves in Lewy body diseases. Application of a kinetic model to previously published data revealed three types of abnormal intraneuronal processes in the Lewy body disease group—(a) attenuated catecholamine biosynthesis via tyrosine hydroxylase and L-aromatic-amino-acid decarboxylase, (b) impaired vesicular sequestration of cytoplasmic catecholamines, reflecting the balance of vesicular uptake versus leakage, and (c) inefficient recycling of released NE by reuptake through the cell membrane NE transporter. ALDH, aldehyde dehydrogenase; AR, aldehyde/aldose reductase; Cys-DA, 5-S-cysteinylDA; Cys-DOPA, 5-S-cysteinyl DOPA; DAc, cytoplasmic DA; DBH, dopamine-β-hydroxylase; DHPG, 3,4-dihydroxyphenylglycol; DOPAc, cytoplasmic DOPA; DOPAC, 3,4-dihydroxyphenylacetic acid; DOPEGAL, 3,4-dihydroxyphenylglycolaldehyde; DOPAL, 3,4-dihydroxyphenylacetaldehyde; DOPET, 3,4-dihydroxyphenylethanol; EPI, epinephrine; LAAAD, L-aromatic-amino-acid decarboxylase; MAO, monoamine oxidase; NEc, cytoplasmic NE; NEe, NE in the extracellular fluid; NESO, NE entering the cardiac venous drainage; TH, tyrosine hydroxylase; TYR, tyrosine; TYRc, cytoplasmic TYR; U1 = Uptake-1, neuronal uptake; U2 = Uptake-2, extraneuronal uptake; VMAT, vesicular monoamine transporter.²³

Until recently there was little information about which of these processes are affected in clinical disease states. Since NE is synthesized withinvesicles by DBHacting on dopamine after vesicular uptake, it is likely that a previously reported finding of an increased tissue dopamine/NE ratio in preterminal idiopathic dilated cardiomyopathy reflected decreased vesicular uptake rather than the inferred decrease in DBH enzyme activity.⁸⁸

We recently used a computational modeling approach to assess comprehensively all the known pathways of NE synthesis, storage, release, reuptake, and metabolism in cardiac sympathetic nerves in Lewy body diseases.²³ Application of a novel kinetic model identified a pattern of dysfunctional steps contributing to NE deficiency. The model identified low rate constants for three types of processes in the Lewy body group—catecholamine biosynthesis via TH and LAAAD, vesicular storage of dopamine and NE, and neuronal NE reuptake via the cell membrane NET (►Fig. 2). Postmortem catechols and catechol ratios confirmed this triad of model-predicted functional abnormalities. Therefore, denervation-independent impairments of neurotransmitter biosynthesis, vesicular sequestration, and NE recycling contribute to the myocardial NE deficiency attending Lewy body diseases.

Catecholamine Autotoxicity and the Catecholaldehyde Hypothesis

Is there a single common cause for the pattern of functional abnormalities in catecholaminergic neurons in Lewy body diseases? Oxidative stress or decreased mitochondrial energy generation would seem likely culprits^110,111; however, for LAAAD to catalyze dopamine synthesis from DOPA requires neither oxygen nor energy, yet LAAAD activity is substantially decreased. Widespread oxidative stress or deficient mitochondrial energy generation would not account easily for the syndromic nature of Lewy body diseases, nor the relatively selective, profound catecholamine deficiencies found in the putamen and heart compared with other regions receiving catecholaminergic innervation.

The catecholamine autotoxicity theory imputes pathologic interactions between catecholamine oxidation products and intracellular proteins in the pathogenesis of diseases involving catecholaminergic neurodegeneration.¹¹²

Many studies have implicated dopamine as an autotoxin. Most have focused on its spontaneous oxidation to dopamine-quinone and then a variety of distal oxidation products^113–139 (►Fig. 3). Some of these are known to be neurotoxic, such as aminochrome,^120,140,141 5-S-cysteinyldopamine,^139,142 and isoquinolines.^143,144 These compounds seem to have in common that they evoke mitochondrial dysfunction.¹²⁷

Fig. 3.

Overview of the sources and fate of intraneuronal catecholamines, with emphasis on spontaneous and enzymatic oxidation of catechols. Dopamine (DA) is synthesized in the neuronal cytoplasmic via tyrosine hydroxylase (TH) acting on tyrosine to form 3,4-dihydroxyphenylalanine (DOPA) and then L-aromatic-amino-acid decarboxylase (LAAAD) acting on DOPA. Most of cytoplasmic DA is taken up into vesicles via the vesicular monoamine transporter (VMAT). Dopamine-β-hydroxylase (DBH) in the vesicles catalyzes the conversion of DA to norepinephrine (NE). DA and NE in the cytoplasm are subject to oxidative deamination catalyzed by monoamine oxidase-A (MAO-A) in the outer mitochondrial membrane to form 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL). DOPAL is converted to 3,4-dihydroxyphenylacetic acid (DOPAC) via aldehyde dehydrogenase (ALDH), and DOPEGAL is converted to 3,4-dihydroxyphenylglycol (DHPG) via aldehyde/aldose reductase (AR). Most of vesicular NE released by exocytosis is taken up into the cytoplasm via the cell membrane NE transporter (NET). DOPA can undergo spontaneous oxidation to DOPA-quinone (DOPA-Q), resulting in formation of 5-S-cysteinylDOPA (Cys-DOPA), and DA can undergo spontaneous oxidation to DA-quinone (DA-Q), resulting in formation of 5-S-cysteinylDA (Cys-DA).

An almost completely independent line of research has centered on DOPAL. DOPAL, an obligate intermediate in neuronal DA metabolism, is formed from the enzymatic oxidation of cytoplasmic dopamine by MAO.

DOPAL is toxic both in vitro and in vivo^145–148 and is the centerpiece of the catecholaldehyde hypothesis. According to the catecholaldehyde hypothesis, DOPAL buildup causes or contributes to the death of catecholaminergic neurons in neurodegenerative diseases such as PD.¹⁴⁸ DOPAL is far more toxic in vivo than is dopamine or its oxidized, reduced, or methylated metabolites.¹⁴⁹ At concentrations as low as 100 ng, injected DOPAL destroys substantia nigra dopaminergic neurons.¹⁴⁹ In energetically compromised mitochondria from PC12 cells, DOPAL induces the permeability transition pore, a harbinger of cell death.¹⁵⁰

The pesticide rotenone, by inhibiting mitochondrial complex 1, decreases formation of NAD+, which is required for ALDH activity. Rotenone therefore builds up DOPAL.⁸⁶ Some of rotenone-induced cytotoxicity in catecholamine-producing cells depends on DOPAL.¹⁵¹ Fungicides inhibit ALDH,^78,152 and thefungicide benomyl blocks ALDHand builds up DOPAL.⁷⁸

The enzymatic oxidative deamination of cytoplasmic dopamine by MAO results in concurrent equimolar formation of hydrogen peroxide and DOPAL. Reaction of DOPAL with hydrogen peroxide yields highly toxic hydroxyl radicals,¹⁵³ and DOPAL oxidation results in formation of superoxide.¹⁵⁴

When DOPAL is incubated with rat brain homogenates, the aldehyde disappears, independent of metabolic enzymes.¹⁵⁵ This finding raises the possibility that DOPAL reacts with proteins, a suspicion that recently has been confirmed amply.¹⁵⁶ DOPAL forms covalent quinone adducts with (“quinonizes”) many PD-related proteins, including TH, LAAAD, VMAT2, glucocerebrosidase, and AS.¹⁵⁶ Quinonization may interfere with the functions of these proteins and thereby with numerous intracellular processes. Thus, DOPAL inhibits activities of TH,^105,157 LAAAD,¹⁵⁶ and ALDH,¹⁵⁸ although whether these effects depend on quinonization is incompletely understood.

Interactions of Dopamine Oxidation Products with α-Synuclein

The catecholamine autotoxicity theory imputes pathologic interactions between catecholamine oxidation products and intracellular proteins in the pathogenesis of diseases involving catecholaminergic neurodegeneration.¹¹²

Oxidized dopamine can interact with AS¹¹⁷ and promote the formation of AS oligomers.^121,129,159 Moreover, aminochrome and 5,6-dihydroxyindole, which are products of dopamine oxidation, can oligomerize AS.^160–162 Most investigations on this topic have not considered the possibility that dopamine-dependent AS oligomerization depends on production of DOPAL from dopamine.^117,121,129

DOPAL potently reacts with AS,^156,163 converting the protein to oligomeric forms^163–167 that potentially are toxic.^168–170 DOPAL-induced AS oligomers impede vesicular functions.¹⁰³ Divalent metal cations—especially Cu(II)—augment DOPAL-induced AS oligomerization.¹⁷¹ Moreover, DOPAL quinonizes AS, probably following spontaneous oxidation of DOPAL to DOPAL-quinone.¹⁵⁶ DOPAL also enhances AS-induced inhibitory effects on tyrosine receptor kinase B, a receptor for brain-derived neurotrophic factor.¹⁷² Meanwhile, AS itself inhibits LAAAD.¹⁰⁶

The literature on dopamine oxidation and synucleinopathy has generally overlooked DOPAL,^{121,137,138,173–176} and the literature on DOPAL and synucleinopathy has generally over-looked dopamine and its spontaneous oxidation to dopamine-quinone.^{156,163,164,177} In the few studies where DOPAL and dopamine have been compared directly in terms of oligomerizing AS, DOPAL has been found to be more potent.^156,163,171

We recently compared DOPAL and dopamine in terms of (1) AS oligomerization and quinonization in test tube experiments; (2) enhancing effects of Cu(II)^171,178 and mitigating effects of antioxidation with N-acetylcysteine (NAC)^{134,156,179,180}; and (3) quinonization of intracellular proteins in cultured cells.^134,156 We found that DOPAL is far more potent than dopamine in both oligomerizing and quinonizing AS.¹⁸¹ Dopamine oxidation evoked by Cu(II) or tyrosinase does not quinonize AS. In cultured MO3.13 human oligodendrocytes, DOPAL, but not dopamine, results in the formation of numerous intracellular quinoproteins that can be visualized by near-infrared microscopy. Therefore, of the two routes by which oxidation of dopamine modifies AS and other proteins (►Fig. 4), that via DOPAL is more prominent.

Fig. 4.

Alternative routes by which dopamine (DA) oxidation may modify α-synuclein. Most of cytoplasmic DA is taken up into vesicles via the vesicular monoamine transporter (VMAT), but a minority undergoes oxidation, by two routes (red numbers in boxes). In route 1, DA is oxidized to form DA-quinone (DA-Q), with subsequent interactions with α-synuclein directly or via various further products of DA-Q, including 5-S-cysteinyldopamine (Cys-DA). In route 2, DA is oxidized enzymatically by monoamine oxidase-A (MAO-A) in the outer mitochondrial membrane to form 3,4-dihydroxyphenylacetaldehyde (DOPAL) and hydrogen peroxide (H2O2). Cu(II) promotes the oxidation of DA and DOPAL. Formation of DA-Q and DOPAL-Q is associated with generation of superoxide radicals (O2−•). DOPAL is metabolized by aldehyde dehydrogenase (ALDH) to form 3,4-dihydroxyphenylacetic acid (DOPAC), which exits the cell.

The differences in potencies of DOPAL and dopamine in oligomerizing and quinonizing AS can be explained by their different chemical structures.¹⁸² Whereas dopamine has a terminal amine group, DOPAL has a reactive aldehyde group that can bind covalently to lysine residues,¹⁸⁰ which are abundant in the AS molecule.^103,164,182 Occupation of lysine residues completely prevents DOPAL-induced oligomerization and quinonization of AS.¹⁵⁶

A specific mechanism has been proposed relating DOPAL oxidation to AS oligomerization. Superoxide, which is generated pari passu with the oxidation of DOPAL, propagates a chain reaction oxidation resulting in a dicatechol pyrrole adduct with lysine (dicatechol pyrrole lysine, DCPL).¹⁶⁵ The same investigators have reported that auto-oxidation of the catechol rings in DCPL produces an intermediate dicatechol isoindole lysine (DCIL) product formed by an intramolecular reaction of the two catechol rings, yielding an unstable tetracyclic structure. DCIL then reacts with a second DCIL to give a dimeric, di-DCIL. DOPAL-catalyzed formation of AS oligomers may therefore be separable into two steps, the first involving generation of DCPL and the second crosslinking of AS molecules via the interadduct reaction.

Potential Vicious Cycles Involving DOPAL

DOPAL-synuclein interactions might lower thresholds for induction of vicious cycles that threaten neuronal homeostasis. First, synucleinopathy impairs vesicular functions.^31,183–188 In particular, DOPAL-induced AS oligomers permeabilize vesicles,¹⁰³ which would interfere with vesicular sequestration,¹⁰³ diverting the fate of cytoplasmic dopamine toward DOPAL.²¹

Second, when MAO acts on cytoplasmic dopamine, hydrogen peroxide and DOPAL are produced concurrently. In the setting of divalent metal cations this generates hydroxyl radicals,¹⁵³ which peroxidate lipid membranes. The lipid peroxidation products inhibit ALDH, and this builds up DOPAL.¹⁸⁹ Indeed, DOPAL may inhibit its own detoxification by ALDH.¹⁵⁸

Third, DOPAL evokes mitochondrial dysfunction,¹⁵⁰ and mitochondrial dysfunction decreases adenosine triphosphate availability; this decreases the efficiency of the proton pump that is required for vesicular storage and builds up DOPAL. Moreover, DOPAL-oligomerized AS enhances the mitochondrial inhibition exerted by DOPAL.¹⁹⁰

The “Smoking Gun”

What links AS with catecholamine deficiency in Lewy body diseases? A straightforward explanation is toxic effects of AS within catecholaminergic neurons. The finding that in Lewy body diseases immunoreactive AS can be present in the same nerve fibers that contain immunoreactive TH seems like a “smoking gun” implicating intraneuronal AS in the death of catecholaminergic neurons.^29,191 We have validated methodology to quantify AS colocalization with TH, a marker of catecholaminergic innervation, and assessed associations of AS-TH colocalization with myocardial NE content and cardiac sympathetic neuroimaging data in patients with nOH. Ganglionic AS-TH colocalization indices are higher and myocardial NE lower in Lewy body than in non-Lewy body nOH.¹⁹² Lewy body nOH is associated with both increased AS-TH colocalization indices in skin biopsies and decreased myocardial 18F-dopamine-derived radioactivity.¹⁹² In this study, all Lewy body nOH patients had elevated colocalization indices in skin biopsies and decreased 18F-dopamine-derived radioactivity, a combination not seen in non-Lewy body nOH patients. Thus, in Lewy body nOH AS deposition in sympathetic noradrenergic nerves is related to in vivo neuroimaging evidence of myocardial noradrenergic deficiency. This association fits with the view that intraneuronal AS deposition plays a pathophysiological role in the myocardial sympathetic neurodegeneration attending Lewy body nOH. Whether intraneuronal AS deposition is associated with NE deficiency in extracardiac tissues has not yet been reported.

Treatment Implications of the Sick-but-not-Dead Phenomenon and Catecholaldehyde Hypothesis

There is no way to treat neurons that are dead; however, sick-but-not-dead neurons might be salvageable. As noted above, both DOPAL and AS inhibit LAAAD. LAAAD therefore offers a promising target for a form of gene therapy to increase LAAAD levels via an adeno-associated virus. This technology is being applied in PD.^193,194

The catecholaldehyde hypothesis predicts that MAO inhibition to decrease DOPAL formation combined with an antioxidant to decrease DOPAL-quinone (DOPAL-Q) formation from DOPAL should prevent autotoxicity from DOPAL-AS interactions and thereby slow catecholaminergic neurodegeneration (►Fig. 5). MAO inhibition alone might not suffice, because although MAO inhibition decreases DOPAL formation,¹⁹⁵ concurrently there is increased formation of dopamine oxidation products, and the oxidation products are toxic—the “MAOI tradeoff.”¹⁹⁵ Concurrent treatment with the antioxidant N-acetylcysteine (NAC) prevents the increase in endogenous Cys-DA levels exerted by the MAO-B inhibitor selegiline without interfering with the decrease in DOPAL levels.¹⁷⁹ NAC also mitigates the protein modification exerted by DOPAL, including oligomerization and quinonization of AS.¹⁵⁶

Fig. 5.

Sites of action of a combination of a monoamine oxidase inhibitor (MAOI) and N-acetylcysteine (NAC) in testing the catecholaldehyde hypothesis. Cytoplasmic DA oxidizes spontaneously to DA-quinone (DA-Q) and then several oxidation products including aminochrome and 5-S-cysteinyldopamine (Cys-DA). DOPAL oxidizes spontaneously to DOPAL-quinone (DOPAL-Q). DA oxidation products are toxic, via mitochondrial and other lesions. DOPAL reacts with hydrogen peroxide and divalent metal cations to form hydroxyl radicals, which peroxidate membrane lipids. The lipid peroxidation products 4-hydroxynonenal and malondialdehyde inhibit ALDH. DOPAL, probably via oxidation to DOPAL-Q, oligomerizes and forms quinoprotein adducts with (“quinonizes”) α-synuclein. DOPAL-induced synucleinopathy impedes vesicular functions. According to the “catecholaldehyde hypothesis,” interactions of DOPAL and α-synuclein set the stage for vicious cycles that challenge homeostasis in catecholaminergic neurons.

Many clinical trials of NAC have been done or are ongoing.¹⁹⁶ The results from a recently completed uncontrolled, unblinded clinical trial support the utility of an oral and intravenous dosing regimen¹⁹⁷ in motor PD. In humans selegiline also inhibits MAO-A in the brain.¹⁹⁸ This is relevant, as MAO-A is the isoform in dopaminergic neurons. Combining NAC with selegiline might protect sick-but-not-dead central dopaminergic and cardiac noradrenergic neurons and test the catecholaldehyde hypothesis for the pathogenesis of PD.

The advantages of MAOI + NAC treatment are that (1) it is likely to be safe, (2) there is a strong hypothesis-driven rationale predicting synergism that is supported by preclinical data, and (3) neurochemical and neuroimaging surrogate biomarkers exist to track the functional status of central and peripheral catecholaminergic neurons. The main weaknesses are that (1) neither treatment is novel; (2) large MAOI trials have failed to slow symptomatic progression of PD¹⁹⁹; (3) the bioavailability of NAC at central dopaminergic neurons is unclear; (4) the catecholaldehyde hypothesis so far has not gained traction, although the situation may be changing²⁰⁰; (5) drugs cannot target disease mechanisms specifically; and (6) the field seems to have moved on from focusing on dopamine deficiency to emphasizing genetic, exotoxic, mitochondrial, inflammatory, microbiome, lysosome, proteasome, and exosome theories that seem distantly related to catecholamine metabolism.

Reviving the view that PD fundamentally involves catecholamine deficiencies in the brain and autonomic nervous system and that those deficiencies are due to autotoxicity involving catecholamine oxidation products, thereby rationalizing MAOI + NAC treatment, seems to be an uphill battle. It is hoped that recognition of the sick-but-not-dead concept in diseases involving catecholaminergic neurodegeneration will be a first step in the ascent.