Neuronal vulnerability, pathogenesis, and Parkinson's disease

Neuronal Vulnerability, Pathogenesis, and Parkinson’s Disease

David Sulzer, Ph.D.1* and D. James Surmeier, Ph.D.2

1Departments of Psychiatry, Neurology, and Pharmacology, Columbia University, New York, New York, USA2Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois USA

ABSTRACT: Although there have been significantadvances, pathogenesis in Parkinson’s disease (PD) isstill poorly understood. Potential clues about pathoge-nesis that have not been systematically pursued aresuggested by the restricted pattern of neuronal pathol-ogy in the disease. In addition to dopaminergic neu-rons in the substantia nigra pars compacta (SNc), asignificant number of other central and peripheral neu-ronal populations exhibit Lewy pathology (LP), pheno-typic dysregulation, or frank degeneration in PDpatients. Drawing on this literature, there appear to bea small number of risk factors contributing to vulner-ability. These include autonomous activity, broad

action potentials, low intrinsic calcium-bufferingcapacity, poorly myelinated long highly branchedaxons and terminal fields, and use of a monoamineneurotransmitter, often with the catecholamine-derivedneuromelanin pigment. Of these phenotypic traits, onlythe physiological ones appear to provide a reachabletherapeutic target at present. VC 2012 Movement Disor-der Society

Key Words: substantia nigra; locus coeruleus; alpha-synuclein; medulla; Lewy body; pedunculopontine; neu-romelanin; enteric; calcium channel; catecholamines;acetylcholine

Although the loss of substantia nigra (SN) dopami-nergic (DA) neurons in Parkinson’s disease (PD) is re-sponsible for its core motor symptoms,1 a variety ofother neurons exhibit signs of pathology in postmor-tem analysis. Indeed, on the basis of symptoms andautopsy results, James Parkinson reasoned that the dis-ease was caused by a primary lesion in the medulla,stating, ‘‘From the impediment to speech, the difficultyin mastication and swallowing, the inability to retain,or freely to eject the saliva, may with propriety beinferred an extension of the morbid change upwardsthrough the medulla spinalis to the medulla oblongata,necessarily impairing the powers of the several nervesderived from that portion into which the morbidchange may have reached.’’2

Fritz Jakob Lewy identified intracellular proteinaggregates in the dorsal motor nucleus of the vagus(DMV), a region within the medulla oblongata, in thebrains of PD patients.3,4 These aggregates becameknown as Lewy bodies (LBs) and Lewy neurites(LNs). The discovery that alpha-synuclein (aSYN) is amajor component of LP has enabled the use of immu-nocytochemical approaches to characterize theseaggregates, which are now often labeled together asLewy pathology (LP). With this assay, pathologistsand the anatomist Heiko Braak have mapped LP inpostmortem samples from PD patients.5

These studies provide important insights, but severalcaveats should be noted. First, the relationshipbetween LP and neuronal dysfunction and death isuncertain. In fact, the existence of LP in a particularcell type could reflect a successful response to proteo-static stress and not a loss of function.6,7 Second, LPis found in other synucleinopathies that have an uncer-tain relationship to PD, particularly patients diagnosedwith dementia with Lewy bodies (DLB) and incidentalLewy body disease (iLBD), the latter of which iswidely suspected by anatomists to be an earlier stageof PD or DLB. Patients diagnosed with multiple sys-tem atrophy (MSA) also show an abnormal aSYNlabel, although the cell types and their distributionpattern, including a virtual lack of peripheral and

------------------------------------------------------------*Correspondence to: Dr. David Sulzer, 650 West 168 St., Black 305,New York, NY 10032, USA; [email protected]

Funding agencies: This work was supported by grants from the JPBFoundation (to D.S. and D.J.S.), NIH (P50NS047085; to D.J.S.), NIH(P50NS38370; to D.S.), the Hartman Foundation (to D.J.S.), and theParkinson’s Disease Foundation (to D.S.).Relevant conflicts of interest/financial disclosures: Nothing to report.Full financial disclosures and author roles may be found in the onlineversion of this article.

Received: 18 April 2012; Revised: 11 May 2012; Accepted: 14 May2012Published online in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/mds.25095

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enteric nervous system pathology, differ from PD,DLB, and iLBD.8 Pathogenesis in these diseasesundoubtedly differs from that in PD, at least in somekey respects, and it appears that aSYN aggregation isreachable by multiple paths. This uncertainty alsomakes it difficult to draw firm conclusions about therelationship between aSYN aggregation in a particulargroup of neurons and PD,9 as patients with PD couldalso be suffering to varying extents from other diseasesthat have such sequelae.In contrast to LP maps, cell loss maps in postmor-

tem PD samples using modern stereologicalapproaches have been rare. This is unfortunatebecause it would allow for a more definitive correla-tion to be established between symptoms and diseasetargets. What is known will be reviewed below.

Vulnerable NeuronalPopulations in PD

Peripheral Nervous System

The peripheral nervous system can be divided into 3parts: sensory, motor, and autonomic. Peripheral sen-sory and motor neurons do not display LP or signs ofdegeneration in PD patients (with the possible excep-tion of the sensory lingual nerve, mentioned below).In contrast, there are signs of pathology in the auto-nomic nervous system (ANS). There are sympatheticand parasympathetic divisions, each of which has pre-ganglionic and postganglionic components.Preganglionic sympathetic neurons arising from the

ventral spinal cord as a rule release acetylcholine andpostganglionic sympathetic neurons release norepi-nephrine (NE). The postganglionic sympathetic neu-rons that innervate sweat glands and releaseacetylcholine had been considered an exception in thatthey release acetylcholine; however, recent work dem-onstrated that a subset of these neurons can expresstyrosine hydroxylase (TH) and the CNS vesicularmonoamine transporter (VMAT2) in human and soare suggested to corelease NE.10

Preganglionic parasympathetic neurons arise fromneurons in the brain stem nuclei, including the DMVand neurons in the sacral spinal cord. These neurons arecholinergic. Postganglionic parasympathetic neuronsare typically in ganglia close to or embedded in the targetorgan. These neurons are largely cholinergic as well, butsome postganglionic parasympathetic neurons expressproteins associated with NE release as well as proteinsassociated with acetylcholine release.10 These neuronsare found in the heart, skin, and salivary glands.In addition to the sympathetic and parasympathetic

neuron of the ANS, there are intrinsic neurons foundin target organs that provide local regulation of func-tion. The largest component of this intrinsic divisionof the ANS is the enteric nervous system (ENS), which

is embedded in the gastrointestinal tract. ENS neuronscan be divided into myenteric (Auerbach’s) or submu-cosal (Meissner’s) plexuses. The myenteric plexusesare located between the inner and outer layers of themuscular layer (muscularis externa) and the submuco-sal plexuses in the adjacent submucosa. ENS neuronshave a wide range of transmitter phenotypes, includ-ing cholinergic, serotonergic (most of the serotonin inthe body is found in the gut11), noradrenergic, and do-paminergic types.12

Enteric Neurons

The ENS displays LP in PD patients, and the origi-nal characterization of PD noted severe constipationin many PD patients.2 It is now widely thought that aloss of DA neurons in the PD myenteric plexus under-lies constipation.13 Enteric LBs were first identified inneurons of the esophagus or colon in 2 of 22 Parkin-son’s disease patients, both of whom also had dyspha-gia; 2 of 8 patients with a primary diagnosis ofdifficulty in swallowing (achalasia) also exhibited LBsin degenerating ganglionic cells of the esophagealmyenteric plexus,14 and it is possible that the achala-sia patients had PD. LP in the enteric neurons of thealimentary tract of a PD patient was confirmed byCote and colleagues.15 Wakabayashi and colleaguesreported LP in both the myenteric and submucosal LPof all 7 PD patients studied, but also in 8 of 24 con-trol age-matched subjects.16 A follow-up study of 3PD patients from that group showed that although inthe sympathetic system, nearly all LBs were found incatecholamine cells, in the alimentary tract, LBs weremostly found in nondopaminergic VIP-secreting neu-rons of the myenteric plexus without accompanyingloss of those neurons.17 However, that conclusion hasbeen challenged in part by recent work on colonicbiopsies from PD patients using phosphorylated alpha-synuclein and TH immunolabel.18 In that study, 60%of neurons of the myenteric plexus with LP wereTHþ, and most of those were also immunolabeled fordopamine-beta hydroxylase and were thus NE neu-rons. It is possible that the 40% of LP neurons thatapparently lacked TH had low expression. There havealso been reports of LP in innervation and regionsproximal to the stomach.16,19

Most of the early reports found a similar distribu-tion and number of THþ cells in the myenteric andsubmucosal plexuses in PD patients and controls,which was interpreted as evidence against overt deathof catecholamine neurons. This view has been chal-lenged by Chandar Singaram and colleagues, whoused a DA-specific monoclonal antibody to demon-strate that the number of DA neurons in the plexusdecreased by more than 90% in 9 of 11 PD patients.20

There was in addition a very striking loss of DA neu-rites in PD enteric tissue, and DA levels, measured in

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the nerve ending regions of the muscularis externa,decreased by 70% (note that DA is also present in NEneurons). There appear to be a large fraction of DAenteric neurons in humans.12,21

These studies show that ENS LP is a frequent butnot necessary concomitant of PD. They also suggestthat DA ENS neurons display LP and can be lost inPD. However, it is not clear whether other transmitterphenotypes such as serotonergic neurons display LPand are lost in PD. Note, in addition, that althoughgastrointestinal dysfunction is common in PDpatients,22 it is also common among aged individualswithout any sign of parkinsonism.23

Sympathetic and Parasympathetic Neurons

The principal divisions of the ANS are also reportedto be affected in PD patients. Orthostatic hypotensionoccurs in a significant fraction of late-stage PDpatients. This symptom has been attributed to a dropin cardiac noradrenaline (NE) innervation.22,24 How-ever, more generalized sympathetic denervation thatimpairs vasogenic reflexes that maintain blood pres-sure could also be a factor.24 In 4 PD patients, therewas profound loss of both TH immunolabel and neu-rofilament immunolabel compared with controls.25

This report was corroborated by another small studyof PD patients.26 Although the loss of TH might inpart reflect loss of phenotypic dysregulation, theextensive loss of neurofilaments suggests that neuronswere lost in these patients. This conclusion is consist-ent with a decreased number of unmyelinated axonsin cardiac sympathetic nerves of PD patients.27 Arecent study found that in 4 of 5 PD patients, allclasses of postganglionic sympathetic NE neurons thatinnervate the heart, including the left and right ven-tricles, the right atrium, and the sinus node, werereduced; this apparent neuronal loss was found in allPD patients who exhibited orthostatic hypotension.28

Three of these patients showed LP and axonal beadingin NE neurons. Loss of cardiac sympathetic NE inner-vation is seen in some iLBD patients but not others.26

In a number of PD and DLB patients, LP is presentin the peripheral vagal nerve and ganglia.29,30 Theseaxons originate in the DMV. There have been reportsof strong LP in the DMV preganglionic parasympa-thetic neurons of nearly all PD patients studied (seebelow). The DMV projects to many areas, includingthe gastrointestinal tract.A subset of parasympathetic neurons in the intrinsic

cardiac ganglia also appear to be at risk in PD.28

These parasympathetic neurons are classically consid-ered cholinergic, but a subset in human were reportedto express VMAT2, suggesting they might coreleaseNE.10 The parasympathetic inferior salivatory nucleus(ninth cranial nerve), which innervates the parotidgland, also features LP in PD,30,31 but neuronal num-

ber even in control patients remains unknown (Kellydel Tredici, personal communication).

Salivary Glands

Reduced salivary secretion (sialopenia; dry mouth)can accompany PD. The submandibular gland (themajor source of saliva) is innervated by parasympa-thetic postganglionic neurons of the submandibularganglion and by sympathetic postganglionic neuronsof the superior cervical ganglion. In PD patients, LPhas been seen in both the submandibular gland andthe superior cervical ganglion.30 Inferior to the sub-mandibular gland within the perviascular stroma, 1 of2 parasympathetic lingual nerves, which are not con-sidered parasympathetic but rather have sensory func-tions, showed LP. Within the superior cervicalganglion, LP was present in all cases, including thecell bodies of the large postganglionic neurons of PDpatients. We are not aware of reports of neuronal lossthere.

Skin

The unmyelinated neurites of the skin are NE-releas-ing sympathetic postganglionic neurons that innervateblood vessels and other structures including sweatglands, which, as above, were previously thought torelease only acetylcholine (ACh) but are now thoughtto also release NE in humans.10 Djaldetti and col-leagues found marked denervation of all autonomicneurites in skin,32 work supported by analyses of skinbiopsies from PD patients.22,33

Adrenal Gland

Although they are not considered neurons, chromaf-fin cells of the adrenal medulla were the first cellsshown to secrete catecholamines (hence, the termadrenaline). den Hartog Jager found that PD patientsdisplay large inclusion bodies in these cells, which hecalled ‘‘adrenal bodies.’’34 LP was subsequentlydescribed as present in adrenal neurons.35 However,later work indicated that adrenal chromaffin cellsthemselves did not show LP, and the adrenal bodiesdescribed by den Jager were not immunoreactive forSYN.35 In contrast, postganglionic sympathetic neu-rons innervating the adrenal gland exhibited LP inapproximately 30% of PD patients,35 although theTH innervation of the adrenal appeared to be normal.

Summary

LP is found in several types of peripheral neurons inPD. The only neurons known to be lost in PD are NEneurons innervating the heart and skin and DA neu-rons of the ENS: the loss of these neurons might be re-sponsible for orthostatic hypotension, sweating

N E U R O N A L V U L N E R A B I L I T Y , P A T H O G E N E S I S , A N D P D


(hyperhydosis), and constipation, which commonlyaccompany PD.

Central Nervous System

The vast majority of the neurons lost or displayingsigns of pathology in early- and mid-stage PD patientsare found in the brain stem. LP and cell loss havebeen reported in the region of the DMV, the medul-lary reticular formation, the raphe nuclei (RN), thelocus coeruleus (LC), the pedunculopontine nuclei(PPN), the substantia nigra pars compacta (SNc), and,to a lesser extent, the ventral tegmental area (VTA)and retrorubral area (RRA). The evidence for theinvolvement of these nuclei will be discussed in turn.SNc and LC neurons that have high levels of neuro-

melanin (NM) pigment appear to be selectively tar-geted. Preganglionic neurons of the DMV do notcontain neuromelanin, but some neurons in regionsneighboring the DMV contain NM, and these aresometimes found to express LP.31 NM is a derivativeof oxidized catecholamine that reacts with lipids andproteins and acts as a reservoir that avidly traps iron,metals, and other toxic substances; NM is sequesteredand accumulated throughout life in autophagic organ-elles in neuronal cell bodies,36 apparently as a neuro-protective response. Although most obvious in theSNc and LC, NM is found in other central catechol-amine neurons37 and by more sensitive chemical meth-ods in additional noncatecholaminergic neurons.38,39

NM autophagic organelles are essentially identical tolipofuscin (LF) organelles, a different pigment derivedfrom oxidized lipids and proteins that accumulatethroughout life within these organelles, save for thecatechol-derived pigments and substances that accrueto them.40 Hence, the presence of NM indicates a his-tory of cytosolic oxyradical formation from catechol-amines, whereas the presence of LF indicates a historyof reactive lipid derivatives. Current research suggeststhat neurons cannot degrade NM and that loss of NMdepends first on cell death, which produces extra-neuronal NM deposits, followed by hydrogen perox-ide–mediated degradation following phagocytosis bymicroglia41; remarkably, this was theorized on the ba-sis of neuropathological observations by Foix and Nic-olesco (1925). The presence of extraneuronal NMremnants and the disappearance of NM neurons there-fore indicates that the neurons have been lost.

DMV

As mentioned, James Parkinson theorized that dam-age to the medulla caused the disease. The caudal me-dulla oblongata includes a number of nucleiimplicated in PD, including some that exhibit NM andLF pigmented areas.37,42 The DMV was one of thefirst nuclei found by Fritz Lewy to express LB.3 TheNM neurons in this region do not appear to be para-

sympathetic preganglionic fibers that project via thevagus nerve. Axons entering the vagus nerve from theDMV can have strong LP,43 whereas LP in the DMVhas been reported in essentially all PD patients exam-ined,31 although other reports found exceptions.44,45

Additional neurons in this region have some variabledegree of LP.31

Neuronal counts demonstrate that DMV neuronsare lost in PD,46 but there are differences in the litera-ture regarding which neurons are lost, probablybecause of differences in stages of the disease. Classi-cal studies reported NM-containing neurons in theDMV region were lost in PD brains, including theoriginal report,47 which stated that ‘‘the pigmentedcells of the dorsal vagal nucleus also degenerated, of-ten with vacuolation, by contrast with the non-pig-mented cells in this nucleus which remained healthy.’’Although this was subsequently reported in a numberof other studies,48 Eadie himself did not confirm thisand reported that motoneurons were lost, consistentwith a later study.46 Saper and colleagues noted thatsome of the most heavily NM-pigmented neurons sur-vive the disease.49 Braak and collaborators42 suggestedthat the preganglionic parasympathetic projection neu-rons are first to degenerate and that loss of NM neu-rons may occur later (Kelly del Tredici, personalcommunication).New work suggests that some DMV neurons,

although cholinergic, also express tyrosine hydroxylaseand aromatic acid decarboxylase, the enzymes thatproduce dopamine, and thus may express cytosoliccatecholamines but not release them because of a lackof vesicular monoamine transporter.50 There is also alocal A2 catecholamine group in this area, and it isnot clear which other DMV neurons may express cate-cholamines and whether these are projection neurons.The brain stem reticular formation also includes sev-

eral nuclei in which LP was observed in PD patients.31

Although neuronal death has not been reported per se,the depigmentation of this area in some patients againindicates that some catecholaminergic neurons mustbe lost.

SNc, VTA, RRF

The loss of DA neurons in the SNc is the best-docu-mented sequela of PD. In 1894, Blocq and Marinescoreported a study of a patient with unilateral parkin-sonism who had a tuberculoma that damaged theSNc.51 The loss of NM in the SNc of PD patients wasreported early in the 20th century4 and confirmed bymany subsequent studies,47,48,52 The loss of NM isseen in all PD patients,53 but not in all patients withparkinsonism.54 LP is nearly always found in the NMneurons in the SNc, particularly in the posterolateralregions.31 LB has been reported in the neighboringVTA and RRF neurons as well.53 DA neuronal loss in



these 2 regions is variable55 but has been estimated tobe as much as 50%.39

Unlike the situation with the LC, neuronal loss inthe SNc appears to be specific to PD. A comparativestudy reported a profound (�80%) loss of NM SNcneurons in PD patients, but only a modest 7% loss inAD patients.56 The most heavily NM pigmented neu-rons lost in the SNc of PD patients are suggested to bethose with lower levels of VMAT2 and with decreasedvesicular accumulation of DA57 as well as lower levelsof calbindin.58,59 Note that although NM neurons areparticularly targeted,39 some of the most highly pig-mented neurons are spared in PD patients.49,60

LC

The neighboring NE neurons in the LC have longbeen known to be lost in PD.47,52 LP is observed inthe larger projection NM containing LC neurons andis absent from non-NM cells in the complex,53 con-firming selective damage to NE neurons. Virtually allPD patients appear to have a substantial loss of LCneurons, with a mean neuronal loss of 83% in laterstages of the disease.56 There is also an average 68%loss of these neurons in Alzheimer’s disease (AD)56

but the extent of LC loss is more variable in ADpatients than in PD patients.

RN

Neurons in the RN are responsible for serotoniner-gic innervation of the CNS. In iLBD and PD, LP ismost apparent in medium-sized neurons of the caudalRN.31 The ventral rostral raphe region around themedial lemniscus also exhibits LB in PD.53 RN neu-rons not only display LB but are lost in PD, withreduction of more than half these neurons in the me-dian raphe and a somewhat smaller fractional lossreported in the raphes obscurus.53,61

PPN

The PPN is rostral to the LC and includes a mixtureof cholinergic, glutamatergic, and GABAergic neurons;the cholinergic and glutamatergic neurons are projec-tion neurons with an array of targets in the mesen-cephalon and diencephalon.62 The PPN was reportedby Braak’s group to exhibit LP in some cases,5,42 andLBs and neuronal loss were reported by Jellinger.63

Death of PPN neurons in PD appears to be highlyvariable. The nucleus itself was smaller in 4 of 6 PDbrains examined by Hirsch and colleagues, as definedby NAPDH diaphorase and the ACh esterase label64;they also estimated that about half the neurons werelost in that subgroup of patients. As in many otherregions, loss does not appear to be specific to PD, asPPN neurons were also dramatically less abundant(�80% reduction) in the brains of individuals with

progressive supranuclear palsy (PSP; Hirsch et al,1987). Jellinger63 attempted to address the specificityissue by comparing the nuclei in patients with PD,PSP, and AD using cresyl violet stain. He found nearlythe same neuronal loss in PSP and PD (�50%–60%)and less loss in AD (�30%). A recent study found a40% drop on average in the number of neurons dis-playing cholinergic markers in the PPNs of PDpatients, and the magnitude of this reduction was pos-itively correlated with symptom severity (Rinne et al,2008). Halliday and collaborators also reported a lossof substance P immunoreactivity in the PPN andneighboring structures in PD brains.61 However, inneither of these studies was it clear whether thisreflects cell loss or phenotypic dysregulation.65

NBM

The nucleus basalis of Meynert (NBM) is a promi-nent site of LP in PD patients. This cholinergic basalforebrain structure supplies the majority of cholinergicinput to the cerebral cortex. Neurons in this region,along with the DMV, were first reported to exhibit LPby Lewy.3 Indeed, the NBM is nearly always found todisplay LP in PD. The specificity of this loss is lessclear, as the NBM also figures prominently in AD.66

This issue was addressed by a comparative study67

that found a loss of about half the NBM neuronsmeasured by cresyl violet label in 11 PD patients,which was significantly greater than in age-matchedcontrol subjects. Not all of these subjects displayedAD-like symptoms, suggesting that there was a symp-tomatic threshold. There was no correlation betweenPD symptom severity and NBM loss in this study. Themost extensive comparative study of PD and ADbrains found similar NBM neuronal losses, also as la-beled by cresyl violet, on average (�40%), but therewas a great deal of variability.56 Thus, LP and neuro-nal loss in the NBM appears to be a common featureof PD and AD.

Olfactory Neurons

There are several other regions of the brain rostralto the mesencephalon that have been reported to ex-hibit signs of pathology in PD. One of these is the ol-factory system. In the olfactory bulb, mitral cellsexhibit LP,68 but this is also seen in AD.69 The ante-rior olfactory nucleus also shows LP in iLBD.31 Theexistence and targeting of neuronal loss in the olfac-tory system is controversial.70,71 This issue is compli-cated by the neurogenesis in this region.72

Additional Areas

Several other regions have been reported to have ei-ther neuronal loss or LP in PD, many in single studies.These include the hypothalamus73 and the



intralaminar nuclei of the thalamus.74 The specificityof these changes is unclear. In the case of the thalamicnuclei, the loss is also seen in PSP.74 In apparent laterstages of PD, LP is also scattered throughout the cere-bral cortex, amygdala, and hippocampus.75,76 RetinalDA also has been reported to decline,77 but LP wasnot identified.

Summary

There are obviously major gaps in the description ofneuronal pathology and loss seen in PD patients, par-ticularly insofar as how frank cell loss contrasts withphenotypic dysregulation. Nevertheless, it is clear thatthe pathology in PD is distributed across both theCNS and PNS. There is well-documented neuronalloss of NMþ catecholamine neurons of the SNc andLC in nearly all PD patients and of DMV neurons inmost patients. Loss of VTA, RRF, PPN, RN, andNBM neurons in PD appears common but quite vari-able. Only the loss in the SNc is specific to PD,although this is in part a circular argument because ofreliance on SN-dependent motor dysfunction to diag-nose the disease, and there are diseases with parkinso-nian symptoms that do not exhibit SNc death.1

Determinants of Vulnerability

The obvious question is what links this seeminglydiverse set of neurons. There are several possible phe-notypic traits that have been proposed to underlie vul-nerability. All are consistent with age being the singlelargest risk factor in PD.

• Lewy pathology. Immunolabel for aSYN aggre-gates is widely used as a marker for PD-relatedpathology, and with the caveat that it is also asso-ciated with several disorders that variably overlapwith PD, it is remarkable that nearly all neuronsthat experience cell death in PD also display LP.The converse, however, does not appear to betrue, as various peripheral and central neurons arefound to express LP without current evidence foreventual neuronal death. It may be that LP resultsfrom a neuroprotective response to stabilize anddetoxify otherwise toxic forms of aSYN,6 as isthought to occur for the intracellular accumula-tion of toxic cysotolic protein aggregates.78 It isnevertheless possible that LP, even if it indicates aprotective stress response, may have deleteriouseffects on intraneuronal function,65 such as axo-nal organelle transport.

• A common reactive neurotransmitter. SNc, LC,RN, enteric DA neurons, and sympathetic post-ganglionic neurons each synthesize a monoamineneurotransmitter, and high levels of cytosolicmonoamines are reactive and hypothesized to

underlie selective neuronal death under severalconditions.79–83 Additional targeted neuronsincluding some vulnerable parasympathetic neu-rons of the DMV appear to possess and/or secretemonoamines.

• Obviously, DA SN and NE LC are the most simi-lar in this regard, and both exhibit NM deposi-tion in humans. These 2 populations display thegreatest loss in PD. VTA and RRF DA neuronsare less pigmented and less vulnerable, althoughstill substantially more than other neurons oftenincluded in lists of targeted neurons; the lowerlevel of neuronal loss than in the SNc has beenhypothesized to stem from lower levels of cyto-solic catecholamines,81 which is consistent withthe lower NM,36 and evidence for lower levels ofvesicular accumulation of DA in the highly tar-geted and more pigmented SNc neurons.57 Never-theless, some highly pigmented A2 catecholamineNM neurons of the DMV do not appear to belost in PD,49 and so NM synthesis per se, whichmay be another neuroprotective response,36,40 isnot sufficient for neuronal death.

• In the periphery, the loss of NE neurons is vari-able, with some sympathetic neurons appearing tobe lost and others not. Moreover, their loss is notspecific to PD, as is also the case for LC neurons.DA neurons in the ENS, which are vulnerable inPD, do not have visible NM, suggesting they maynot experience the cytosolic oxidant stress typicalof SNc and LC neurons. Two other observationsmilitate against a reactive monoamine transmitterhypothesis in a simple form. One is that thereclearly is pathology and loss of neurons that donot use monoamines. Targeted neurons in the PPNand NBM use either acetylcholine or glutamate asneurotransmitters. It must be acknowledged thatthe loss of neurons in PPN and NBM is not specificto PD, raising the possibility that their loss has adifferent origin and is not really a reflection of PDper se; it has recently been suggested that loss ofcholinergic neurons is a downstream response toprevious loss of monoamine neurons.84 Anotherobservation of relevance is that use of L-dopa—theprecursor for DA—to treat PD does not acceleratethe progression of the disease, as might be expectedif DA was the toxic agent in the disease.1

• A long, highly branched axon with multiplerelease sites. SNc, LC, RN, PPN, and NBM neu-rons all have unusually long highly branchedaxons that are unmyelinated or thinly myelin-ated.27,85 This feature is particularly well docu-mented for SNc DA neurons. Single SNc axonsterminating in the striatum are highly branched

and possess as many as several hundred thousandsynaptic release sites.86 This is an order of magni-tude (or more) greater than most neurons thathave been carefully studied. Interestingly, theseterminals do not appear to have an elevated mito-chondrial oxidant stress.87 However, maintaininga massive terminal field is very likely to create ametabolic and proteostatic burden on the cellbody. Mitochondrial trafficking could prove par-ticularly problematic; in fact, mitochondrial den-sity in the somatodendritic region of SNc DAneurons is low,88 possibly reflecting the need totraffic mitochondria to axons. It is worth notingthat aSYN is thought to be a presynaptic regula-tor of synaptic vesicle exocytosis89–91; the proteo-static burden it creates could scale with thenumber of synaptic release sites and may contrib-ute to mishandling of presynaptic mitochondria.92

It is unclear whether VTA and RRF DA neuronshave as extensive an axonal field as do SNc DAneurons. Matsuda et al did not report profounddifferences in dorsal and ventral striatal terminalfields, which should correspond to SNc and VTA;as a consequence, the differences in vulnerabilitybetween these regions would have to be explainedby other factors. The LC also forms very long andcomplex projections. Based on the distance trav-eled and terminal field, the axons of DMV neu-rons are also long and highly branched, and DAenteric neurons are also highly branched.

• A common physiological phenotype. An extendeddiscussion of this hypothesis has recently beenpublished.93 PD is a disease of neurons, not of theliver, kidney, or heart. An implication of this isthat 1 or more of the features distinguishing neu-rons from these other cell types must contributein a seminal way to pathogenesis. A cardinal fea-ture of neurons that separates them from nearlyall other cell types is excitability. Neurons usesteep electrochemical gradients across theirplasma membrane to perform computations onincoming chemical signals from other neuronsand to pass the outcome of this computation toother cells. Each step in this process expendsenergy. Action potentials (or spikes) and synaptictransmission dissipate the ionic gradients for so-dium, potassium, calcium, and chloride that aremaintained by ATP-dependent pumps andexchangers. Although all neurons share this basicset of properties, the parameters of spikes andsynaptic transmission vary dramatically. Thephysiological phenotype of neurons ranges fromwhat might be called a ‘‘wallflower,’’ or quies-cent, phenotype to a ‘‘chatter box’’ phenotypethat never stops spiking. SNc, LC, RN, NBM,PPN, and DMV neurons all fall into the chatter-

box phenotype. That is, all of them spike continu-ously in vivo during the waking state.94–99 SNc,LC, DMV, and PPN neurons are autonomouspacemakers (they spike on their own in the ab-sence of synaptic input). Moreover, each of theseneurons has very broad spikes that further dissi-pate ionic gradients, particularly those for cal-cium. Although less well studied, many of theneurons in the ANS, particularly those in theENS, also are spontaneously active and havebroad spikes.100–102

Calcium entry is energetically expensive because itmust be pumped out of the cell against a much steeperelectrochemical gradient than any of the other ions.Many neurons ameliorate this burden by expressingspecialized calcium-binding proteins that effectivelybuffer calcium and minimize the metabolic cost ofpumping it back across the plasma membrane. Theexpression of calcium-binding proteins in the ANSand ENS varies from cell type to cell type.60,103,104

SNc, LC, and DMV neurons express relatively little ofthese calcium-binding proteins105 (D.J.S., unpublishedresults). (RN, PPN, and NBM have not been rigor-ously characterized in this regard to our knowledge.)In contrast, VTA neurons robustly express the cal-cium-binding protein calbindin; most other, relativelyPD-resistant autonomous pacemakers in the brain alsoexpress calcium-binding proteins (eg, Purkinje neu-rons, globus pallidus neurons, striatal cholinergicinterneurons).It is worth noting that autonomous pacemaking

neurons spend all their time at relatively depolarizedmembrane potentials, where NMDA receptors arerelieved of their magnesium block, creating anotherpoint of sodium and calcium entry during excitatorysynaptic transmission; excitotoxicity is perhaps one ofthe oldest theories of pathogenesis in PD.106,107 Highcytosolic calcium levels also act to increase cytosolicDA in SNc neurons,81 possibly because of an effect onsynthesis, and can lead to aSYN-dependent neuronaldeath. Thus, although there certainly are gaps in ourphysiological characterization of PD-vulnerable neu-rons, at this point it appears that they all are a variantof the chatterbox phenotype: continuous generation ofslow, broad spikes and low intrinsic calcium-bufferingcapacity.This physiological phenotype creates a sustained

metabolic burden carried largely by mitochondria. Inmost neurons, the metabolic burden associated withactivity and synaptic transmission is thought to signifi-cantly diminish the respiratory reserve of mitochon-dria.108 In SNc DA neurons and others of its kind,this reserve should be even smaller. In fact, there is ameasurable increase in the oxidation of mitochondrialthiol proteins when SNc DA neurons are simply



pacemaking.109 Unpublished work by our group(D.J.S.) has revealed that LC and DMV neurons havesimilar (albeit smaller) mitochondrial oxidant stress.Mitochondrial dysfunction is widely viewed as a piv-otal step in PD pathogenesis.110 Sustained mitochon-drial oxidant stress in principle should lead to theaccumulation of mitochondrial DNA mutations andimpaired complex I function seen in the SNc withaging and PD.111 Genetic mutations, environmentaltoxins, and proteostatic stress that further challengemitochondria could synergize with this cell-type-spe-cific stress to produce a bioenergetic crisis, resulting inimpaired protein degradation and ultimatelydegeneration.This scenario does not exclude a role for the trans-

mitter phenotype or axonal branching in pathogenesis.In fact, these traits, particularly the axonal one, read-ily fit into this scenario. The translational question ishow to devise a therapeutic strategy to stop or slowthe progression of PD. Clearly, changing a neuron’stransmitter phenotype or axonal branching is not anoption. Strategies could be developed to amelioratethe consequences of these traits, but what these mightbe is not yet obvious. In contrast, there is a way inwhich the consequences of the chatterbox phenotypeon mitochondrial stress could be diminished. One ofthe ion channels contributing to the basal metabolicstress in SNc DA neurons is the L-type calcium chan-nel. There are FDA-approved antagonists (dihydropyr-idines) of these channels that have an excellent safetyrecord in humans. Moreover, there is epidemiologicalevidence that sustained use of brain-penetrant dihy-dropyridines reduces the observed risk of PD.112,113

This effect is surprising given the relatively low affinityof most dihydropyridines for the subtype of L-typecalcium channel responsible for most of the calciumentry in SNc DA neurons, that is, one with a Cav1.3pore-forming subunit.93,114

Acknowledgments: We thank in particular Dr. Kelly del Tredicifor thorough comments and personal communication on her findings.We thank Drs. Jaime Guzman, Javier Sanchez Paul Schumacker, RobertBurke, Luigi Zecca, Oren Levy, Michael Gershon, and Lloyd Greene forhelpful discussion and Carolina Cebri�an for translations from the Frenchliterature.

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Neuronal vulnerability, pathogenesis, and Parkinson's disease