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??-Synuclein in the olfactory system in Parkinson's disease: Role of neural

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Article  in  Brain Structure and Function · October 2013

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REVIEW

a-Synuclein in the olfactory system in Parkinson’s disease: roleof neural connections on spreading pathology

Isabel Ubeda-Banon • Daniel Saiz-Sanchez •

Carlos de la Rosa-Prieto • Alino Martinez-Marcos

Received: 6 June 2013 / Accepted: 4 October 2013 / Published online: 18 October 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract Parkinson’s disease (PD) is a neurodegenera-

tive disease characterized by bradykinesia, rigidity, resting

tremor, and postural instability. Neuropathologically,

intracellular aggregates of a-synuclein in Lewy bodies and

Lewy neurites appear in particular brain areas according to

a sequence of stages. Clinical diagnosis is usually estab-

lished when motor symptoms are evident (corresponding to

Braak stage III or later), years or even decades after onset

of the disease. Research at early stages is therefore

essential to understand the etiology of PD and improve

treatment. Although classically considered as a motor

disease, non-motor symptoms have recently gained inter-

est. Olfactory deficits are among the earliest non-motor

features of PD. Interestingly, a-synuclein deposits are

present in the olfactory bulb and anterior olfactory nucleus

at Braak stage I. Several lines of evidence have led to

proposals that PD pathology spreads by a prion-like

mechanism via the olfactory and vagal systems to the

substantia nigra. In this context, current data on the tem-

poral appearance of a-synuclein aggregates in the olfactory

system of both humans and transgenic mice are of partic-

ular relevance. In addition to the proposed retrograde nigral

involvement via brainstem nuclei, olfactory pathways

could potentially reach the substantia nigra, and the pos-

sibility of centrifugal progression warrants investigation.

This review analyzes the involvement of a-synuclein in

different elements of the olfactory system, in both humans

and transgenic models, from the hodological perspective of

possible anterograde and/or retrograde progression of this

proteinopathy within the olfactory system and beyond—to

the substantia nigra and the remainder of the central and

peripheral nervous systems.

Keywords Lewy body � Premotor � Prion-like

hypothesis � Synucleinopathy

Abbreviation

PD Parkinson’s disease

Parkinson’s disease

Parkinson’s disease (PD) is the second most prevalent

neurodegenerative disorder, with a lifetime risk of devel-

oping the disease of 1.5 %, and is characterized by bra-

dykinesia, rigidity, resting tremor, and postural instability

(Lees et al. 2009). Although a small percentage of cases

(so-called familial PD) are linked to mutations in known

genes such as a-synuclein (SNCA; Farrer et al. 1999;

Kruger et al. 1998; Polymeropoulos et al. 1997; Proukakis

et al. 2013; Zarranz et al. 2004), leucine-rich repeat kinase-

2 (LRRK-2), and glucocerebrosidase (GBA; Hardy 2010),

most patients suffer from idiopathic PD (Morley and Hurtig

2010).

PD is neuropathologically characterized by intracellular

aggregates of a-synuclein and ubiquitin in Lewy bodies

and Lewy neurites (Spillantini et al. 1997). It has been

proposed that the appearance of Lewy bodies and neurites

in particular brain areas takes place according to six

neuropathological stages (Braak et al. 2003a; Braak and

Del Tredici 2008; Dickson et al. 2009). Clinical diagnosis

is established when motor symptoms appear owing to the

I. Ubeda-Banon � D. Saiz-Sanchez � C. de la Rosa-Prieto �A. Martinez-Marcos (&)

Laboratorio de Neuroplasticidad y Neurodegeneracion,

Departamento de Ciencias Medicas, Facultad de Medicina de

Ciudad Real, Centro Regional de Investigaciones Biomedicas,

Universidad de Castilla-La Mancha, Avda. de Moledores s/n,

13071 Ciudad Real, Spain

e-mail: Alino.Martinez@uclm.es

123

Brain Struct Funct (2014) 219:1513–1526

DOI 10.1007/s00429-013-0651-2

loss of dopaminergic cells in substantia nigra (Tolosa et al.

2006). In PD, Lewy bodies in the substantia nigra starts to

appear in Braak stage III, although the pathological process

is likely to have begun years or even decades before

(Claassen et al. 2010; Hawkes et al. 2010). This ana-

tomopathological staging has been queried (Parkkinen

et al. 2011), as has its correlation with clinical stages

(Burke et al. 2008; Jellinger 2010). At the time of diag-

nosis, between 30 and 50 % of nigral dopaminergic neu-

rons have already degenerated (Cheng et al. 2010; Fearnley

and Lees 1991; Ma et al. 1997; Riederer and Wuketich

1976). At the same time a reduction of up to 80 % of

dopamine levels was observed at striatal synapses (Bern-

heimer et al. 1973; Cheng et al. 2010; Riederer and

Wuketich 1976), which would be in favor of a ‘dying-back’

disease progression mechanism. Studies on the early PD

brain are therefore crucial to understanding the etiology of

PD and for the development of more effective treatments

(Berg et al. 2013; Lees 2009; Toulouse and Sullivan 2008).

Prodromal PD

Evidence is accumulating for several non-motor symptoms

in PD (Chaudhuri and Schapira 2009; Chaudhuri and Odin

2010; Lim and Lang 2010), some of which appear early

during a long-lasting prodromal (preclinical) period (Ferrer

et al. 2011; Olanow and Obeso 2012; Tolosa et al. 2009).

These early symptoms include hyposmia, dysphagia, con-

stipation, depression, and rapid-eye-movement sleep

behavior disorder (Hawkes 2008; Noyce et al. 2012;

Siderowf and Stern 2008; Stiasny-Kolster et al. 2005).

Hyposmia appears to be one of the most reliable bio-

markers (Bohnen et al. 2010; Doty 2012a, b; Haehner et al.

2007; Kranick and Duda 2008), and olfactory dysfunction

has been detected in *90 % of early-stage sporadic PD

cases (Doty et al. 1988). It has also been associated with an

increased risk of developing PD of at least 10 % (Ponsen

et al. 2004), and can pre-date clinical PD by at least 4 years

(Ross et al. 2008).

Interestingly, these first signs have been suggested to

correlate with pathological involvement of specific neuro-

anatomical structures during the initial stages. Hyposmia,

dysphagia, and constipation in stage I have been proposed

to be associated, respectively, with involvement of the

olfactory bulb and anterior olfactory nucleus, and also of

the dorsal motor glossopharyngeal and vagal nuclei, and

Auerbach’s and Meissner’s plexuses (Braak et al. 2006a;

Del Tredici and Braak 2012; Tolosa and Pont-Sunyer 2011;

Wolters and Braak 2006). The involvement of such struc-

tures appears to include some particularly vulnerable neu-

ronal types (Braak et al. 2003b) in which aggregation of a-

synuclein appears to spread either anterogradely (Hawkes

et al. 1999) and/or retrogradely (Braak et al. 2003b; Del

Tredici and Braak 2012).

Therefore, progressive appearance of a-synuclein aggre-

gates in known neuronal circuits, together with other data—

such as the postmortem presence of Lewy bodies in dopa-

minergic fetal cells grafted into the substantia nigra of Par-

kinson’s patients, and pathological a-synuclein transmission

leading PD-like neurodegeneration in mice—have led to

proposals that a-synucleinopathy propagates in PD brain via

a prion-like mechanism (Angot et al. 2010; Brundin et al.

2008; Olanow and Brundin 2013; Prusiner 2012).

a-Synuclein spreading

a-Synuclein is a soluble, 140 amino acid protein of

unknown function (Olanow and Brundin 2013) that is

predominantly localized to presynaptic terminals in asso-

ciation with synaptic vesicles (Lee and Trojanowski 2006).

In PD, conformational transformation refolds native

a-helical a-synuclein into pathology-associated b-sheet

a-synuclein (Li et al. 2002) which can efficiently form

Lewy bodies and neurite fibrils (Fig. 1a, b), as also dem-

onstrated in vitro and in vivo (Luk et al. 2009, 2012a, b).

Intracellular a-synuclein aggregates can be also reproduced

in transgenic mouse models (Fig. 1c, d). It remains unclear,

however, whether the intracellular aggregation of a-syn-

uclein is the primary cause of neuronal loss, a protective

mechanism, or is an irrelevant epiphenomenon (Duda

2010; Halliday and McCann 2008). In this sense, the

olfactory system displays early a-synucleinopathy, and

a-synuclein aggregation correlates with olfactory deficits.

Early reports pointing to anosmia as an early symptom

associated with PD were published in the 1970s (Ansari and

Johnson 1975; Constantinidis and de Ajuriaguerra 1970;

Kissel and Andre 1976), but were only firmly established a

decade later (Doty et al. 1988, 1989; Hawkes and Shephard

1993; Quinn et al. 1987). By that time ‘the olfactory vector

hypothesis’ was proposed to explain both olfactory losses and

the etiology of several neurologic diseases as a result of the

transit of an environmental virus, toxin, or xenobiotic agent

from the nasal cavity into the brain via the olfactory fila (Doty

et al. 1991; Ferreyra-Moyano and Barragan 1989; Harrison

1990; Mattock et al. 1988; Pearson et al. 1985; Roberts 1986;

Rohn and Catlin 2011; Yamada 1996). This fact, together with

the finding of Lewy bodies in the olfactory bulb and anterior

olfactory nucleus (Daniel and Hawkes 1992; Pearce et al.

1995), led to the proposal that the initial causative event in

idiopathic PD may start in the olfactory system prior to damage

in the basal ganglia (Hawkes et al. 1999).

Detailed neuropathological studies identified neural

structures affected by Lewy pathology according to a

constant and predictable series of stages (Braak et al.

1514 Brain Struct Funct (2014) 219:1513–1526

123

2003a). Further, because early symptoms, including

hyposmia and glossopharyngeal and vagal dysfunction,

appear to have a direct neuropathological correlate in

Braak stage I—Lewy pathology in the olfactory bulb,

anterior olfactory nucleus and enteric plexuses, and glos-

sopharyngeal and vagal nuclei—a ‘dual-hit hypothesis’

was proposed (Hawkes et al. 2007). This states that a

neurotropic pathogen, probably viral, enters the brain via

two routes: nasal, with anterograde progression into the

temporal lobe; and also gastric, secondary to swallowing

nasal secretions in saliva and retrograde propagation

through the vagal and glossopharyngeal nerves (Hawkes

et al. 2009; Fig. 2). The evidence for and against this

hypothesis has been thoroughly reviewed (Doty 2008).

Fig. 1 Horizontal (a, b) and

sagittal (c, d) Nissl-counterstained

sections showing a-synuclein

immunohistochemical labeling

in the human bulbar anterior

olfactory nucleus (AONb)

(a, b) and in the A53T

transgenic mouse olfactory

bulb (OB) (c, d). b and d are

high power microphotographs

of a and c, respectively.

Calibration bar for a, c 160 lm;

b, d 20 lm

Fig. 2 Scheme showing the

possible routes for a-

synucleinopathy progression

through the peripheral and

central nervous system (a) and

the location of structures

involved by a-synucleinopathy

(b). G gigantocellular reticular

nucleus, LC locus coeruleus, OB

olfactory bulb, OC olfactory

cortex, OE olfactory epithelium,

P pontine nuclei, PP peduncle

pontine nucleus, Ro nucleus

raphe obscurus, Rp nucleus

raphe pallidus, SN substantia

nigra, IX glossopharyngeal

nerve, IX/X glossopharyngeal/

vagal dorsal motor nucleus,

X vagus nerve

Brain Struct Funct (2014) 219:1513–1526 1515

123

At the same time, data appeared that PD pathology can

spread to intrastriatal grafts of young, healthy neurons

(Brundin et al. 2008). Specifically, two reports described

that a fraction (5 %) of implanted fetal dopaminergic

neurons in PD patients developed a-synuclein- and ubiq-

uitin-positive Lewy bodies more than a decade after

transplantation (Kordower et al. 2008; Li et al. 2008). This

observation was not corroborated in patients that survived

less than 10 years after transplantation (Mendez et al.

2008), suggesting that at least one decade is required for

the development of Lewy bodies in young and previously

healthy neurons. These reports could support the idea that

a-synuclein pathology might spread through a prion-like

mechanism (Angot et al. 2010; Brundin et al. 2008;

Olanow and Brundin 2013; Prusiner 2012).

The route of transmission, however, is far from clear.

Different mechanisms for anterograde and/or retrograde

transfer of a-synucleinopathy between cells (exocytosis

and endocytosis, exosomes, tunneling nanotubes, passive

diffusion, or receptor-mediated internalization) have been

proposed. Likewise, the seeding mechanism leading native

a-synuclein to misfold into a pathological isoform is

unknown (Dunning et al. 2012; Hansen and Li 2012).

It was recently demonstrated that intracerebral inocula-

tion of pathological a-synuclein initiates a rapidly pro-

gressive neurodegenerative a-synucleinopathy in mice.

Young asymptomatic a-synuclein transgenic mice intra-

cerebrally injected with brain homogenates from older

transgenic mice exhibiting a-synuclein pathology showed

accelerated formation of intracellular Lewy body-like

inclusions as well as accelerated onset of neurological

symptoms. a-Synucleinopathy propagated along major

central nervous system pathways to regions far beyond the

injection sites. Further, synthetic a-synuclein was solely

sufficient to initiate Lewy body-like inclusions and to

transmit the disease in mice overexpressing human a-

synuclein (Luk et al. 2012b). Further, the same group has

demonstrated that Lewy-like a-synuclein aggregates also

formed after a single injection of a-synuclein fibrils into

the striatum or cortex of wild-type animals. Moreover, after

intrastriatal injections some nigral dopaminergic neurons

had a-synuclein inclusions and no longer stained for

tyrosine hydroxylase, consistent with retrograde transport

of toxic a-synuclein from the injection site (Luk et al.

2012a) and dying-back disease progression. Lewy-like

pathology only developed in brain regions anatomically

connected to the site of injection. For instance, at 30 days

post-injection, Lewy-like accumulations were exclusively

ipsilateral to the injection site, with the exception of the

amygdala, to which the striatum connects bilaterally. At 90

and 180 days post-injection, Lewy-like pathology was

present in the contralateral neocortex (Luk et al. 2012a),

thus showing a time-dependent dissemination through the

white-matter tracts, which is however in contrast to

spreading routes in PD patients (Braak et al. 2003a).

Taken together, these data suggest that several peripheral

and autonomic nervous system structures show early PD

pathological changes that probably spread retrogradely to the

brain. By contrast, in the olfactory system this progression

seems to occur anterogradely (Del Tredici and Braak 2012).

However, the pattern of pathological progression within the

olfactory system, the temporal involvement of primary,

secondary, and tertiary olfactory structures, the cell types

involved by Lewy pathology, and whether this pathway

could reach the substantia nigra are not known.

The olfactory system

Structure of the olfactory system

Although the olfactory system has traditionally been

neglected from a clinical point of view, broad acceptance

of hyposmia as an early symptom in neurodegenerative

diseases, particularly Alzheimer disease (AD) and PD,

together with pathological hallmarks in olfactory structures

in the initial stages of disease, has focused interest on

changes in this system as a neurological sign (Albers et al.

2006; Benarroch 2010; Doty 2012b; Duda 2010; Hawkes

and Doty 2009; Ruan et al. 2012). However, it remains

unclear whether olfactory dysfunction results from

involvement of the olfactory epithelium, the olfactory bulb,

or tertiary olfactory structures, and to what extent a-syn-

ucleinopathy in the olfactory system contributes to the

pathoetiology of the disease.

The olfactory system has several unique traits that dif-

ferentiate it from other sensory systems. (1) The olfactory

mucosa is directly exposed to the environment, thus pro-

viding a primary route for entry into the brain; (2) there are

several hundred genes that encode olfactory receptors

(Buck and Axel 1991); (3) sensory cells are true neurons

with dendrites, axons, and action potentials; (4) these cells

undergo turnover through a neurogenic process during

adulthood (Bermingham-McDonogh and Reh 2011); (5)

there is a second potential neurogenic source from the

anterior subventricular zone to the olfactory bulb (Lepou-

sez et al. 2013)—which has been suggested to be altered in

PD and could underlie olfactory deficits (Marxreiter et al.

2013), although postnatal neurogenesis in this area is

controversial in humans (Bergmann et al. 2012; Curtis

et al. 2007); (6) there is no thalamic relay in the olfactory

system, and second-order neurons instead synapse directly

in the cortex; and (7), the olfactory system is not organized

in an odorant-specific (odotopic) manner; instead, a spatial

and temporal pattern of activation appears to codify odor-

ant information (Mori and Sakano 2011; Murthy 2011).

1516 Brain Struct Funct (2014) 219:1513–1526

123

The human olfactory system begins with sensory neu-

rons in the dorsocaudal portion of the nasal cavity. Sensory

cells are bipolar neurons whose dendritic cilia contain

olfactory receptors exposed to the mucus layer; at the other

pole, axons converge to form fila olfatoria that enter the

skull. These axons make synapses with apical dendrites of

mitral and tufted cells in neuropil spheres termed glome-

ruli. Axons of mitral and tufted cells project to several

tertiary olfactory structures: the anterior olfactory nucleus,

olfactory tubercle, piriform cortex, periamygdaloid cortex,

and the rostral entorhinal cortex (Mohedano-Moriano et al.

2005; Fig. 3).

Fig. 3 Scheme illustrating the

primary (yellow), secondary

(pink) and tertiary (blue)

olfactory structures in the

human brain. AONb bulbar

anterior olfactory nucleus,

AONc cortical anterior olfactory

nucleus, AONi intrapeduncular

anterior olfactory nucleus,

AONr retrobulbar anterior

olfactory nucleus, C claustrum,

cc corpus callosum, Cd caudate,

ECo olfactory entorhinal cortex,

ic internal capsule, OB olfactory

bulb, OP olfactory peduncle, ot

olfactory tract, ox optic chiasm,

PAC periamygdaloid cortex,

PirF frontal piriform cortex,

PirT temporal piriform cortex,

Pu putamen, Tu olfactory

tubercle

Brain Struct Funct (2014) 219:1513–1526 1517

123

The human olfactory bulb is composed of seven layers

including the glomerular and mitral and tufted cell layers

(Smith et al. 1993). Among tertiary olfactory-recipient

structures (van Hartevelt and Kringelbach 2011), the

anterior olfactory nucleus is composed of at least seven

subdivisions, including bulbar, intrapeduncular, retrobul-

bar, and cortical anterior and posterior portions, with

medial and lateral components at both sides of the olfactory

tract (Fig. 3b–d; Saiz-Sanchez et al. 2010a). It is far from

clear whether all these subdivisions correspond to a single

structure or to individual nuclei. Therefore, in the human

brain the term ‘anterior olfactory nucleus’ is to be used

with caution. The olfactory tubercle is located in the frontal

lobe and contains the islands of Calleja (Fig. 3e, f). The

piriform cortex includes frontal and temporal domains at

both sides of the limen insulae (Fig. 3e–g). The peri-

amygdaloid cortex comprises different structures along the

cortical surface of the amygdaloid complex (Fig. 3f, g).

Finally, only a fraction of the rostral entorhinal cortex, the

olfactory entorhinal cortex, receives olfactory inputs

(Fig. 3e–g; Insausti et al. 1995, 2002).

The rodent olfactory system shows a similar scheme but,

given its macrosmatic nature, is comparatively hypertro-

phic compared to human (Halpern and Martinez-Marcos

2003; Martinez-Marcos 2009; Fig. 4). The rodent anterior

olfactory nucleus is located posterolateral to the olfactory

bulb and contains several subdivisions (Fig. 4c, d), as well

as being organized in a somewhat different manner from

the human equivalent (Brunjes et al. 2005), to the point that

strict comparison to the human structure is difficult (Saiz-

Sanchez et al. 2010a). The olfactory tubercle is located in

the ventral portion of the hemisphere (Fig. 4e), whereas the

piriform cortex occupies a large portion of the lateral

hemisphere containing the olfactory tract (Fig. 4e, f). The

olfactory amygdala includes the anterior cortical and pos-

terolateral cortical amygdaloid nuclei (Fig. 4f), somewhat

comparable to the human periamygdaloid cortex. Finally, a

comparatively large portion of the entorhinal cortex, the

lateral entorhinal cortex, receives olfactory inputs in

rodents (Fig. 4g; Insausti 1993; Insausti et al. 2002; Mar-

tinez-Marcos and Halpern 2006).

Connections of the olfactory system

Given the structures of the human and rodent olfactory

systems, it is interesting to analyze connections that may be

relevant in the context of PD. It should be taken into

account that data in humans are inferred from comparative

studies. Primary olfactory projections are those originating

in the olfactory epithelium to the ipsilateral olfactory bulb

(yellow arrow in Fig. 5). Secondary olfactory projections

include projections of mitral and tufted cells of the olfac-

tory bulb to the remaining olfactory structures—predomi-

nantly ipsilaterally (pink arrows in Fig. 5). Tertiary

olfactory projections are those originating in tertiary

olfactory structures including contralateral, centrifugal, and

associational projections out of the olfactory system (blue

arrows in Fig. 5). Non-olfactory projections are herein

focused on the connections of the central amygdala in view

of its pivotal position in aggregation spreading (green

arrows in Fig. 5). Intra-brainstem projections are restricted

to those connecting the dorsal motor glossopharyngeal and

vagal nuclei of the medulla to the mesencephalic substantia

Fig. 4 Scheme illustrating the primary (yellow), secondary (pink) and

tertiary (blue) olfactory structures in the mouse brain. AON anterior

olfactory nucleus, CA1 hippocampal CA1, cc corpus callosum, DG

dentate gyrus, ECl lateral entorhinal cortex, ic internal capsule, OA

olfactory amygdala, OB olfactory bulb, ot olfactory tract, Pir piriform

cortex, rf rhinal fissure, Tu olfactory tubercle

1518 Brain Struct Funct (2014) 219:1513–1526

123

nigra through the pons via pontine and raphe nuclei and the

locus coeruleus (violet arrows in Fig. 5). Finally, ascending

modulatory projections from the locus coeruleus and raphe

nuclei are considered (black arrows in Fig. 5), leaving

apart the substantia nigra.

The anterior olfactory nucleus shows multiple connec-

tions that place it as a nodal point in the olfactory system,

and this could potentially underlie its early and preferential

involvement by a-synuclein (Ubeda-Banon et al. 2010a)

and in other proteinopathies such as AD (Saiz-Sanchez

et al. 2010b). The anterior olfactory nucleus receives direct

projections from the olfactory bulb and, in turn, projects

back to the olfactory bulb, including ipsi- and contralateral

centrifugal projections through the anterior commissure,

thus mediating interhemispheric communication (Mohed-

ano-Moriano et al. 2012). In addition, it projects to the

majority of secondary olfactory structures, including the

olfactory tubercle and piriform and entorhinal cortices—

part of the so-called associational connections within and

among olfactory structures (Luskin and Price 1983), and

receives projections from at least 27 ‘non-olfactory’

regions (Fig. 5a; Brunjes et al. 2005). In primates, for

example, the anterior olfactory nucleus is reciprocally

connected not only with the remaining secondary olfactory

structures, but also with the superior temporal sulcus, an

area of multimodal integration (Mohedano-Moriano et al.

2005).

The olfactory tubercle receives direct projections from the

olfactory bulb (Martinez-Marcos 2009), and also projects

centrifugally back to the olfactory bulb (Mohedano-Moriano

et al. 2012). The olfactory tubercle also receives connections

from olfactory structures (Ubeda-Banon et al. 2007) and,

interestingly, is reciprocally connected with the substantia

nigra (Fig. 5b, d; Newman and Winans 1980). The piriform

Fig. 5 Scheme representing different anatomical connections as

possible pathways for a-synucleinopathy spreading within the brain.

AONb bulbar anterior olfactory nucleus, AONc cortical anterior

olfactory nucleus, AONi intrapeduncular anterior olfactory nucleus,

C claustrum, Cd caudate, Ce central amygdala, ECo olfactory

entorhinal cortex, G gigantocellular reticular nucleus, ic internal

capsule, IC inferior colliculus, IO inferior olive, LC locus coeruleus,

OB olfactory bulb, OE olfactory epithelium, P pontine nuclei, PAC

periamygdaloid cortex, pc pars compacta, PirF frontal piriform

cortex, PirT temporal piriform cortex, PP peduncle pontine nucleus,

pr pars reticulate, Pu putamen, R pontine raphe nucleus, Ro raphe

obscurus nucleus, Rp raphe pallidus nucleus, SN substantia nigra, Tu

olfactory tubercle, IX glossopharyngeal nerve, X vagus nerve

Brain Struct Funct (2014) 219:1513–1526 1519

123

and entorhinal cortices are also connected with the olfactory

bulb (Martinez-Marcos 2009; Mohedano-Moriano et al.

2012), the anterior olfactory nucleus (Brunjes et al. 2005),

and the olfactory tubercle (Newman and Winans 1980)

through associational connections among olfactory struc-

tures (Fig. 5b, c; Luskin and Price 1983). The entorhinal

cortex also receives direct projections from the substantia

nigra (Fig. 5c, d; Loughlin and Fallon 1984).

In addition to these intra-olfactory connections and con-

nections with the substantia nigra, it is interesting to note that

structures such as the central nucleus of the amygdala are

connected not only with the piriform and entorhinal cortices

but also with the substantia nigra and the dorsal motor

nucleus of the vagus nerve (Fig. 5c, d, f; Volz et al. 1990).

Similarly, other neuromodulatory systems, such as seroto-

nergic inputs from the raphe nuclei and noradrenergic inputs

from the locus coeruleus, also project to the olfactory bulb

(Fig. 5a, e; Shipley and Adamek 1984).

Therefore, the olfactory system displays a complex

system of centripetal, centrifugal, commissural, and asso-

ciational connections, as well as reciprocal direct and

indirect connections with the substantia nigra, amygdala,

and brainstem nuclei involved in PD. Analyzing this hod-

ological context is essential to understanding a-synuclein

spreading in PD.

a-Synuclein in the olfactory system and beyond

In the context of neuroanatomical circuits, data on the

temporal appearance of aggregates of a-synuclein and/or

their progression in the olfactory system and other struc-

tures involved in PD, both in humans and in transgenic

models, are of particular relevance to understanding both

the etiology of the disease and the progression of neuro-

pathology in the brain. Accordingly, several important

questions need to be addressed. Are there predominantly

two independent routes of spreading of pathology through

the vagal and olfactory systems? Does progression occur

anterogradely and/or retrogradely? Are these two routes

interconnected? How does such spreading relate to the

asymmetry of pathology that is often seen in PD?

The hypothesis of an anterograde progression through

the olfactory system would predict that olfactory receptor

neurons would be involved first. First-order receptor neu-

rons undergo continuous cell turnover with an average

lifetime between 1 and 2 months (Bermingham-McDonogh

and Reh 2011). It is therefore likely that, even if patho-

logical a-synuclein might involve these cells, this pro-

teinopathy would not be detected (Duda et al. 1999)

because Lewy bodies take at least 6 months to form,

according to an estimate carried out in aged nigral cells of

PD patients (Greffard et al. 2010). Studies on olfactory

epithelium amyloid-b and paired helical filament/tau

pathology in AD conclude that these two pathologies in the

olfactory epithelium are present in the majority of cases

with pathologically verified AD, and that they correlate

with disease development. In this same report, however, a-

synuclein expression was observed in only one case from

seven PD cases analyzed (Arnold et al. 2010). Accordingly,

the presence/absence of a-synuclein in the olfactory epi-

thelium cannot be used to evaluate potential anterograde or

retrograde progression through the olfactory system.

Following the rationale of an anterograde spreading

through the olfactory system, second-order neurons would

also be primarily involved. Early neuropathological reports

described the occasional presence of Lewy bodies in mitral

cells (Daniel and Hawkes 1992), as confirmed in later

investigations (Braak et al. 2003a; Del Tredici et al. 2002;

Hubbard et al. 2007; Sengoku et al. 2008; Ubeda-Banon

et al. 2010a). Interestingly, studies of colocalization in the

olfactory bulb have revealed that, apart from scattered

mitral cells 54, 43, and 68 % of neighboring interneurons

expressing calcium-binding proteins (such as calbindin,

calretinin, and parvalbumin) display a-synuclein pathol-

ogy, respectively; whereas only 6 and 8 % of cells

expressing tyrosine hydroxylase (presumptively dopami-

nergic) or somatostatin coexpress a-synuclein, respectively

(Ubeda-Banon et al. 2010a). Interestingly, an increased

number of dopaminergic periglomerular neurons in the

olfactory bulb has been reported in PD, AD, and fronto-

temporal dementia patients (Mundinano et al. 2011). In this

regard, it has been demonstrated that mesencephalic

dopaminergic cells express genes such as HNF3a, synapt-

otagmin I, and Ebf3 that distinguish them from bulbar

dopaminergic cells (Thuret et al. 2004). These data raise

the issue of the low rate of a-synucleinopathy observed in

mitral cells versus adjacent interneurons. It is possible that

mitral cells are less vulnerable to a-synuclein aggregation,

or indeed that mitral cells bearing Lewy bodies disappear

as consequence of normal aging (Bhatnagar et al. 1987).

The presence of a-synuclein aggregates in the olfactory

bulb, however, is densest in the bulbar anterior olfactory

nucleus, which correspond to third-order neurons (Braak

et al. 2003a; Del Tredici et al. 2002; Hubbard et al. 2007;

Pearce et al. 1995; Sengoku et al. 2008; Ubeda-Banon et al.

2010a). Indeed, a-synuclein aggregates are particularly

conspicuous along the different subdivisions of the anterior

olfactory nucleus (Ubeda-Banon et al. 2010a), to the point

that olfactory bulb biopsies have been proposed as a means

to confirm diagnosis in PD subjects being assessed for

surgical therapy (Beach et al. 2009). The fact that the

densest labeling in the olfactory system occurs in the

anterior olfactory nucleus cannot be exclusively explained

from olfactory bulb afferent connections, but it is probably

due to its multiple centripetal, centrifugal, commissural,

1520 Brain Struct Funct (2014) 219:1513–1526

123

associational, and non-olfactory connections. This fact

should be taken into account to understand early a-syn-

uclein spreading in PD.

Regarding the remaining structures receiving projec-

tions from the olfactory bulb, thereby containing third-

order neurons (the olfactory tubercle, the piriform cortex,

the olfactory amygdala, and the rostral portion of the en-

torhinal cortex) these show only moderate a-synuclein

aggregation (Hubbard et al. 2007; Ubeda-Banon et al.

2010a), although these aggregates are somewhat more

abundant in the temporal piriform cortex (Silveira-Moriy-

ama et al. 2009) and cortical amygdala (Braak et al. 1994,

2003a; Harding et al. 2002). Compared to adjacent non-

olfactory structures, however, the labeling is not compar-

atively much higher—thus suggesting that the mechanism

of spreading of a-synucleinopathy through the olfactory

system is much more complex than two synaptic antero-

grade jumps, and that many other possibilities must also be

considered.

Despite their limitations, transgenic mouse models offer

unique possibilities for addressing specific questions

regarding PD (Magen and Chesselet 2010; Smith et al.

2012). Data from our laboratory using A53T (Prnp–

SNCA*, 83Vle/J) mice, in which a-synuclein is expressed

under prion promoter control, indicate moderate a-synuc-

lein aggregation in all layers of the olfactory bulb starting

at 2 months of age (early adulthood), but this increased

significantly at age 6–8 months (Ubeda-Banon et al.

2010b) when motor symptoms appear in this model

(Giasson et al. 2002). As in humans (Sengoku et al. 2008;

Ubeda-Banon et al. 2010a), dopaminergic bulbar inter-

neurons appear not to be substantially involved, in contrast

to calcium-binding protein- and glutamate-expressing

neurons, including mitral cells (Ubeda-Banon et al. 2010b).

Regarding structures receiving projections from the olfac-

tory bulb, the level of aggregation is comparatively low

during the first 8 months of age, but a significant increase

takes place at 6–8 months in the piriform cortex and cor-

tical amygdala (Ubeda-Banon et al. 2012), which is remi-

niscent of the human pattern. Tract-tracing experiments

using anterograde tracers in the olfactory bulb of wild-type

and transgenic mice revealed reduced olfactory projections

in the latter. Further, terminal axons of mitral cells have

been observed in the piriform cortex in close proximity to

third-order neurons expressing a-synuclein, thus suggest-

ing a potential site for protein spreading (Ubeda-Banon

et al. 2012). Taken together, data in this transgenic mice

model reveal a temporal pattern of aggregation from

peripheral to central olfactory structures, with involvement

of cell types similar to those observed in humans, and thus

point to the piriform cortex as a site in which a-synuclein

aggregation could be increased in part owing to the prox-

imity of axonal collaterals of mitral cells.

Data in other rodent models have been helpful to shed

light on the role of the olfactory system in PD. Prediger

et al. (2009, 2010, 2011) have developed a rodent model

after single 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP) intranasal administration causing olfactory, emo-

tional, and motor impairments as well as loss of tyrosine

hydroxylase expression in the olfactory bulb and substantia

nigra, thus supporting the propensity of the olfactory sys-

tem to transport toxins into the central nervous system.

New a-synuclein transgenic rat models study in depth the

neurotoxic conversion of a-synuclein from soluble to

insoluble and fibrillar inclusions (Nuber et al. 2013), as

well as its role on cell proliferation in the subventricular

zone and olfactory bulb (Lelan et al. 2011). Finally, it has

been demonstrated, using a novel mouse model displaying

conditional bulbar-specific expression of a-synuclein, that

there is a reduction of dopamine signaling in olfactory bulb

interneurons, but increased dopaminergic tone in midbrain

regions (Nuber et al. 2011), thus supporting the close link

between a-synuclein and dopamine shown ex vitro and

in vitro to underpin the selective vulnerability of dopami-

nergic neurons (Lee et al. 2011; Nakaso et al. 2013;

Yamakawa et al. 2010).

Taken together, data on neuroanatomical connections

and changes in a-synuclein aggregation over time, both in

humans and transgenic models, permit some conclusions to

be reached. Regarding the question—where does PD

pathology begin in the brain?—it seems clear that the

predictable sequence generally begins in olfactory struc-

tures and in the dorsal motor vagal and glossopharyngeal

nerves (Braak et al. 2006a) and nuclei (Del Tredici et al.

2002). Regarding the question—where does PD pathology

end in the brain?—it has been assumed that, during retro-

grade caudorostral progression (stages I–II), pathology is

confined to the medulla oblongata, pontine tegmentum, and

anterior olfactory structures. In stages III–IV, pathology

reaches the mid- and forebrain—including the mesocor-

tex—and in stages V–VI the pathology is seen in neocor-

tical association areas. However, in a small percentage of

cases the distribution pattern of Lewy bodies and neurites

diverges from this staging scheme; instead there is pre-

dominant involvement of olfactory structures and the

amygdala in the virtual absence of brainstem pathology.

This is indicative of anterograde rostrocaudal progression.

However, the majority of such divergent cases had con-

comitant AD pathology (Braak et al. 2006b), raising con-

troversies regarding Braak staging in these individuals

(Kalaitzakis et al. 2008).

Regarding possible neuroanatomical pathways for

anterograde and or retrograde progression (Fig. 5), it is

plausible to imagine a retrograde pathway including two or

three synaptic relays from the vagal nucleus to the locus

coeruleus, gigantocellular and raphe nuclei, then extending

Brain Struct Funct (2014) 219:1513–1526 1521

123

to the substantia nigra (violet arrows in Fig. 5; Braak et al.

2003b). Alternatively, perhaps a better explanation for the

divergent cases commented above is that a trisynaptic relay

pathway could extend both anterogradely from the olfac-

tory tubercle (Newman and Winans 1980) as well as an-

terogradely and retrogradely from the entorhinal cortex

(Loughlin and Fallon 1984) to the substantia nigra (blue

arrows in Fig. 5). In this context, the anterior olfactory

nucleus is an early and preferential site of pathology and,

interestingly, is centripetally and centrifugally connected

with the remainder of the olfactory structures, and also

with at least 27 ‘non-olfactory’ structures (Brunjes et al.

2005); this suggests that this nucleus could be the center of

confluence of a-synucleinopathy due to its particular con-

nections (Fig. 5a). This may be relevant to the controversy

regarding temporal Braak staging because it is possible that

a-synuclein does not aggregate linearly in different struc-

tures, and instead follows an anterograde or retrograde

pathway through modulatory systems via the locus coeru-

leus and raphe nuclei to the olfactory bulb and anterior

olfactory nucleus (black arrows in Fig. 5; Shipley and

Adamek 1984)—as has been demonstrated after intranasal

administration of horseradish peroxidase and transport to

these neuromodulatory nuclei (Shipley 1985). In this sense,

it has been hypothesized that early degeneration of nor-

adrenergic and serotonergic inputs to the olfactory bulb

leads to an increase of dopaminergic bulbar interneurons as

a compensatory mechanism (Mundinano et al. 2011). An

alternative pathway could be also hypothesized between

the dorsal nucleus of the vagus or substantia nigra to the

piriform or entorhinal cortices via the central nucleus of the

amygdala (green arrows in Fig. 5; Volz et al. 1990).

Regarding asymmetry in neuropathology and motor man-

ifestation in PD (Hobson 2012), it should be taken into

account that retrograde vagal pathways are predominantly

ipsilateral, whereas most olfactory structures, particularly

the anterior olfactory nucleus, show (in addition to ipsi-

lateral connections) important contralateral and commis-

sural projections. All these potential neuroanatomical

pathways, and many others not discussed here, should be

considered when analyzing the temporal progression of a-

synuclein within the nervous system as well as its possible

relationship with asymmetry in PD.

Conclusions

We have addressed several questions. Are there predomi-

nantly two independent routes of spreading of pathology

through the vagal and olfactory systems? Does progression

occur anterogradely and/or retrogradely? Are these two

routes interconnected? How does such spreading relate to

the asymmetry of pathology that is often seen in PD? A

growing body of evidence supports the hypothesis that a-

synuclein is a prion-like protein and PD a prion-like dis-

order. Spreading of a-synucleinopathy through the

peripheral and central nervous system occurs through

specific neuronal pathways but, in most pathways, it is far

from clear whether this occurs anterogradely and/or retro-

gradely. Many data support the idea of retrograde poly-

synaptic pathways to the substantia nigra via the vagus

nerve and brainstem nuclei. The olfactory system shows

early a-synucleinopathy involvement, but data on Lewy

pathology across time are not conclusive regarding a sim-

ple linear anterograde progression. Progression of a-syn-

uclein aggregation through the olfactory system, however,

may help to explain PD cases not matching retrograde

vagal spreading. Among olfactory structures, the anterior

olfactory nucleus is primarily and preferentially involved

by a-synucleinopathy, likely due its multiple and particular

connections in and out the olfactory system. Alternative

pathways including the central amygdala and neuromodu-

latory serotonergic and noradrenergic pathways to the

olfactory bulb should be considered for spreading of a-

synucleinopathy. Therefore, although vagal (retrograde)

and olfactory (anterograde) routes appear to be indepen-

dent, the data reviewed suggest that both pathways may be

interconnected at several levels, including bidirectional

spreading. Asymmetry observed in PD should be analyzed

under the perspective of ipsilateral, contralateral, and

commissural projections. The possibility of multiple and

simultaneous focus for a-synuclein dissemination, includ-

ing vagal, olfactory, and some other routes, should be

further investigated.

Acknowledgments Supported by the Spanish Ministry of Science

and Innovation (current Ministry of Economy and Competitiveness)/

FEDER (BFU2010-15729). The assistance of International Science

Editing in revising the English version of the manuscript is

acknowledged.

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