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??-Synuclein in the olfactory system in Parkinson's disease: Role of neural
connections on spreading pathology
Article in Brain Structure and Function · October 2013
DOI: 10.1007/s00429-013-0651-2 · Source: PubMed
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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: [email protected]
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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