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Olfactory system pathology as a model of Lewy neurodegenerative disease
Journal of the Neurological Sciences 289 (2010) 49–54
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Olfactory system pathology as a model of Lewy neurodegenerative disease John E. Duda ⁎ Parkinson's Disease Research, Education and Clinical Center/MS #127, Philadelphia VA Medical Center, 3900 Woodland Avenue, Philadelphia, PA 19104, United States Department of Neurology, University of Pennsylvania School of Medicine, United States
a r t i c l e
i n f o
Available online 23 September 2009 Keywords: Olfaction Parkinson's disease Lewy bodies Neurodegeneration Pathophysiology
a b s t r a c t Olfactory dysfunction has gained recognition as an early and nearly universal feature of Lewy body Parkinson's disease (PD). Recently, research efforts have focused on the use of early non-motor symptoms of PD as early biomarkers and have suggested that investigating neurodegeneration in the aspects of the nervous system subserving these symptoms may offer important insights into the pathophysiology of Lewy body PD. Therefore, there has been interest in characterizing the pathology observed in the olfactory bulb and system of patients with PD, dementia with Lewy bodies and perhaps more importantly, in subjects with incidental Lewy pathology, defined as people with Lewy pathology without evidence of Parkinsonism or dementia during life. The olfactory bulb may be ideally suited to investigations into the pathophysiology of the Lewy body disorders as it is one of the few areas of the brain wherein the entirety of neurons susceptible to Lewy neurodegeneration, including the dendritic arborization, cell soma, axon and synaptic terminals, can be examined in the same preparation. Interestingly, there is a lack of Lewy neurodegeneration in the dopaminergic neurons of the olfactory bulb and paradoxically, an apparent increase in dopaminergic neurons in some PD patients compared to controls. In this report, the known neuropathology of the olfactory system in PD will be reviewed and the advantages of investigating degeneration of the olfactory bulb as a model of Lewy neurodegeneration will be discussed. Published by Elsevier B.V.
1. Introduction Although they have been recognized as a pathologic hallmark of PD for nearly 100 years, the role that Lewy bodies (LBs); intracytoplasmic peri-nuclear protein aggregates), play in the neurodegenerative cascade of the Lewy body disorders, including PD and dementia with Lewy bodies (DLB) is still unclear. Indeed, it is not clear if the aggregation of alpha-synuclein into insoluble intra-cellular aggregates is one of the primary causes of neuronal demise, a protective mechanism against a second deleterious pathophysiological process or a relatively inconsequential epiphenomenon. The answer to this question will be instrumental in guiding the development of future therapeutic strategies for PD and other related synucleinopathies. Olfactory dysfunction is increasingly recognized as a consistent feature of the symptomatology of PD. Indeed, current estimates of the prevalence of olfactory dysfunction of up to 96% suggest that it is more common than tremor, at approximately 70%, one of the well-accepted cardinal symptoms of PD [1–3]. While olfactory dysfunction has been recognized as a feature of PD for almost 30years, there has been relatively limited investigation into the underlying pathophysiology of this prominent aspect of the disease. Existing reports have demonstrated several alterations in the olfactory bulb and tract in PD including the ⁎ Parkinson's Disease Research, Education and Clinical Center/MS #127, Philadelphia VA Medical Center, 3900 Woodland Avenue, Philadelphia, PA 19104, United States. E-mail address: [email protected]. 0022-510X/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jns.2009.08.042
existence of LBs and Lewy neurites (LNs), a decrease in the number of anterior olfactory nucleus neurons, and a paradoxical increase in the number of periglomerular dopaminergic neurons in some patients. However, very little is known about the relationship between these alterations, and few of these investigations focused on the pathological alterations that occur in preclinical or early PD, during the early phases of the degenerative process. The olfactory bulb and tract do offer a unique system in which to study neurodegeneration in PD because of a well-described cytoarchitecture and the acknowledgement that a simple dissection enables the assessment of a population of neurons, (including dendritic arborization, cell soma, axon and synaptic terminals) that are susceptible to Lewy neurodegeneration, within the sample. 2. Neuroanatomy of human olfactory bulb Having long served as a subject of experimental investigation into the anatomy and physiology of the nervous system, the olfactory bulb remains one of the most studied and best characterized aspects of the nervous system. While less well-organized in its cytoarchitectural appearance than in many other mammalian species, the human olfactory bulb maintains a fundamentally similar 6 layer organization including, from outermost to innermost, the olfactory nerve layer, glomerular layer, external plexiform layer, mitral cell layer, internal plexiform layer and granule cell layer [4,5] (Fig. 1). The outermost layer of the olfactory bulb, the olfactory nerve layer, is made up of
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Fig. 1. Olfactory bulb neuroanatomy schematic. Bipolar olfactory receptor neurons of the olfactory epithelium project axons through the cribriform plate that diverge upon entering the olfactory bulb to form the olfactory nerve layer and coalesce with axons from olfactory receptor neurons with identical olfactory receptor genes to form glomeruli. In the glomeruli, olfactory receptor neurons synapse with mitral (M) and tufted (T) cells and are modulated by periglomerular neurons and centrifugal axons from brainstem nuclei. Granule cells (G) project from the granular layer to modulate mitral and tufted cells. Mitral and tufted cells are the main output neurons of the olfactory bulb, projecting to the intrabulbar portion of the anterior olfactory nucleus as well as the primary olfactory cortices via the lateral olfactory tract. See text for additional details.
unmyelinated axons from the bipolar olfactory receptor neurons of the olfactory epithelium. These fibers fasciculate into bundles as they traverse the cribriform plate and, upon entering the olfactory bulb, disperse to form the olfactory nerve layer. The terminals from these fibers synapse upon the branched dendrites of mitral and tufted cells in the olfactory glomeruli of the glomerular layer. Mitral and tufted cells are the primary efferent projection neurons from the olfactory bulb and have one primary dendrite that projects to a single glomerulus and several secondary dendrites that project laterally to terminate in the external plexiform layer (EPL) and contribute significantly to the neuropil of the EPL. The mitral cells predominantly occupy a narrow layer deep to the external plexiform layer referred to as the mitral cell layer. Tufted cells however, are spread throughout the external plexiform layer and in general are smaller than mitral cells and have distinct projection patterns. Mitral and tufted cell axons project centripetally to innervate the anterior olfactory nucleus (AON) and extend through the lateral olfactory tract to primary olfactory cortices. Deep to the mitral cell layer is another narrow layer, the internal plexiform layer (IPL) that is made up primarily of axon collaterals from mitral and tufted cells. Below the IPL is the granule cell layer, the most densely populated layer made up of the cell bodies of axonless granule cells whose primary dendrites project out to the external plexiform layer. Unlike other vertebrates, a component of the AON is found within the olfactory bulb, deep to the granule cell layer, as the intrabulbar AON. Neurons of the AON receive the first synapse from axons leaving the olfactory bulb and are thought to reinforce impulses received from ORNs of the olfactory mucosa.
In addition to granule cells there is another class of intrinsic neurons, the periglomerular (PG) cells, which also modulate the output of the mitral and tufted cells. A subset of PG cells represents the primary population of dopaminergic neurons within the olfactory bulb. The cell bodies of PG cells surround the glomeruli and send short dendrites into a glomerulus where they intermingle with the axons of the ORNs and dendrites of mitral and tufted cells. PG cell axons then project laterally within the glomerular layer to modulate the output of adjacent glomeruli.
3. Neuropathological studies of olfaction in PD Just as clinical studies of olfaction in PD are limited in their ability to discern the characteristics of olfactory dysfunction in the early stages of the illness, current understanding of the pathophysiology of olfactory dysfunction in PD relies primarily on studies of late stage illness. Of course, pathological studies of early PD are rare in general, due to the prolonged survival of most patients before autopsy. However, what is known about the pathology of late stage PD may enable the identification of patients with early or preclinical PD at autopsy. While the pathological basis of the motor dysfunction in PD has been studied extensively for decades, concerted efforts to understand the pathological basis of olfactory dysfunction span little more than 10 years. Nonetheless, several avenues of research have uncovered pathological alterations in the olfactory system in PD that now suggest multiple causes of olfactory dysfunction in PD.
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As the only component of the olfactory system available for biopsy, the olfactory epithelium (OE) of patients with a wide variety of neurodegenerative diseases was examined for evidence of diseasespecific pathology that could be used as a biomarker [6]. In this report, antibodies specific for α-, β-, and γ-synuclein were used to examine the olfactory mucosa of patients with PD, DLB, Alzheimer's disease (AD), multiple system atrophy (MSA), and controls without a neurological disorder. Although antibodies to α- and β-synuclein detected abnormal dystrophic neurites in the OE of patients with neurodegenerative disorders, similar pathology was also seen in the OE of controls. More significantly, it was shown for the first time that α-, β-, and γ-synuclein are differentially expressed in cells of the OE and respiratory epithelium and that α-synuclein is the most abundant synuclein in the olfactory mucosa, where it is prominently expressed in ORNs. Moreover, α- and γ-synuclein were prominent in the OE basal cells, which include the progenitor cells of the ORNs in the OE. Thus, the synucleins may play a role in the regeneration and plasticity of ORNs in the adult human OE. However, there was no evidence of disease-specific pathology at this first step of the olfactory pathway. One of the first reports of disease-specific pathology in the olfactory system in PD was a brief but important report by Daniel and Hawkes that suggested that ubiquitin immunostaining of the olfactory bulb and tract could reliably distinguish PD patients at autopsy [7]. The olfactory bulb and tract from a series of 18 cases (9 patients with a clinical diagnosis of PD and 9 controls) were examined blindly and then compared with the results of the full autopsy. In 8 of the 9 PD patients, the examiners found LBs throughout the olfactory bulb and tract sufficient to make a diagnosis of probable PD. Two of the patients had many LBs as well as distended ubiquitinated neurites that would now be referred to as Lewy neurites (LNs; intracytoplasmic protein aggregates occurring within the dendritic or axonal processes of neurons) and are also recognized as a pathological hallmark of PD. Also, one patient was found to have occasional glial cytoplasmic inclusions (GCIs) in the olfactory bulb and tract, which are the pathological hallmark of another neurodegenerative disorder characterized by Parkinsonism, MSA. In contrast, none of the control patients had similar pathology. Interestingly, full autopsies confirmed the 8 cases that were diagnosed with probable PD on the basis of olfactory bulb and tract examination alone, and found diffuse cortical LBs in the two cases with the highest burden of LBs and LNs in the olfactory system. In addition, the one brain that had GCIs in the olfactory system was confirmed to have MSA by full autopsy. Therefore, this study demonstrated that one of the pathological hallmarks of PD, the formation of LBs and LNs, is a commonplace occurrence within the olfactory system and may contribute to the olfactory dysfunction in PD. After Polymeropoulos and colleagues identified a mutation in the α-synuclein gene on chromosome 4q21–23 that segregated with PD in a large Italian kindred and three Greek kindreds [8], research focused on understanding the role of α-synuclein in sporadic PD. It was soon realized that while α-synuclein is a normal component of presynaptic terminals, it is also robustly expressed in LBs and LNs, collectively referred to as Lewy pathology (LP), in all forms of LB disease (PD, DLB, LB variant of Alzheimer's disease) and is the most sensitive marker of LP [9,10]. Therefore, researchers began using antibodies to α-synuclein to demonstrate the full extent of LP in the olfactory system. Recently, Beach and colleagues examined 58 cases of PD, 35 cases of DLB, 21 cases with incidental LBs (ILBs; defined as the presence of LBs in a patient with no clinical history of neurodegenerative disease), 69 elderly controls and AD cases with and without LP. In this cohort, they observed that the overwhelming majority of cases of PD and DLB had olfactory bulb LP [11]. In addition 2/3rds of cases with ILBs elsewhere in the brain also had them in the olfactory bulb while only 5/64 elderly cases had LP confined to the olfactory bulb. The region of the bulb most frequently involved was the intrabulbar AON followed by the IPL which contained only LNs. In
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comparing frequencies between patient subgroups, the authors found that olfactory bulb LP was > 90% sensitive and specific for distinguishing PD or DLB patients from elderly controls and olfactory bulb LP density correlated well with LP density in other brain regions. In addition to confirming the presence of α-synuclein pathology in the form of LBs and LNs in the olfactory bulb, a report by Tsuboi and colleagues presented the unexpected finding of tau-positive neurofibrillary degeneration in the AON in cases with Lewy body pathology, particularly those with concomitant AD pathology [12]. The tau pathology also unexpectedly correlated with cortical LB counts. While the relationship between α-synuclein and tau pathology is unclear, it is known that there is an abnormal accumulation of tau aggregates in familial PD [13] and there is growing evidence to support a synergistic effect in aggregation [14,15]. The significance of LP, in the olfactory system is strengthened with the realization that it may be one of the few LP ‘induction sites’ in the brain. In a comprehensive assay of LP in the brains of 110 patients including 41 with clinical PD and 69 with ILBs, Braak and colleagues developed a staging system of the progression of LP and determined that the olfactory system is one of 3 ‘induction sites’ for LP [16,17]. That is, LP seems to appear independently in the dorsal motor nucleus of the vagus and intermediate reticular zone of the medulla as well as the olfactory system (including the olfactory bulb and AON) and progresses throughout the brain from these areas. In addition, Braak and colleagues recognized that the earliest evidence of LP is the deposition of LNs, which precedes the formation of LBs in nearly all areas studied. Therefore, the olfactory system seems to be particularly susceptible to Lewy neurodegeneration, is virtually always involved in the earliest stages of the disease and appears to be an attractive target for further investigation. In addition, Lerner and Bagic recently reviewed anatomic connections between the olfactory system and brainstem nuclei and suggested a hypothesis for the spread of Lewy pathology from the olfactory system to many of the selectively vulnerable brainstem nuclei [18]. They propose that a preponderance of pathways leading to the dorsal motor nucleus of the vagus may engender selective vulnerability to these neurons in a topdown fashion that obviates the need for transmission from the enteric nervous system as has been proposed elsewhere [19]. Neuronal loss, another pathological hallmark of PD, was confirmed in the olfactory system in further studies by Hawkes and colleagues [20]. In this report, the olfactory bulbs and tracts from 7 patients with histologically-proven PD and 7 age-matched controls were examined for gross atrophy, LB and LN accumulation and neuronal counts in the AON. Significant atrophy was grossly apparent in several of the PD specimens and quantification of bulb thickness revealed a statistically significant difference between the PD patients and controls. Moreover, a mean reduction in the total number of AON neurons was found in the PD patients compared to controls, with only one count in a PD patient overlapping the values in the control group. Significantly, a strong correlation was found between AON neuronal counts and disease duration in the PD patients, with dramatically reduced numbers in the patients with longest duration, suggesting an ongoing neurodegenerative process. In addition to LP deposition and neuronal loss in the olfactory bulb, degenerative changes have been demonstrated in the olfactoryrelated cortices in PD and in patients with ILBs. Silveira-Moriyama and colleagues examined 10 cases of PD, 7 cases of with ILBs and 5 healthy controls for Lewy pathology in the primary olfactory cortices including the olfactory tubercle, the frontal piriform cortex and the temporal piriform cortex [21]. They observed Lewy pathology in each area in all cases of ILBs and PD, but none of the controls. Interestingly, although Braak and colleagues recognized that LP spreads from the olfactory bulb and AON into olfactory cortices including the olfactory tubercle, periamygdaloid, piriform and entorhinal cortices, it did not seem to spread from there to adjacent cortices unrelated to olfactory processing [17]. In the material that Braak examined, it seems that spread into non-olfactory neocortical regions was only possible after
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LP affected the anteromedial temporal mesocortex and magnocellular nuclei of the basal forebrain, which raises intriguing questions about the mechanisms of LP progression throughout the brain. Two novel observations concerning the pathology of olfactory dysfunction in PD arose in separate reports from Huisman and colleagues in the past few years [22–24]. In 2003, they reported their findings concerning misrouted olfactory fibers and ectopic olfactory glomeruli in 20 normal adults, 12 AD patients and 16 Parkinsonian patients [22]. As discussed previously, in the normal mammalian olfactory system, the axons of ORNs of the OE project through the cribriform plate in the olfactory nerves, into the olfactory nerve layer of the olfactory bulb, and synapse on the dendrites of mitral and tufted neurons in the olfactory glomeruli of the glomerular layer. In the adult human, they were able to demonstrate the presence of misrouted olfactory fibers, defined as olfactory marker protein (OMP) labeled ORN axons that penetrated deeper into the bulb than the glomerular layer. These misrouted olfactory fibers were found in all patient groups including normal adults and did not correlate with age or gender, suggesting that they were part of the normal aging process in the olfactory bulb. In addition, in the Parkinsonian patients, which included not only patients with autopsy-proven PD, but patients with other causes of Parkinsonism including progressive supranuclear palsy (PSP), MSA and vascular disease, they found ectopic glomeruli, defined as more or less spherical structures containing OMP-positive fibers that were located outside of the glomerular layer. They found no
correlation with duration of illness and the number of ectopic olfactory glomeruli. Since all patients with a variety of causes of Parkinsonism displayed ectopic glomeruli, the authors concluded that this phenomenon was not related to the specific pathophysiology of PD, but must be secondary to degeneration of the substantia nigra, regardless of the mechanism. Therefore, neither misrouted olfactory fibers nor ectopic glomeruli seem to offer significant insight into the etiology of the olfactory dysfunction of PD. The same group, in 2004, published results of an examination of the number of dopaminergic PG neurons in the glomerular layer of the olfactory bulb in 10 patients with autopsy-proven PD and 10 control patients [23]. Interestingly, the authors, using stereological quantitative methods, demonstrated a paradoxical 100% increase in the number of tyrosine hydroxylase-containing PG neurons, that previous studies suggest are dopaminergic [25], in the PD patients compared to controls. While no correlation between the duration of PD and the number of dopaminergic periglomerular neurons was demonstrated, the mean duration of disease was 12 years and the authors admit that a relationship may have been obscured by only examining the final stage of the disease. However, in a follow-up study examining twice as many subjects, the same authors recently reported that there was no difference in the number of dopaminergic neurons between male and female PD patients and male control subjects [24]. Female control subjects did have significantly less dopaminergic neurons than male controls for unclear reasons but there was significant variability, even
Fig. 2. Neuritic dystrophy hypothesis of Lewy neurodegeneration. α-synuclein is produced in the cell soma, transported down the axon and located at the synaptic terminal (A). In this model, the aggregation of α-synuclein begins as LNs are formed within axons of neurons (B). These axonal aggregates enlarge by the sequestration of additional α-synuclein, other proteins and subcellular elements, until interruption of axonal transport occurs (B/C). The axonal transport blockade causes an accumulation of anterogradely transported subcellular elements proximal to the occlusion and a supersaturation of the cell soma with α-synuclein (C). The supersaturation of α-synuclein predisposes the neuron to the formation of somatic α-synuclein aggregates, known as Lewy bodies (LBs) (C). LB growth continues until the normal somatic architecture is disrupted, with nuclear displacement, and α-synuclein aggregates form within proximal dendrites as LNs (D). Finally, continued axonal transport blockade prohibits distal axonal viability, leading to axonal degeneration (D) with the possibility of resultant neuronal degeneration. Reproduced from reference [38] with permission from S. Karger AG, Basel.
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within controls, with as few as zero and as many as 60,000 dopaminergic neurons in the olfactory bulb. The authors originally speculated, based on animal studies, a mechanism of olfactory dysfunction in PD due to an increased dopaminergic inhibition of the neurotransmission between the ORNs and mitral cells in the glomeruli, although the recent study casts doubt on this hypothesis. Although the role that PG dopaminergic neurons play in the olfactory dysfunction seen in PD is now in question, gender-specific alterations in the density of these neurons represents an opportunity to investigate the pathophysiological impact of gender on PD risk [26,27]. 4. Olfactory bulb as model of adult neurogenesis Another exciting aspect of investigating the olfactory system in PD is the possibility of gaining further understanding of the mechanisms of neurogenesis in the adult human nervous system. The presence of new neurons in the olfactory bulb of the adult human was suggested recently in two immunohistochemical analyses using markers of proliferation and immature neuronal state to confirm the existence of neurons in the olfactory bulb with an immature neuronal phenotype [28,29]. In a study by Bedard and Parent, neurons expressing immature neuronal markers were found to be granular and periglomerular dopaminergic neurons and provided strong evidence for adult neurogenesis of these cell types in humans. However, these studies were carried out on patients who had no evidence of neurodegeneration. In contrast, Höglinger and colleagues examined 4 patients with autopsy-confirmed PD and compared the number of progenitor cells in the subependymal zone and olfactory bulb to the corresponding numbers in matched control patients [30]. In both areas, the number of neurons was decreased in the PD patients and suggests that chronic impairment of neural regeneration might also contribute to the olfactory dysfunction in PD.
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by the neuron. Eventually, the blockade of axonal transport disallows distal axonal viability and ultimately leads to axonal degeneration with the possibility of resultant neuronal degeneration. While most pathological systems make testing this hypothesis difficult at best because of the long distances between the dendritic aborizations, cell bodies and synaptic terminals of most neurons susceptible to Lewy neurodegeneration, the olfactory bulb and tract are the exception. They provide an easily dissectible preparation that includes all of these cellular components in a population of mitral and tufted cells that project from the glomerular layer to synapse in the AON. By addressing the sequence in which pathological alterations occur in the olfactory bulb in early Lewy neurodegeneration, and in particular, assessing whether LB and LN formation precedes frank neuronal death, evidence supporting or contradicting the neuritic dystrophy hypothesis would be generated. In addition, because of the carefully defined cytoarchitecture of the olfactory bulb, investigation of Lewy neurodegeneration in this system may offer insight into another hypothesis regarding the progression of LP throughout the brain, i.e. the ‘prion-like’ theory of Lewy neurodegeneration [18,40]. In this hypothesis, cellular vulnerability is transmitted in a ‘prion-like’ manner via a conformationally abnormal form of a protein such as α-synuclein. If this hypothesis is correct, then the sequence of vulnerable neurons should be ORNs followed by mitral and tufted cells, followed by AON neurons, etc. Braak and colleagues have also identified additional features that most neurons susceptible to Lewy neurodegeneration share [40]. These include the characteristic of being of the class of projection neurons rather than local circuit neurons and having long, mostly unmyelinated axons. To date, in the olfactory bulb, LP has been demonstrated in projection neurons including mitral cells and AON neurons, but has not been demonstrated in local circuit
5. Olfactory bulb pathology as a model of Lewy neurodegeneration Importantly, among the Parkinsonian disorders, the alteration in ability to smell is relatively specific to PD. Thus, decreased ability to smell is absent, or present infrequently or only to a minor degree, in several of the other common causes of Parkinsonism including PSP [31], MSA [32], essential tremor [33,34], and parkinsonism induced by MPTP [35]. Additionally, earlier studies found that subtle differences in olfactory dysfunction exist among subtypes of PD. That is, slightly greater dysfunction is present in patients with postural instability-gait predominant PD than with tremor-predominant PD [36]. More recently, Ondo and Lai found that tremor-predominant PD patients with a family history of tremor had less olfactory dysfunction than other PD groups [37]. In light of these findings, examining the substrate of olfactory dysfunction in PD may provide new insight into the pathophysiological processes occurring in brain areas involved in smell perception, that are specific to PD and presumably, independent of nigrostriatal degeneration. Indeed, investigation of the pathophysiology of olfactory dysfunction in PD should lead to important insight into the mechanisms of neurodegeneration in PD throughout the brain. One hypothesis that may be tested by investigation of olfactory system neurodegeneration involves the role of neuritic dystrophy in Lewy neurodegeneration [38,39]. In this hypothetical model (Fig. 2), various environmental, genetic or intrinsic factors cause a conformational change in the structure of α-synuclein to β-pleated sheet and a propensity to aggregate into pathologic inclusions. These inclusions start in the axonal compartment as LNs and grow by accumulating additional α-synuclein and trapping other proteins and/or organelles. These inclusions expand until they congest the axonal compartment, resulting in interruption of axonal transport. This cessation of transport may produce abnormally high concentrations of α-synuclein in the cell soma, possibly triggering LB formation, either as a consequence of supersaturation or as a physiologic response
Fig. 3. Lewy pathology of the olfactory bulb. Immunostaining with an antibody specific to aggregated alpha-synuclein [41] demonstrates Lewy neurite and Lewy body pathology in the olfactory bulb of a patient with Parkinson's disease. Note the dense accumulation of Lewy pathology in the internal plexiform layer and anterior olfactory nucleus. Scale bar=100 μm. Abbreviations: ONL: olfactory nerve layer; GLOM: glomerular layer; EPL: external plexiform layer; MCL: mitral cell layer; IPL: internal plexiform layer; GRAN: granular layer; iAON: intrabulbar portion of anterior olfactory nucleus.
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neurons such as PG cells and granule cells. Further examination of neuronal vulnerabilities in the olfaction system will lead to a greater understanding of particular neuronal characteristics conveying vulnerability to Lewy neurodegeneration. 6. Conclusion As elsewhere in the brain, there is compelling reason to believe that LP plays a detrimental role in the neurodegenerative process affecting neurons of the olfactory system in PD and related disorders. As an early induction site, the olfactory system is particularly enticing for investigation into the early stages of the illness, presumably represented in the brains of people with ILBs. Due to the availability of easily dissectible preparations including whole neurons susceptible to Lewy neurodegeneration (Fig. 3), the olfactory system is particularly compelling as a model of how the formation of LNs and LBs affects host cells. Acknowledgements Dr. Duda is supported by grants from the Biomedical Laboratory Research and Development Service of the United States Department of Veterans Affairs. The author is grateful to Andrew Sheller for assistance in the development of Fig. 1 and to Joseph V. Noorigian for assistance with Fig. 3. References [1] Hawkes CH, Shephard BC, Daniel SE. Olfactory dysfunction in Parkinson's disease. J Neurol Neurosurg Psychiatry 1997;62:436–46. [2] Doty RL, Deems DA, Stellar S. Olfactory dysfunction in parkinsonism: a general deficit unrelated to neurologic signs, disease stage, or disease duration. Neurology 1988;38:1237–44. [3] Hower RE, Rick JH, Balderston CC, Weintraub D, Sidenvall B, Stern MB, et al. Olfactory dysfunction and Parkinson's disease progression. Neurology 2008;70:A397. [4] Kratskin IL, Belluzzi O. Anatomy and neurochemistry of the olfactory bulb. In: Doty RL, editor. Handbook of olfaction and gustation. New York: Marcel Dekker; 2003. p. 235–76. [5] Shepherd GM, Greer CA. Olfactory bulb. In: Shepherd GM, editor. The synaptic organization of the brain. New York: Oxford University Press; 1997. p. 159–203. [6] Duda JE, Shah U, Arnold SE, Lee VM, Trojanowski JQ. The expression of alpha-, beta-, and gamma-synucleins in olfactory mucosa from patients with and without neurodegenerative diseases. Exp Neurol 1999;160:515–22. [7] Daniel SE, Hawkes CH. Preliminary diagnosis of Parkinson's disease by olfactory bulb pathology. Lancet 1992;340:186. [8] Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 1997;276:2045–7. [9] Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alphasynuclein in Lewy bodies. Nature 1997;388:839–40. [10] Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. Alpha-synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc Natl Acad Sci U S A 1998;95:6469–73. [11] Beach TG, White III CL, Hladik CL, Sabbagh MN, Connor DJ, Shill HA, et al. Olfactory bulb alpha-synucleinopathy has high specificity and sensitivity for Lewy body disorders. Acta Neuropathol 2009;117:169–74. [12] Tsuboi Y, Wszolek ZK, Graff-Radford NR, Cookson N, Dickson DW. Tau pathology in the olfactory bulb correlates with Braak stage, Lewy body pathology and apolipoprotein epsilon4. Neuropathol Appl Neurobiol 2003;29:503–10. [13] Duda JE, Giasson BI, Mabon ME, Miller DC, Golbe LI, Lee VM, et al. Concurrence of alpha-synuclein and tau brain pathology in the Contursi kindred. Acta Neuropathol (Berl) 2002;104:7–11.
[14] Frasier M, Wolozin B. Following the leader: fibrillization of alpha-synuclein and tau. Exp Neurol 2004;187:235–9. [15] Lee VM, Giasson BI, Trojanowski JQ. More than just two peas in a pod: common amyloidogenic properties of tau and alpha-synuclein in neurodegenerative diseases. Trends Neurosci 2004;27:129–34. [16] Braak H, Del Tredici K, Bratzke H, Hamm-Clement J, Sandmann-Keil D, Rub U. Staging of the intracerebral inclusion body pathology associated with idiopathic Parkinson's disease (preclinical and clinical stages). J Neurol 2002;249(Suppl 3):III/1–5. [17] Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 2003;24:197–211. [18] Lerner A, Bagic A. Olfactory pathogenesis of idiopathic Parkinson disease revisited. Mov Disord 2008;23:1076–84. [19] Hawkes CH, Del Tredici K, Braak H. Parkinson's disease: a dual-hit hypothesis. Neuropathol Appl Neurobiol 2007;33:599–614. [20] Pearce RK, Hawkes CH, Daniel SE. The anterior olfactory nucleus in Parkinson's disease. Mov Disord 1995;10:283–7. [21] Silveira-Moriyama L, Holton J, Kingsbury A, Ayling H, Petrie A, Sterlacci W, et al. Regional differences in the severity of Lewy body pathology across the olfactory cortex. Neurosic Lett 2009;453:77–80. [22] Hoogland PV, van den BR, Huisman E. Misrouted olfactory fibres and ectopic olfactory glomeruli in normal humans and in Parkinson and Alzheimer patients. Neuropathol Appl Neurobiol 2003;29:303–11. [23] Huisman E, Uylings HB, Hoogland PV. A 100% increase of dopaminergic cells in the olfactory bulb may explain hyposmia in Parkinson's disease. Mov Disord 2004;19:687–92. [24] Huisman E, Uylings HB, Hoogland PV. Gender-related changes in increase of dopaminergic neurons in the olfactory bulb of Parkinson's disease patients. Mov Disord 2008;23:1407–13. [25] Smeets WJ, Gonzalez A. Catecholamine systems in the brain of vertebrates: new perspectives through a comparative approach. Brain Res Brain Res Rev 2000;33:308–79. [26] Van Den Eeden SK, Tanner CM, Bernstein AL, Fross RD, Leimpeter A, Bloch DA, et al. Incidence of Parkinson's disease: variation by age, gender, and race/ethnicity. Am J Epidemiol 2003;157:1015–22. [27] Shulman LM. Gender differences in Parkinson's disease. Gend Med 2007;4:8–18. [28] Bedard A, Parent A. Evidence of newly generated neurons in the human olfactory bulb. Brain Res Dev Brain Res 2004;151:159–68. [29] Liu Z, Martin LJ. Olfactory bulb core is a rich source of neural progenitor and stem cells in adult rodent and human. J Comp Neurol 2003;459:368–91. [30] Höglinger GU, Rizk P, Muriel MP, Duyckaerts C, Oertel WH, Caille I, et al. Dopamine depletion impairs precursor cell proliferation in Parkinson disease. Nat Neurosci 2004;7:726–35. [31] Doty RL, Golbe LI, McKeown DA, Stern MB, Lehrach CM, Crawford D. Olfactory testing differentiates between progressive supranuclear palsy and idiopathic Parkinson's disease. Neurology 1993;43:962–5. [32] Wenning GK, Shephard B, Hawkes C, Petruckevitch A, Lees A, Quinn N. Olfactory function in atypical parkinsonian syndromes. Acta Neurol Scand 1995;91:247–50. [33] Busenbark KL, Huber SJ, Greer G, Pahwa R, Koller WC. Olfactory function in essential tremor. Neurology 1992;42:1631–2. [34] Louis ED, Bromley SM, Jurewicz EC, Watner D. Olfactory dysfunction in essential tremor: a deficit unrelated to disease duration or severity. Neurology 2002;59:1631–3. [35] Doty RL, Singh A, Tetrud J, Langston JW. Lack of major olfactory dysfunction in MPTP-induced parkinsonism. Ann Neurol 1992;32:97–100. [36] Stern MB, Doty RL, Dotti M, Corcoran P, Crawford D, McKeown DA, et al. Olfactory function in Parkinson's disease subtypes. Neurology 1994;44:266–8. [37] Ondo WG, Lai D. Olfaction testing in patients with tremor-dominant Parkinson's disease: is this a distinct condition? Mov Disord 2005;20:471–5. [38] Duda JE. Pathology and neurotransmitter abnormalities of dementia with Lewy bodies. Dement Geriatr Cogn Disord 2004;17(Suppl 1):3–14. [39] Duda JE, Giasson BI, Lee VM-Y, Trojanowski JQ. Is the initial insult in Parkinson's disease and dementia with Lewy bodies a neuritic dystrophy? Ann NY Acad Sci 2003;991:295–7. [40] Braak H, Rub U, Gai WP, Del Tredici K. Idiopathic Parkinson's disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen. J Neural Transm 2003;110:517–36. [41] Duda JE, Giasson BI, Mabon ME, Lee VM, Trojanowski JQ. Novel antibodies to synuclein show abundant striatal pathology in Lewy body diseases. Ann Neurol 2002;52:205–10.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Olfactory system pathology as a model of Lewy neurodegenerative disease John E. Duda ⁎ Parkinson's Disease Research, Education and Clinical Center/MS #127, Philadelphia VA Medical Center, 3900 Woodland Avenue, Philadelphia, PA 19104, United States Department of Neurology, University of Pennsylvania School of Medicine, United States
a r t i c l e
i n f o
Available online 23 September 2009 Keywords: Olfaction Parkinson's disease Lewy bodies Neurodegeneration Pathophysiology
a b s t r a c t Olfactory dysfunction has gained recognition as an early and nearly universal feature of Lewy body Parkinson's disease (PD). Recently, research efforts have focused on the use of early non-motor symptoms of PD as early biomarkers and have suggested that investigating neurodegeneration in the aspects of the nervous system subserving these symptoms may offer important insights into the pathophysiology of Lewy body PD. Therefore, there has been interest in characterizing the pathology observed in the olfactory bulb and system of patients with PD, dementia with Lewy bodies and perhaps more importantly, in subjects with incidental Lewy pathology, defined as people with Lewy pathology without evidence of Parkinsonism or dementia during life. The olfactory bulb may be ideally suited to investigations into the pathophysiology of the Lewy body disorders as it is one of the few areas of the brain wherein the entirety of neurons susceptible to Lewy neurodegeneration, including the dendritic arborization, cell soma, axon and synaptic terminals, can be examined in the same preparation. Interestingly, there is a lack of Lewy neurodegeneration in the dopaminergic neurons of the olfactory bulb and paradoxically, an apparent increase in dopaminergic neurons in some PD patients compared to controls. In this report, the known neuropathology of the olfactory system in PD will be reviewed and the advantages of investigating degeneration of the olfactory bulb as a model of Lewy neurodegeneration will be discussed. Published by Elsevier B.V.
1. Introduction Although they have been recognized as a pathologic hallmark of PD for nearly 100 years, the role that Lewy bodies (LBs); intracytoplasmic peri-nuclear protein aggregates), play in the neurodegenerative cascade of the Lewy body disorders, including PD and dementia with Lewy bodies (DLB) is still unclear. Indeed, it is not clear if the aggregation of alpha-synuclein into insoluble intra-cellular aggregates is one of the primary causes of neuronal demise, a protective mechanism against a second deleterious pathophysiological process or a relatively inconsequential epiphenomenon. The answer to this question will be instrumental in guiding the development of future therapeutic strategies for PD and other related synucleinopathies. Olfactory dysfunction is increasingly recognized as a consistent feature of the symptomatology of PD. Indeed, current estimates of the prevalence of olfactory dysfunction of up to 96% suggest that it is more common than tremor, at approximately 70%, one of the well-accepted cardinal symptoms of PD [1–3]. While olfactory dysfunction has been recognized as a feature of PD for almost 30years, there has been relatively limited investigation into the underlying pathophysiology of this prominent aspect of the disease. Existing reports have demonstrated several alterations in the olfactory bulb and tract in PD including the ⁎ Parkinson's Disease Research, Education and Clinical Center/MS #127, Philadelphia VA Medical Center, 3900 Woodland Avenue, Philadelphia, PA 19104, United States. E-mail address: [email protected]. 0022-510X/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jns.2009.08.042
existence of LBs and Lewy neurites (LNs), a decrease in the number of anterior olfactory nucleus neurons, and a paradoxical increase in the number of periglomerular dopaminergic neurons in some patients. However, very little is known about the relationship between these alterations, and few of these investigations focused on the pathological alterations that occur in preclinical or early PD, during the early phases of the degenerative process. The olfactory bulb and tract do offer a unique system in which to study neurodegeneration in PD because of a well-described cytoarchitecture and the acknowledgement that a simple dissection enables the assessment of a population of neurons, (including dendritic arborization, cell soma, axon and synaptic terminals) that are susceptible to Lewy neurodegeneration, within the sample. 2. Neuroanatomy of human olfactory bulb Having long served as a subject of experimental investigation into the anatomy and physiology of the nervous system, the olfactory bulb remains one of the most studied and best characterized aspects of the nervous system. While less well-organized in its cytoarchitectural appearance than in many other mammalian species, the human olfactory bulb maintains a fundamentally similar 6 layer organization including, from outermost to innermost, the olfactory nerve layer, glomerular layer, external plexiform layer, mitral cell layer, internal plexiform layer and granule cell layer [4,5] (Fig. 1). The outermost layer of the olfactory bulb, the olfactory nerve layer, is made up of
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Fig. 1. Olfactory bulb neuroanatomy schematic. Bipolar olfactory receptor neurons of the olfactory epithelium project axons through the cribriform plate that diverge upon entering the olfactory bulb to form the olfactory nerve layer and coalesce with axons from olfactory receptor neurons with identical olfactory receptor genes to form glomeruli. In the glomeruli, olfactory receptor neurons synapse with mitral (M) and tufted (T) cells and are modulated by periglomerular neurons and centrifugal axons from brainstem nuclei. Granule cells (G) project from the granular layer to modulate mitral and tufted cells. Mitral and tufted cells are the main output neurons of the olfactory bulb, projecting to the intrabulbar portion of the anterior olfactory nucleus as well as the primary olfactory cortices via the lateral olfactory tract. See text for additional details.
unmyelinated axons from the bipolar olfactory receptor neurons of the olfactory epithelium. These fibers fasciculate into bundles as they traverse the cribriform plate and, upon entering the olfactory bulb, disperse to form the olfactory nerve layer. The terminals from these fibers synapse upon the branched dendrites of mitral and tufted cells in the olfactory glomeruli of the glomerular layer. Mitral and tufted cells are the primary efferent projection neurons from the olfactory bulb and have one primary dendrite that projects to a single glomerulus and several secondary dendrites that project laterally to terminate in the external plexiform layer (EPL) and contribute significantly to the neuropil of the EPL. The mitral cells predominantly occupy a narrow layer deep to the external plexiform layer referred to as the mitral cell layer. Tufted cells however, are spread throughout the external plexiform layer and in general are smaller than mitral cells and have distinct projection patterns. Mitral and tufted cell axons project centripetally to innervate the anterior olfactory nucleus (AON) and extend through the lateral olfactory tract to primary olfactory cortices. Deep to the mitral cell layer is another narrow layer, the internal plexiform layer (IPL) that is made up primarily of axon collaterals from mitral and tufted cells. Below the IPL is the granule cell layer, the most densely populated layer made up of the cell bodies of axonless granule cells whose primary dendrites project out to the external plexiform layer. Unlike other vertebrates, a component of the AON is found within the olfactory bulb, deep to the granule cell layer, as the intrabulbar AON. Neurons of the AON receive the first synapse from axons leaving the olfactory bulb and are thought to reinforce impulses received from ORNs of the olfactory mucosa.
In addition to granule cells there is another class of intrinsic neurons, the periglomerular (PG) cells, which also modulate the output of the mitral and tufted cells. A subset of PG cells represents the primary population of dopaminergic neurons within the olfactory bulb. The cell bodies of PG cells surround the glomeruli and send short dendrites into a glomerulus where they intermingle with the axons of the ORNs and dendrites of mitral and tufted cells. PG cell axons then project laterally within the glomerular layer to modulate the output of adjacent glomeruli.
3. Neuropathological studies of olfaction in PD Just as clinical studies of olfaction in PD are limited in their ability to discern the characteristics of olfactory dysfunction in the early stages of the illness, current understanding of the pathophysiology of olfactory dysfunction in PD relies primarily on studies of late stage illness. Of course, pathological studies of early PD are rare in general, due to the prolonged survival of most patients before autopsy. However, what is known about the pathology of late stage PD may enable the identification of patients with early or preclinical PD at autopsy. While the pathological basis of the motor dysfunction in PD has been studied extensively for decades, concerted efforts to understand the pathological basis of olfactory dysfunction span little more than 10 years. Nonetheless, several avenues of research have uncovered pathological alterations in the olfactory system in PD that now suggest multiple causes of olfactory dysfunction in PD.
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As the only component of the olfactory system available for biopsy, the olfactory epithelium (OE) of patients with a wide variety of neurodegenerative diseases was examined for evidence of diseasespecific pathology that could be used as a biomarker [6]. In this report, antibodies specific for α-, β-, and γ-synuclein were used to examine the olfactory mucosa of patients with PD, DLB, Alzheimer's disease (AD), multiple system atrophy (MSA), and controls without a neurological disorder. Although antibodies to α- and β-synuclein detected abnormal dystrophic neurites in the OE of patients with neurodegenerative disorders, similar pathology was also seen in the OE of controls. More significantly, it was shown for the first time that α-, β-, and γ-synuclein are differentially expressed in cells of the OE and respiratory epithelium and that α-synuclein is the most abundant synuclein in the olfactory mucosa, where it is prominently expressed in ORNs. Moreover, α- and γ-synuclein were prominent in the OE basal cells, which include the progenitor cells of the ORNs in the OE. Thus, the synucleins may play a role in the regeneration and plasticity of ORNs in the adult human OE. However, there was no evidence of disease-specific pathology at this first step of the olfactory pathway. One of the first reports of disease-specific pathology in the olfactory system in PD was a brief but important report by Daniel and Hawkes that suggested that ubiquitin immunostaining of the olfactory bulb and tract could reliably distinguish PD patients at autopsy [7]. The olfactory bulb and tract from a series of 18 cases (9 patients with a clinical diagnosis of PD and 9 controls) were examined blindly and then compared with the results of the full autopsy. In 8 of the 9 PD patients, the examiners found LBs throughout the olfactory bulb and tract sufficient to make a diagnosis of probable PD. Two of the patients had many LBs as well as distended ubiquitinated neurites that would now be referred to as Lewy neurites (LNs; intracytoplasmic protein aggregates occurring within the dendritic or axonal processes of neurons) and are also recognized as a pathological hallmark of PD. Also, one patient was found to have occasional glial cytoplasmic inclusions (GCIs) in the olfactory bulb and tract, which are the pathological hallmark of another neurodegenerative disorder characterized by Parkinsonism, MSA. In contrast, none of the control patients had similar pathology. Interestingly, full autopsies confirmed the 8 cases that were diagnosed with probable PD on the basis of olfactory bulb and tract examination alone, and found diffuse cortical LBs in the two cases with the highest burden of LBs and LNs in the olfactory system. In addition, the one brain that had GCIs in the olfactory system was confirmed to have MSA by full autopsy. Therefore, this study demonstrated that one of the pathological hallmarks of PD, the formation of LBs and LNs, is a commonplace occurrence within the olfactory system and may contribute to the olfactory dysfunction in PD. After Polymeropoulos and colleagues identified a mutation in the α-synuclein gene on chromosome 4q21–23 that segregated with PD in a large Italian kindred and three Greek kindreds [8], research focused on understanding the role of α-synuclein in sporadic PD. It was soon realized that while α-synuclein is a normal component of presynaptic terminals, it is also robustly expressed in LBs and LNs, collectively referred to as Lewy pathology (LP), in all forms of LB disease (PD, DLB, LB variant of Alzheimer's disease) and is the most sensitive marker of LP [9,10]. Therefore, researchers began using antibodies to α-synuclein to demonstrate the full extent of LP in the olfactory system. Recently, Beach and colleagues examined 58 cases of PD, 35 cases of DLB, 21 cases with incidental LBs (ILBs; defined as the presence of LBs in a patient with no clinical history of neurodegenerative disease), 69 elderly controls and AD cases with and without LP. In this cohort, they observed that the overwhelming majority of cases of PD and DLB had olfactory bulb LP [11]. In addition 2/3rds of cases with ILBs elsewhere in the brain also had them in the olfactory bulb while only 5/64 elderly cases had LP confined to the olfactory bulb. The region of the bulb most frequently involved was the intrabulbar AON followed by the IPL which contained only LNs. In
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comparing frequencies between patient subgroups, the authors found that olfactory bulb LP was > 90% sensitive and specific for distinguishing PD or DLB patients from elderly controls and olfactory bulb LP density correlated well with LP density in other brain regions. In addition to confirming the presence of α-synuclein pathology in the form of LBs and LNs in the olfactory bulb, a report by Tsuboi and colleagues presented the unexpected finding of tau-positive neurofibrillary degeneration in the AON in cases with Lewy body pathology, particularly those with concomitant AD pathology [12]. The tau pathology also unexpectedly correlated with cortical LB counts. While the relationship between α-synuclein and tau pathology is unclear, it is known that there is an abnormal accumulation of tau aggregates in familial PD [13] and there is growing evidence to support a synergistic effect in aggregation [14,15]. The significance of LP, in the olfactory system is strengthened with the realization that it may be one of the few LP ‘induction sites’ in the brain. In a comprehensive assay of LP in the brains of 110 patients including 41 with clinical PD and 69 with ILBs, Braak and colleagues developed a staging system of the progression of LP and determined that the olfactory system is one of 3 ‘induction sites’ for LP [16,17]. That is, LP seems to appear independently in the dorsal motor nucleus of the vagus and intermediate reticular zone of the medulla as well as the olfactory system (including the olfactory bulb and AON) and progresses throughout the brain from these areas. In addition, Braak and colleagues recognized that the earliest evidence of LP is the deposition of LNs, which precedes the formation of LBs in nearly all areas studied. Therefore, the olfactory system seems to be particularly susceptible to Lewy neurodegeneration, is virtually always involved in the earliest stages of the disease and appears to be an attractive target for further investigation. In addition, Lerner and Bagic recently reviewed anatomic connections between the olfactory system and brainstem nuclei and suggested a hypothesis for the spread of Lewy pathology from the olfactory system to many of the selectively vulnerable brainstem nuclei [18]. They propose that a preponderance of pathways leading to the dorsal motor nucleus of the vagus may engender selective vulnerability to these neurons in a topdown fashion that obviates the need for transmission from the enteric nervous system as has been proposed elsewhere [19]. Neuronal loss, another pathological hallmark of PD, was confirmed in the olfactory system in further studies by Hawkes and colleagues [20]. In this report, the olfactory bulbs and tracts from 7 patients with histologically-proven PD and 7 age-matched controls were examined for gross atrophy, LB and LN accumulation and neuronal counts in the AON. Significant atrophy was grossly apparent in several of the PD specimens and quantification of bulb thickness revealed a statistically significant difference between the PD patients and controls. Moreover, a mean reduction in the total number of AON neurons was found in the PD patients compared to controls, with only one count in a PD patient overlapping the values in the control group. Significantly, a strong correlation was found between AON neuronal counts and disease duration in the PD patients, with dramatically reduced numbers in the patients with longest duration, suggesting an ongoing neurodegenerative process. In addition to LP deposition and neuronal loss in the olfactory bulb, degenerative changes have been demonstrated in the olfactoryrelated cortices in PD and in patients with ILBs. Silveira-Moriyama and colleagues examined 10 cases of PD, 7 cases of with ILBs and 5 healthy controls for Lewy pathology in the primary olfactory cortices including the olfactory tubercle, the frontal piriform cortex and the temporal piriform cortex [21]. They observed Lewy pathology in each area in all cases of ILBs and PD, but none of the controls. Interestingly, although Braak and colleagues recognized that LP spreads from the olfactory bulb and AON into olfactory cortices including the olfactory tubercle, periamygdaloid, piriform and entorhinal cortices, it did not seem to spread from there to adjacent cortices unrelated to olfactory processing [17]. In the material that Braak examined, it seems that spread into non-olfactory neocortical regions was only possible after
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LP affected the anteromedial temporal mesocortex and magnocellular nuclei of the basal forebrain, which raises intriguing questions about the mechanisms of LP progression throughout the brain. Two novel observations concerning the pathology of olfactory dysfunction in PD arose in separate reports from Huisman and colleagues in the past few years [22–24]. In 2003, they reported their findings concerning misrouted olfactory fibers and ectopic olfactory glomeruli in 20 normal adults, 12 AD patients and 16 Parkinsonian patients [22]. As discussed previously, in the normal mammalian olfactory system, the axons of ORNs of the OE project through the cribriform plate in the olfactory nerves, into the olfactory nerve layer of the olfactory bulb, and synapse on the dendrites of mitral and tufted neurons in the olfactory glomeruli of the glomerular layer. In the adult human, they were able to demonstrate the presence of misrouted olfactory fibers, defined as olfactory marker protein (OMP) labeled ORN axons that penetrated deeper into the bulb than the glomerular layer. These misrouted olfactory fibers were found in all patient groups including normal adults and did not correlate with age or gender, suggesting that they were part of the normal aging process in the olfactory bulb. In addition, in the Parkinsonian patients, which included not only patients with autopsy-proven PD, but patients with other causes of Parkinsonism including progressive supranuclear palsy (PSP), MSA and vascular disease, they found ectopic glomeruli, defined as more or less spherical structures containing OMP-positive fibers that were located outside of the glomerular layer. They found no
correlation with duration of illness and the number of ectopic olfactory glomeruli. Since all patients with a variety of causes of Parkinsonism displayed ectopic glomeruli, the authors concluded that this phenomenon was not related to the specific pathophysiology of PD, but must be secondary to degeneration of the substantia nigra, regardless of the mechanism. Therefore, neither misrouted olfactory fibers nor ectopic glomeruli seem to offer significant insight into the etiology of the olfactory dysfunction of PD. The same group, in 2004, published results of an examination of the number of dopaminergic PG neurons in the glomerular layer of the olfactory bulb in 10 patients with autopsy-proven PD and 10 control patients [23]. Interestingly, the authors, using stereological quantitative methods, demonstrated a paradoxical 100% increase in the number of tyrosine hydroxylase-containing PG neurons, that previous studies suggest are dopaminergic [25], in the PD patients compared to controls. While no correlation between the duration of PD and the number of dopaminergic periglomerular neurons was demonstrated, the mean duration of disease was 12 years and the authors admit that a relationship may have been obscured by only examining the final stage of the disease. However, in a follow-up study examining twice as many subjects, the same authors recently reported that there was no difference in the number of dopaminergic neurons between male and female PD patients and male control subjects [24]. Female control subjects did have significantly less dopaminergic neurons than male controls for unclear reasons but there was significant variability, even
Fig. 2. Neuritic dystrophy hypothesis of Lewy neurodegeneration. α-synuclein is produced in the cell soma, transported down the axon and located at the synaptic terminal (A). In this model, the aggregation of α-synuclein begins as LNs are formed within axons of neurons (B). These axonal aggregates enlarge by the sequestration of additional α-synuclein, other proteins and subcellular elements, until interruption of axonal transport occurs (B/C). The axonal transport blockade causes an accumulation of anterogradely transported subcellular elements proximal to the occlusion and a supersaturation of the cell soma with α-synuclein (C). The supersaturation of α-synuclein predisposes the neuron to the formation of somatic α-synuclein aggregates, known as Lewy bodies (LBs) (C). LB growth continues until the normal somatic architecture is disrupted, with nuclear displacement, and α-synuclein aggregates form within proximal dendrites as LNs (D). Finally, continued axonal transport blockade prohibits distal axonal viability, leading to axonal degeneration (D) with the possibility of resultant neuronal degeneration. Reproduced from reference [38] with permission from S. Karger AG, Basel.
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within controls, with as few as zero and as many as 60,000 dopaminergic neurons in the olfactory bulb. The authors originally speculated, based on animal studies, a mechanism of olfactory dysfunction in PD due to an increased dopaminergic inhibition of the neurotransmission between the ORNs and mitral cells in the glomeruli, although the recent study casts doubt on this hypothesis. Although the role that PG dopaminergic neurons play in the olfactory dysfunction seen in PD is now in question, gender-specific alterations in the density of these neurons represents an opportunity to investigate the pathophysiological impact of gender on PD risk [26,27]. 4. Olfactory bulb as model of adult neurogenesis Another exciting aspect of investigating the olfactory system in PD is the possibility of gaining further understanding of the mechanisms of neurogenesis in the adult human nervous system. The presence of new neurons in the olfactory bulb of the adult human was suggested recently in two immunohistochemical analyses using markers of proliferation and immature neuronal state to confirm the existence of neurons in the olfactory bulb with an immature neuronal phenotype [28,29]. In a study by Bedard and Parent, neurons expressing immature neuronal markers were found to be granular and periglomerular dopaminergic neurons and provided strong evidence for adult neurogenesis of these cell types in humans. However, these studies were carried out on patients who had no evidence of neurodegeneration. In contrast, Höglinger and colleagues examined 4 patients with autopsy-confirmed PD and compared the number of progenitor cells in the subependymal zone and olfactory bulb to the corresponding numbers in matched control patients [30]. In both areas, the number of neurons was decreased in the PD patients and suggests that chronic impairment of neural regeneration might also contribute to the olfactory dysfunction in PD.
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by the neuron. Eventually, the blockade of axonal transport disallows distal axonal viability and ultimately leads to axonal degeneration with the possibility of resultant neuronal degeneration. While most pathological systems make testing this hypothesis difficult at best because of the long distances between the dendritic aborizations, cell bodies and synaptic terminals of most neurons susceptible to Lewy neurodegeneration, the olfactory bulb and tract are the exception. They provide an easily dissectible preparation that includes all of these cellular components in a population of mitral and tufted cells that project from the glomerular layer to synapse in the AON. By addressing the sequence in which pathological alterations occur in the olfactory bulb in early Lewy neurodegeneration, and in particular, assessing whether LB and LN formation precedes frank neuronal death, evidence supporting or contradicting the neuritic dystrophy hypothesis would be generated. In addition, because of the carefully defined cytoarchitecture of the olfactory bulb, investigation of Lewy neurodegeneration in this system may offer insight into another hypothesis regarding the progression of LP throughout the brain, i.e. the ‘prion-like’ theory of Lewy neurodegeneration [18,40]. In this hypothesis, cellular vulnerability is transmitted in a ‘prion-like’ manner via a conformationally abnormal form of a protein such as α-synuclein. If this hypothesis is correct, then the sequence of vulnerable neurons should be ORNs followed by mitral and tufted cells, followed by AON neurons, etc. Braak and colleagues have also identified additional features that most neurons susceptible to Lewy neurodegeneration share [40]. These include the characteristic of being of the class of projection neurons rather than local circuit neurons and having long, mostly unmyelinated axons. To date, in the olfactory bulb, LP has been demonstrated in projection neurons including mitral cells and AON neurons, but has not been demonstrated in local circuit
5. Olfactory bulb pathology as a model of Lewy neurodegeneration Importantly, among the Parkinsonian disorders, the alteration in ability to smell is relatively specific to PD. Thus, decreased ability to smell is absent, or present infrequently or only to a minor degree, in several of the other common causes of Parkinsonism including PSP [31], MSA [32], essential tremor [33,34], and parkinsonism induced by MPTP [35]. Additionally, earlier studies found that subtle differences in olfactory dysfunction exist among subtypes of PD. That is, slightly greater dysfunction is present in patients with postural instability-gait predominant PD than with tremor-predominant PD [36]. More recently, Ondo and Lai found that tremor-predominant PD patients with a family history of tremor had less olfactory dysfunction than other PD groups [37]. In light of these findings, examining the substrate of olfactory dysfunction in PD may provide new insight into the pathophysiological processes occurring in brain areas involved in smell perception, that are specific to PD and presumably, independent of nigrostriatal degeneration. Indeed, investigation of the pathophysiology of olfactory dysfunction in PD should lead to important insight into the mechanisms of neurodegeneration in PD throughout the brain. One hypothesis that may be tested by investigation of olfactory system neurodegeneration involves the role of neuritic dystrophy in Lewy neurodegeneration [38,39]. In this hypothetical model (Fig. 2), various environmental, genetic or intrinsic factors cause a conformational change in the structure of α-synuclein to β-pleated sheet and a propensity to aggregate into pathologic inclusions. These inclusions start in the axonal compartment as LNs and grow by accumulating additional α-synuclein and trapping other proteins and/or organelles. These inclusions expand until they congest the axonal compartment, resulting in interruption of axonal transport. This cessation of transport may produce abnormally high concentrations of α-synuclein in the cell soma, possibly triggering LB formation, either as a consequence of supersaturation or as a physiologic response
Fig. 3. Lewy pathology of the olfactory bulb. Immunostaining with an antibody specific to aggregated alpha-synuclein [41] demonstrates Lewy neurite and Lewy body pathology in the olfactory bulb of a patient with Parkinson's disease. Note the dense accumulation of Lewy pathology in the internal plexiform layer and anterior olfactory nucleus. Scale bar=100 μm. Abbreviations: ONL: olfactory nerve layer; GLOM: glomerular layer; EPL: external plexiform layer; MCL: mitral cell layer; IPL: internal plexiform layer; GRAN: granular layer; iAON: intrabulbar portion of anterior olfactory nucleus.
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neurons such as PG cells and granule cells. Further examination of neuronal vulnerabilities in the olfaction system will lead to a greater understanding of particular neuronal characteristics conveying vulnerability to Lewy neurodegeneration. 6. Conclusion As elsewhere in the brain, there is compelling reason to believe that LP plays a detrimental role in the neurodegenerative process affecting neurons of the olfactory system in PD and related disorders. As an early induction site, the olfactory system is particularly enticing for investigation into the early stages of the illness, presumably represented in the brains of people with ILBs. Due to the availability of easily dissectible preparations including whole neurons susceptible to Lewy neurodegeneration (Fig. 3), the olfactory system is particularly compelling as a model of how the formation of LNs and LBs affects host cells. Acknowledgements Dr. Duda is supported by grants from the Biomedical Laboratory Research and Development Service of the United States Department of Veterans Affairs. The author is grateful to Andrew Sheller for assistance in the development of Fig. 1 and to Joseph V. Noorigian for assistance with Fig. 3. References [1] Hawkes CH, Shephard BC, Daniel SE. Olfactory dysfunction in Parkinson's disease. J Neurol Neurosurg Psychiatry 1997;62:436–46. [2] Doty RL, Deems DA, Stellar S. Olfactory dysfunction in parkinsonism: a general deficit unrelated to neurologic signs, disease stage, or disease duration. Neurology 1988;38:1237–44. [3] Hower RE, Rick JH, Balderston CC, Weintraub D, Sidenvall B, Stern MB, et al. Olfactory dysfunction and Parkinson's disease progression. Neurology 2008;70:A397. [4] Kratskin IL, Belluzzi O. Anatomy and neurochemistry of the olfactory bulb. In: Doty RL, editor. Handbook of olfaction and gustation. New York: Marcel Dekker; 2003. p. 235–76. [5] Shepherd GM, Greer CA. Olfactory bulb. In: Shepherd GM, editor. The synaptic organization of the brain. New York: Oxford University Press; 1997. p. 159–203. [6] Duda JE, Shah U, Arnold SE, Lee VM, Trojanowski JQ. The expression of alpha-, beta-, and gamma-synucleins in olfactory mucosa from patients with and without neurodegenerative diseases. Exp Neurol 1999;160:515–22. [7] Daniel SE, Hawkes CH. Preliminary diagnosis of Parkinson's disease by olfactory bulb pathology. Lancet 1992;340:186. [8] Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 1997;276:2045–7. [9] Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alphasynuclein in Lewy bodies. Nature 1997;388:839–40. [10] Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. Alpha-synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc Natl Acad Sci U S A 1998;95:6469–73. [11] Beach TG, White III CL, Hladik CL, Sabbagh MN, Connor DJ, Shill HA, et al. Olfactory bulb alpha-synucleinopathy has high specificity and sensitivity for Lewy body disorders. Acta Neuropathol 2009;117:169–74. [12] Tsuboi Y, Wszolek ZK, Graff-Radford NR, Cookson N, Dickson DW. Tau pathology in the olfactory bulb correlates with Braak stage, Lewy body pathology and apolipoprotein epsilon4. Neuropathol Appl Neurobiol 2003;29:503–10. [13] Duda JE, Giasson BI, Mabon ME, Miller DC, Golbe LI, Lee VM, et al. Concurrence of alpha-synuclein and tau brain pathology in the Contursi kindred. Acta Neuropathol (Berl) 2002;104:7–11.
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