MD Kurt A Jellinger
email: kurt.jellinger@univie.ac.at

A critical evaluation of current staging of α-synuclein pathology in Lewy body disorders

Keywords: a-synucleinopathies, dementia with Lewy bodies, Parkinson disease, disease staging, Braak Lewy body stages, DLB consensus pathologic guidelines

The two most frequent synucleinopathies, Parkinson disease (PD) or brainstem predominant type of Lewy body disease, and dementia with Lewy bodies (DLB), are neurodegenerative multisystem disorders with widespread occurrence of a-synuclein containing deposits in the central and peripheral autonomic systems. For both Lewy bodyrelated disorders staging/classification systems based on semiquantitative assessment of the distribution and progression pattern of a-synuclein pathology are used that are considered to be linked to clinical dysfunctions. In PD a six-stage system is suggested to indicate a predictable sequence of lesions with ascending progression from medullary and olfactory nuclei to the cortex, the first two presymptomatic stages related to incidental Lewy body disease, stages 3 and 4 presenting with motor symptoms and the last two (cortical) stages frequently associated with cognitive impairment. DLB, according to consensus pathologic guidelines, by semiquantitative scoring of a-synuclein pathology (Lewy body density and distribution) in specific brain regions, is distinguished into three phenotypes (brainstem, transitory/limbic and diffuse cortical), also considering concomitant Alzheimer-related pathology. Recent retrospective clinico-pathologic studies, although largely confirming the staging system, particularly for younger onset PD with long duration, have shown that between 6.3 and 43% of cases did not follow the proposed caudo-rostral progression pattern of a-synuclein pathology. In 7 to 8.3% of clinically manifested PD cases with synuclein inclusions in midbrain and cortex corresponding to LB stages 4-5 the medullary nuclei were spared, whereas mild parkinsonian symptoms were already observed in stages 2 and 3. There is considerable clinical and pathologic overlap between PD (with or without dementia) and DLB, corresponding to Braak LB stages 5 and 6, both frequently associated with variable Alzheimer-type pathology. Dementia often does not correlate with progressed stages of Lewy body pathology, but often is related to concomitant Alzheimer lesions or mixed pathologies. The relation between cortical Lewy body lesions and cognitive impairment is under discussion. On the other hand, in large unselected autopsy series 30 to 55% of elderly subjects with widespread a-synuclein pathology (Braak stages 5-6) revealed no definite neuropsychiatric symptoms or were not classifiable, indicating compensatory mechanisms of the brain. The causes and molecular basis of rather frequent deviations from the proposed caudo-rostral progression of a-synuclein pathology in PD, its relation to the onset of classical parkinsonian symptoms, the causes for the lack of definite clinical symptoms despite widespread a-synuclein pathology in the nervous system, their relations to Alzheimer-type lesions, and the pathophysiologic impact of both pathologies remain to be further elucidated.

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M A N U S C R I P T Introduction Among proteinopathies, a heterogenous group of neurodegenerative disorders, characterized by intra-and extracellular accumulation of abnormal filament proteins, Lewy body (LB)-associated disorders, such as Parkinson disease (PD) and dementia with Lewy bodies (DLB) show protein inclusions in neurons and neurites (LBs and Lewy neurites/LNs), glia, and presynaptic terminals [START_REF] Kramer | Presynaptic a-synuclein aggregates, not Lewy bodies, cause neurodegeneration in dementia with Lewy bodies[END_REF], with a-synuclein (aSyn) as their major component [for rev see 2]). Given the fundamental nature of the aSyn containing lesions, these and other disorders, such as autonomic failure, LB dysphagia, and multiple system atrophy (MSA) have been summarized as a-synucleinopathies [START_REF] Jellinger | a-Synuclein pathology in Parkinson and Alzheimer disease brain: incidence and topographic distribution -a pilot study[END_REF][START_REF] Galvin | Synucleinopathies: clinical and pathological implications[END_REF]. PD or brainstem type of LB disease [START_REF] Mckeith | Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop[END_REF][START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium[END_REF] is the most common neurodegenerative movement disorder in the elderly, with progressive degeneration of the dopaminergic nigrostriatal system and other neuronal networks caused by loss of pigmented neurons in the substantia nigra compacta (SNc) and many other subcortical nuclei associated with widespread occurrence of LBs and LNs in the brain and specific nuclei of the spinal cord. PD-related lesions are not confined to the central nervous system, but also involve autonomic nuclei, sympathetic ganglia, cardiac and pelvic plexuses, and nerves as well as adrenal medulla, salvary gland and skin [START_REF] Amino | Profound cardiac sympathetic denervation occurs in Parkinson disease[END_REF][START_REF] Braak | Parkinson's disease: lesions in dorsal horn layer I, involvement of parasympathetic and sympathetic pre-and postganglionic neurons[END_REF][START_REF] Braak | Stages in the development of Parkinson's disease-related pathology[END_REF][START_REF] Braak | Pathology associated with sporadic Parkinson's disease -where does it end?[END_REF][START_REF] Bloch | a-Synuclein pathology of the spinal and peripheral autonomic nervous system in neurologically unimpaired elderly subjects[END_REF][START_REF] Den Hartog | The distribution of Lewy bodies in the central and autonomic nervous systems in idiopathic paralysis agitans[END_REF][START_REF] Hishikawa | Clinical and neuropathological correlates of Lewy body disease[END_REF][START_REF] Iwanaga | Lewy body-type degeneration in cardiac plexus in Parkinson's and incidental Lewy body diseases[END_REF][START_REF] Murayama | Systemic pathology of Lewy-related Parkinson changes in aging. The experience of Tokyo Metropolitan Brain Bank for Aging Research[END_REF][START_REF] Orimo | Cardiac sympathetic denervation precedes neuronal loss in the sympathetic ganglia in Lewy body disease[END_REF][START_REF] Oyanagi | Lewy bodies in the lower sacral parasympathetic neurons of a patient with Parkinson's disease[END_REF][START_REF] Probst | New insights into the pathology of Parkinson's disease: does the peripheral autonomic system become central?[END_REF][START_REF] Takeda | Parkinson's disease with involvement of the parasympathetic ganglia[END_REF][START_REF] Wakabayashi | Parkinson's disease: the presence of Lewy bodies in Auerbach's and Meissner's plexuses[END_REF][START_REF] Wakabayashi | Parkinson's disease: an immunohistochemical study of Lewy body-containing neurons in the enteric nervous system[END_REF][START_REF] Wakabayashi | Lewy bodies in the visceral autonomic nervous system in Parkinson's disease[END_REF][START_REF] Wakabayashi | Neuropathology of autonomic nervous system in Parkinson's disease[END_REF], clearly indicating that PD is a multisystem disorder [for rev. see [START_REF] Jellinger | Lewy body disorders[END_REF][START_REF] Braak | Parkinson's disease: lesions in dorsal horn layer I, involvement of parasympathetic and sympathetic pre-and postganglionic neurons[END_REF]. DLB, the second most frequent cause of dementia in the elderly after Alzheimer disease (AD), with the core neuropsychiatric features of fluctuating consciousness, visual hallucinations and parkinsonism, is morphologically featured by a variable burden of a synucleinopathy with (often widespread) cortical LBs and various degrees of AD-related pathology [START_REF] Mckeith | Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop[END_REF][START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium[END_REF].

The distribution pattern of aSyn pathology within selectively vulnerable neuronal populations is considered to be intimately linked to the clinical dysfunctions seen in both disorders, aSyn lesions in the brainstem been mainly claimed to be responsibe for extrapyramidal motor symptoms, whereas cognitive impairment has been attributed to the limbic and neocortical spread of LB lesions [START_REF] Apaydin | Parkinson disease neuropathology: later-developing dementia and loss of the levodopa response[END_REF][START_REF] Kovari | Lewy body densities in the entorhinal and anterior cingulate cortex predict cognitive deficits in Parkinson's disease[END_REF][START_REF] Hurtig | a-Synuclein cortical Lewy bodies correlate with dementia in Parkinson's disease[END_REF][START_REF] Mattila | Neuritic degeneration in the hippocampus and amygdala in Parkinson's disease in relation to Alzheimer pathology[END_REF][START_REF] Mattila | a-Synucleinimmunoreactive cortical Lewy bodies are associated with cognitive impairment in Parkinson's disease[END_REF]. Thus, PD and DLB are believed to represent phenotypes in a continuum within the spectrum of LB disorders, wherein the clinical manifestations predominantly depend on the anatomical distribution and load of aSyn pathology [START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium[END_REF][START_REF] Hishikawa | Clinical and neuropathological correlates of Lewy body disease[END_REF][START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Ince | Dementia with Lewy bodies. A distinct non-Alzheimer dementia syndrome?[END_REF][START_REF] Kosaka | Diffuse type of Lewy body disease: progressive dementia with abundant cortical Lewy bodies and senile changes of varying degree -a new disease?[END_REF]. Despite clinical, biochemical and morphological similarities and dissimilarities, the relations between the two disorders have been discussed controversially [START_REF] Jellinger | Lewy body disorders[END_REF][START_REF] Aarsland | Are Parkinson's disease with dementia and dementia with Lewy bodies the same entity?[END_REF][START_REF] Lippa | DLB and PDD boundary issues: diagnosis, treatment, molecular pathology, and biomarkers[END_REF][START_REF] Mori | Pathological substrate of dementia in Parkinson's disease -its relation to DLB and DLBD[END_REF][START_REF] Mosimann | Dementia with Lewy bodies and Parkinson's disease dementia -two synucleinopathies[END_REF][START_REF] Richard | Parkinson's disease and dementia with Lewy bodies: one disease or two?[END_REF][START_REF] Tsuboi | Dementia with Lewy bodies and Parkinson's disease with dementia: are they different?[END_REF]. Two morphological staging/classification systems are currently used for the assessment of the progressive regional distribution of aSyn pathology in PD and DLB [START_REF] Mckeith | Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop[END_REF][START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium[END_REF][START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Braak | Cognitive status correlates with neuropathologic stage in Parkinson disease[END_REF], the applicability and clinical relevance of which has been critically discussed recently [START_REF] Alafuzoff | Assessment of a-synuclein pathology: a study of the BrainNet Europe Consortium[END_REF]. Both staging systems, the Braak hypothesis of LB staging in PD [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF] and the

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DLB consensus guidelines [START_REF] Mckeith | Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop[END_REF][START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium[END_REF] were developed in non-population-based cohorts. The essentials and validity of these two systems will be critically evaluated based on recent literature and personal studies.

Staging of aSyn inclusion body pathology in sporadic PD

Based on semiquantitative assessment of aSyn-positive inclusions in 413 postmortem cases including 41 PD cases, 69 with aSyn inclusions and and 58 aged-matched controls, a hypothetic staging system of brain pathology indicating a predictable sequence with increasing severity throughout the brain and ascending progression has been proposed [START_REF] Braak | Stages in the development of Parkinson's disease-related pathology[END_REF][START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Braak | Pattern of brain destruction in Parkinson's and Alzheimer's diseases[END_REF][START_REF] Braak | The staging procedure for the inclusion body pathology associated with sporadic Parkinson's disease reconsidered[END_REF][START_REF] Del Tredici | Where does Parkinson disease pathology begin in the brain?[END_REF]. aSyn pathology was divided into six successive stages: The earliest lesions were seen in the lower medulla oblongata with a few LNs in the dorsal motor nucleus of the vagus nerve (DMV), in anterior olfactory structures, chiefly affecting the anterior olfactory nucleus embedded in the olfactory tract, and some aSyn-positive aggregates in preganglionic vagal axons [START_REF] Braak | Pathology associated with sporadic Parkinson's disease -where does it end?[END_REF], with the nucleus basalis of Meynert (NBM) and midbrain regions being preserved (Stage 1). More severe lesions in the DMV with extension to the caudal raphe nuclei and gigantocellular portions of the adjoining reticular formation, and some LNs in the noradrenergic locus ceruleus (LC) are seen in stage 2. These initial stages (observed in 7% of the cohort) were considered a(pre)symptomatic and may explain early non-motor (autonomic and olfactory) symptoms that preceed the somato-motor dysfunctions [START_REF] De Lau | Subjective complaints precede Parkinson disease: the rotterdam study[END_REF][START_REF] Halliday | Critical appraisal of brain pathology staging related to presymptomatic and symptomatic cases of sporadic Parkinson's disease[END_REF][START_REF] Przuntek | Diagnostic staging of Parkinson's disease: conceptual aspects[END_REF][START_REF] Wolters | Parkinson's disease: premotor clinico-pathological correlations[END_REF]. In stage 3, the LC, the central nucleus of the amygdala, the tegmental pedunculopontine nucleus and cholinergic nuclei of the basal forebrain including the NMB are the focus of cytoskeletal changes and neuronal depletions, the posterolateral and posteromedial parts of SNc showing pale bodies (precursors of LBs) [START_REF] Dale | Relationships between Lewy bodies and pale bodies in Parkinson's disease[END_REF] and LBs without neuronal loss, while the allocortex and neocortex are preserved. In stage 4, the anteromedial temporal limbic (transentorhinal) and mesocortex and amygdala are additionally affected. Stages 3 and 4 have been correlated with clinical motor symptoms (11% of their cohort). In stage 5, the lesions in the temporal mesocortex are more striking and from there progress to adjoining association fields of the temporal and prefrontal neocortex, while in stage 6, the lesions involve the neocortex, first affecting the high-order sensory association cortex and prefrontal areas, later progressing to the primary sensory and motor areas or involving the whole neocortex (Fig. 1). The cases with severe pathology (stage 5-6) accounting for 6% of their cohort, frequently show cognitive decline that relates to the severity of the neuropathologic stage [START_REF] Braak | Pathology associated with sporadic Parkinson's disease -where does it end?[END_REF][START_REF] Braak | Cognitive status correlates with neuropathologic stage in Parkinson disease[END_REF]. In another retrospective study of 25 individuals (17 with PD, 8 with incidental aSyn inclusions) LB pathology and glial cytoplasmic inclusions in striatum were seen for the first time in stage 3 in

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the medium-sized GABA-ergic spiny projection neurons that reach both the pallidum and SN reticulata (SNr), and at stages 5 -6 in the large cholinergic interneurons [START_REF] Mori | a-Synuclein pathology in the neostriatum in Parkinson's disease[END_REF].

Consensus pathologic guidelines for the diagnosis of DLB

According to the initial consensus pathologic guidelines, LBs are scored semiquantiatively according to the severity and anatomical distribution, separating brainstem predominant (PD), limbic/transitional and diffuse neocortical types, depending on the anatomical distribution of the aSyn-positive structures [START_REF] Mckeith | Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop[END_REF]. The revised consensus guidelines [START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium[END_REF] proposed semiquantitative assessment of LB density, based on aSyn immunohistochemistry, in five cortical regions (Table 1a). However, the stage of AD pathology is usually not associated with the particular pattern of aSyn lesions [START_REF] Zaccai | Patterns and stages of asynucleinopathy: Relevance in a population-based cohort[END_REF]. A third pattern concentrated on aSyn pathology in cortical areas [START_REF] Kosaka | Diffuse Lewy body disease[END_REF]. Considering the significance and clinical impact of concomitant ADrelated pathology frequently seen in aged subjects with and without dementia, the revised consensus criteria for DLB have recommended to take it into account seriously [START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium[END_REF] (Table 1b).

This protocol was simplified by excluding the frontal region because of the common occurrence of occasional LBs in this regions in PD in the absence of dementia [START_REF] Harding | Cortical Lewy body pathology in the diagnosis of dementia[END_REF].

These guidelines did not provide definitie diagnostic criteria as it is sometimes mistakenly assumed, and were not included in the CERAD protocol, which is used for the semiquantitative evaluation of neuritic plaques and neurofibrillary tangles [START_REF] Mirra | The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease[END_REF].

Studies to evaluate the reliability of aSyn-related stagings

The proposed staging procedure for aSyn pathology in PD rested, in part, on the assumption that incidental LB pathology is the first step along a disease continuum [START_REF] Del Tredici | Where does Parkinson disease pathology begin in the brain?[END_REF], but that sporadic PD, like most neurodegenerative disorders, is not a static but a dynamic biologic process and that so-called incidental lesions (LBs/LNs seen in subjects without PD related signs and symptoms) represent presymptomatic (subclinical) correlates of a pathologic condition ultimately leading to a manifest clinical disease [START_REF] Braak | Pathology associated with sporadic Parkinson's disease -where does it end?[END_REF][START_REF] Forno | Concentric hyalin intraneuronal inclusions of Lewy type in the brains of elderly persons (50 incidental cases): relationship to parkinsonism[END_REF][START_REF] Gibb | The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson's disease[END_REF][START_REF] Dickson | Identification of normal and pathological aging in prospectively studied nondemented elderly humans[END_REF].

Incidental LB disease (iLBD) is the term when LBs are found in the nervous system in subjects without clinically documented parkinsonism or cognitive impairment [START_REF] Gibb | The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson's disease[END_REF][START_REF] Forno | Neuropathology of Parkinson's disease[END_REF].

Epidemiological studies indicated that autonomic symptoms, REM sleep behavioral disorder and olfactory dysfunction may precede overt extrapyramidal motor symptoms by years [START_REF] Abbott | Bowel movement frequency in late-life and incidental Lewy bodies[END_REF][START_REF] Stiasny-Kolster | Combination of 'idiopathic' REM sleep behaviour disorder and olfactory dysfunction as possible indicator for a-synucleinopathy demonstrated by dopamine transporter FP-CIT-SPECT[END_REF][START_REF] Iranzo | Rapid-eye-movement sleep behaviour disorder as an early marker for a neurodegenerative disorder: a descriptive study[END_REF], resulting from LBs and LNs in the enteric plexus [START_REF] Braak | Pathology associated with sporadic Parkinson's disease -where does it end?[END_REF][START_REF] Wakabayashi | Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism[END_REF], affection of lower brainstem nuclei [START_REF] Boeve | Insights into REM sleep behavior disorder pathophysiology in brainstem-predominant Lewy body disease[END_REF][START_REF] Marion | Is REM sleep behaviour disorder (RBD) a risk factor of dementia in idiopathic Parkinson's disease?[END_REF] and the sympathetic cardiac nervous system [START_REF] Mitsui | Pathology of the sympathetic nervous system corresponding to the decreased cardiac uptake in 123I-metaiodobenzylguanidine (MIBG) scintigraphy in a patient with Parkinson disease[END_REF]. Recent studies of iLBD cases

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have shown a distribution of LBs similar to that in PD, involving one or multiple brain areas, some also with sparse LBs in limbic or temporal cortex (average Braak stage 2.7 ± 0.3) in comparison to definite PD cases with more numerous LBs in all regions and significantly higher Braak PD stage (average 4.4 ± 0.3). When both groups were taken together, a significant inverse correlation existed between neuronal densities in the three anatomical regions studied and the Braak PD stage [START_REF] Dickson | Evidence that incidental Lewy body disease is pre-symptomatic Parkinson's disease[END_REF]. Furthermore, there was decreased TH immunoreactivity in both the striatum and sympathetic epicardial nerve fibers in cases with incidental LB pathology compared to normal controls but not in the same extent as in PD [START_REF] Dickson | Evidence that incidental Lewy body disease is pre-symptomatic Parkinson's disease[END_REF][START_REF] Beach | Reduced striatal tyrosine hydroxylase in incidental Lewy body disease[END_REF]. On the other hand, incidental aSyn pathology may affect solely the LC and SN without affection of the medullary nuclei [START_REF] Jellinger | Lewy body-related a-synucleinopathy in the aged human brain[END_REF][START_REF] Attems | The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease[END_REF][START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF][START_REF] Parkkinen | Regional distribution of a-synuclein pathology in unimpaired aging and Alzheimer disease[END_REF]. Incidental LBs are not considered as a normal aging phenomenon, whereas neurofibrillary tangles increase with age in the SN [START_REF] Frigerio | Substantia nigra Lewy bodies, neurofibrillary tangles, and aging[END_REF],

and are associated with increasing neuritic AD stages [START_REF] Attems | Tau and a-synuclein brainstem pathology in Alzheimer disease: relation with extrapyramidal signs[END_REF]. These findings suggest that iLBD is likely a precursor to or preclinical form of PD, and the lack of symptoms is due to subthreshold aSyn pathology.

Some recent studies have largely confirmed this staging of LB-related pathology in sporadic PD, showing that all brains of subjects with clinical PD revealed aSyn-positive inclusions and neuronal losses in medullary and pontine nuclei and SN, and additional lesions in NBM (90-98 %), limbic cortex (50-60%), cingulate area (32-46%), frontal cortex (29-31%), and amygdala (25%), corresponding to LB stages 4 to 6 [START_REF] Jellinger | a-Synuclein pathology in Parkinson and Alzheimer disease brain: incidence and topographic distribution -a pilot study[END_REF][START_REF] Jellinger | Lewy body-related a-synucleinopathy in the aged human brain[END_REF]. In a previous study, LBs in SN were found in 99.2% of clinico-pathologically confirmed PD cases [START_REF] Jellinger | Overview of morphological changes in Parkinson's disease[END_REF]. LB pathology first appears in the ventrolateral part of SNc, spreads to the paranigral nucleus and then to the medial part, and finally to the dorsal part of SNc [START_REF] Wakabayashi | Progression patterns of neuronal loss and Lewy body pathology in the substantia nigra in Parkinson's disease[END_REF]. There is severe depletion of melanized neurons (45-66%) and of dopaminergic neurons immunoreactive for tyrosine hydroxylase (TH) (60-85%) in the A-9 group of SNc, particularly in the ventrolateral tier (91%) projecting to striosomal compartments of the striatum [START_REF] Feigenbaum-Langer | Distinct nigrostriatal projection systems innervate striosomes and matrix in the primate striatum[END_REF], followed by the ventromedial (71%) and dorsal parts (56%) [START_REF] Fearnley | Pathology of Parkinson's disease[END_REF]. The calbindin (CAB)-rich compartments show greater cell loss in the caudal and mediolateral region (98%) than the adjacent matrix.

From there, it spreads to other nigrosomes and finally to the matrix along a caudorostral, lateromedial and ventrodorsal progression [START_REF] Damier | The substantia nigra of the human brain. I. Nigrosomes and the nigral matrix, a compartmental organization based on calbindin D(28K) immunohistochemistry[END_REF]. These changes differ from age-related lesions in the dorsal tier of SNc that is involved only in late stages of PD [START_REF] Fearnley | Pathology of Parkinson's disease[END_REF][START_REF] Halliday | Midbrain neuropathology in idiopathic Parkinson's disease and diffuse Lewy body disease[END_REF]. The A-10 group of dopaminergic neurons -ventral tegmental area, nucleus parabrachialis, and parabrachialis pigmentosus -projecting to the striatal matrix, thalamus [START_REF] Sanchez-Gonzalez | The primate thalamus is a key target for brain dopamine[END_REF], cortical and limbic areas (mesocortico-limbic system) [START_REF] Berger | Dopaminergic innervation of the cerebral cortex: unexpected differences between rodents and primates[END_REF] in PD show less severe involvement (40-50% cell loss), the retrorubral A-8 region containing only few dopaminergic but CAB-rich neurons, and the

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central periventricular gray suffer little or no degeneration [START_REF] Mcritchie | Specific A10 dopaminergic nuclei in the midbrain degenerate in Parkinson's disease[END_REF]. Others reported greater cell loss in LC (area A-6) than in SN in both PD and AD [START_REF] Zarow | Neuronal loss is greater in the locus coeruleus than nucleus basalis and substantia nigra in Alzheimer and Parkinson diseases[END_REF]. Morphometric studies showed a 35-41% reduction of pigmented SN cells, with severe loss of dopamine transporter (DAT)immunoreactive neurons in older persons [START_REF] Ma | Dopamine transporter-immunoreactive neurons decrease with age in the human substantia nigra[END_REF] and increase in the volume of these cells [START_REF] Cabello | Ageing of substantia nigra in humans: cell loss may be compensated by hypertrophy[END_REF].

Some studies have estimated the neuronal loss as 4.3% per decade [START_REF] Cabello | Ageing of substantia nigra in humans: cell loss may be compensated by hypertrophy[END_REF], while others have reported almost 10% [START_REF] Ma | Unbiased morphometrical measurements show loss of pigmented nigral neurones with ageing[END_REF]. Recent morphometric stereologic studies of the human SN revealed a significant loss of pigmented (-28.3%) and TH-positive (-36.2%) neurons in older controls compared to younger subjects, with hypertrophy of cells in older controls, interpreted as a compensatory mechanism to allow normal motor function despite cell loss. PD showed a massive loss of SN neurons with significant atrophy of remaining cells (20% of controls), but most of the examined cases were in the end stage of the disease [START_REF] Rudow | Morphometry of the human substantia nigra in ageing and Parkinson's disease[END_REF].

A prerequisite of the proposed staging system of aSyn inclusion pathology is that the extent and severity of lesions increase as the disease progresses [START_REF] Braak | The staging procedure for the inclusion body pathology associated with sporadic Parkinson's disease reconsidered[END_REF]. A study of 21 PD brains by six observers from five different institutions examining 11 different brain areas revealed highly significant inter-and intra-rater reliability and supported the suitability of the staging procedure of aSyn pathology for application in routie neuropathology and brain banking [START_REF] Müller | Staging of sporadic Parkinson disease-related a-synuclein pathology: inter-and intra-rater reliability[END_REF].

However, the reliability of this staging system has recently been challenged [START_REF] Jellinger | a-Synuclein pathology in Parkinson and Alzheimer disease brain: incidence and topographic distribution -a pilot study[END_REF][START_REF] Zaccai | Patterns and stages of asynucleinopathy: Relevance in a population-based cohort[END_REF][START_REF] Jellinger | Lewy body-related a-synucleinopathy in the aged human brain[END_REF][START_REF] Attems | The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease[END_REF][START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF][START_REF] Kalaitzakis | The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease: a critical analysis of asynuclein staging[END_REF][START_REF] Parkkinen | Alafuzoff, a-Synuclein pathology does not predict extrapyramidal symptoms or dementia[END_REF]. In Braak's cohort of 301 cases (including 176 clinical PD and 106 with incidental LB pathology), only 6.3% of PD brains diverged from the hypothetical staging scheme of aSyn pathology, with predominant involvement of olfactory structures and amygdala, and advanced concomitant AD-related neuritic pathology [START_REF] Braak | Pathology associated with sporadic Parkinson's disease -where does it end?[END_REF][START_REF] Braak | The staging procedure for the inclusion body pathology associated with sporadic Parkinson's disease reconsidered[END_REF]. However, among 71 cases of PD from the London brain tissue bank only 53% showed a distribution pattern of aSyn compatible with the caudo-rostral spreading suggested by Braak et al [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF] and 43% did not fit the predicted spread of aSyn inclusion pathology. The most frequently affected regions were SN and NBM (100 and 98.5%, respectively), followed by LC and DMV (97 and 92.9%, respectively). In 7% the DMV was not affected, although aSyn inclusions were found in SN and/or cortical regions [START_REF] Kalaitzakis | The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease: a critical analysis of asynuclein staging[END_REF]. On the other hand, in a were the olfactory bulb (70%), CA 2/3 sector of hippocampus (39%), previously regarded as a means to differentiate diffuse DLB from PD [START_REF] Dickson | Immunoreactivity profile of hippocampal CA2/3 neurites in diffuse Lewy body disease[END_REF], and cingulate gyrus and/or frontal cortex (31.6%) [START_REF] Attems | The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease[END_REF]. In general, DMV and SN were found to be equally susceptible nuclei, but structures even earler affected by aSyn pathology include the spinal cord, DMV, olfactory bulb and amygdala [START_REF] Bloch | a-Synuclein pathology of the spinal and peripheral autonomic nervous system in neurologically unimpaired elderly subjects[END_REF][START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Del Tredici | Where does Parkinson disease pathology begin in the brain?[END_REF][START_REF] Hamilton | Lewy bodies in Alzheimer's disease: a neuropathological review of 145 cases using a-synuclein immunohistochemistry[END_REF][START_REF] Klos | a-Synuclein pathology in the spinal cords of neurologically asymptomatic aged individuals[END_REF][START_REF] Uchikado | Alzheimer disease with amygdala Lewy bodies: a distinct form of a-synucleinopathy[END_REF]. The fact that between 6.3 and even 47% of all clinical and autopsy-confirmed PD cases obviously do not strictly follow the proposed staging system of aSyn inclusion pathology and that in 7 to 8.3% the DMV was not involved despite definite aSyn pathology in higher brainstem or even cortical regions [START_REF] Attems | The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease[END_REF][START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF][START_REF] Kalaitzakis | The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease: a critical analysis of asynuclein staging[END_REF] suggests that the proposed ascending pathway may not be the only possible route of disease progression and simultaneous involvement of subcortical and cortical regions appears to be possible. In very few cases of DLB with severe AD-pathology, the amygdala develops aSyn pathology and neuronal loss prior to the brainstem nuclei, while depletion of cardiac nerves is not necessarily seen [START_REF] Yokota | Lewy body variant of Alzheimer's disease or cerebral type Lewy body disease? Two autopsy cases of presenile onset with minimal involvement of the brainstem[END_REF]. aSyn pathology beginning in the amygdala associated with AD [START_REF] Hamilton | Lewy bodies in Alzheimer's disease: a neuropathological review of 145 cases using a-synuclein immunohistochemistry[END_REF][START_REF] Uchikado | Alzheimer disease with amygdala Lewy bodies: a distinct form of a-synucleinopathy[END_REF][START_REF] Braak | Amygdala pathology in Parkinson's disease[END_REF] may show rostro-caudal spread to the entorhinal cortex and midbrain/brainstem regions. However, the reasons for such deviations from the frequently but not consistently observed caudorostral propagation and for sparing of medullar nuclei even in clinically manifested PD await further elucidation.

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A recent study on the progression of pathology in longitudinally followed PD patients verified three different clinicopathologic groups: (1.) In a group of younger onset patients presenting with long clinical duration and LB distributions consistent with the Braak staging, brainstem LBs dominated in those surviving up to 5 years; by 13 years 50% of cases had a limbic distribution of LBs, and by 18 years, all had at least this pathologic subtype. (2.) About 25% of cases had an early malignant, dementia-dominant syndrome and severe neocortical disease consistent with DLB. (3.) The last group with older onset, shorter survival and more complex disease course showed higher LB loads, suggesting that widespread LB pathology either occurs at the onset of clinical disease or rapidly infiltrates the brain with frequent concomitant lesions, in particular more plaque pathology, supporting a more aggressive and linked phenotype. These data suggest that the different phenotypes cannot be differentiated by pathology alone and are also not consistent with a unitary concept of the pathogenesis of LB disease [START_REF] Halliday | The progression of pathology in longitudinally followed patients with Parkinson's disease[END_REF].

a-Syn pathology in the nervous system and clinical manifestations

Lewy body pathology in the central and peripheral autonomic system in a neurologically unremarkable elderly human population is not an infrequent finding [START_REF] Jellinger | a-Synuclein pathology in Parkinson and Alzheimer disease brain: incidence and topographic distribution -a pilot study[END_REF][START_REF] Bloch | a-Synuclein pathology of the spinal and peripheral autonomic nervous system in neurologically unimpaired elderly subjects[END_REF][START_REF] Zaccai | Patterns and stages of asynucleinopathy: Relevance in a population-based cohort[END_REF][START_REF] Forno | Concentric hyalin intraneuronal inclusions of Lewy type in the brains of elderly persons (50 incidental cases): relationship to parkinsonism[END_REF][START_REF] Jellinger | Lewy body-related a-synucleinopathy in the aged human brain[END_REF][START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF][START_REF] Parkkinen | Alafuzoff, a-Synuclein pathology does not predict extrapyramidal symptoms or dementia[END_REF][START_REF] Alafuzoff | a-Synuclein in ageing and Alzheimer's disease[END_REF][START_REF] Parkkinen | Alafuzoff, a-Synuclein pathology is highly dependent on the case selection[END_REF][START_REF] Parkkinen | Widespread and abundant asynuclein pathology in a neurologically unimpaired subject[END_REF], which was detected only in comprehensive studies of the whole nervous system.

Comparisons between such studies should account on case selection and the methods used for detecting LBs [START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF][START_REF] Parkkinen | Alafuzoff, a-Synuclein pathology does not predict extrapyramidal symptoms or dementia[END_REF][START_REF]Pathological correlates of late-onset dementia in a multicentre, community-based population in England and Wales[END_REF][START_REF] Wakisaka | Age-associated prevalence and risk factors of Lewy body pathology in a general population: the Hisayama study[END_REF][START_REF] Perry | Lewy body prevalence in the aging brain: relationship to neuropsychiatric disorders, Alzheimer-type pathology and catecholaminergic nuclei[END_REF][START_REF] Ince | Neuropathological diagnoses in elderly patients in Oslo: Alzheimer's disease, Lewy body disease, vascular lesions[END_REF]. Earlier studies showed LBs in the brains of 50 elderly persons without extrapyramidal symptoms [START_REF] Forno | Concentric hyalin intraneuronal inclusions of Lewy type in the brains of elderly persons (50 incidental cases): relationship to parkinsonism[END_REF], aSyn pathology in SNc in about 10% of neurologically unimpaired elderly persons [START_REF] Parkkinen | Alafuzoff, a-Synuclein pathology is highly dependent on the case selection[END_REF] and in midbrain and limbic cortex in 31% of asymptomatic aged controls with a mean age of 82.0 years. These were referred to as iLBD.

while multifocal aSyn pathology in10% of them corresponded to Braak LB stages 4 [START_REF] Jellinger | Lewy body-related a-synucleinopathy in the aged human brain[END_REF].

Among (DLB transitional form, 2.1%), and stage 5 (neocortical DLB, 1.9%) [START_REF] Saito | Lewy body-related a-synucleinopathy in aging[END_REF], while in a single neurologically unimpaired subject, widespread and abundant aSyn pathology was detected [START_REF] Parkkinen | Widespread and abundant asynuclein pathology in a neurologically unimpaired subject[END_REF], which was suggested to predict neither extrapyramidal symptoms (EPS) nor dementia.

Among 98 elderly autopsy cases without PD-associated symptoms, aSyn pathology was found in the brain, spinal cord and peripheral autonomic system in 17.3% [START_REF] Bloch | a-Synuclein pathology of the spinal and peripheral autonomic nervous system in neurologically unimpaired elderly subjects[END_REF]. Among 208

A C C E P T E D M A N U S C R I P T

autopsy cases from the MRC CFAS brain donor cohort aged 65+ years (almost 75% over 80 years old, 50% of them demented), 76 brains (36.5%) showed LBs. Only 51% of these conformed to the Braak LB staging, while 17% had aSyn pathology in a higher region which was absent in a lower region. A further 29% showed amygdala-predominant aSyn pathology.

Six brains presented predominant neocortical aSyn pathology with minor involvement of amygdala and SN [START_REF] Zaccai | Patterns and stages of asynucleinopathy: Relevance in a population-based cohort[END_REF]. This population-based study showed that 80% of those with aSyn pathology conformed either to a DLB consensus/Braak stage pattern (51%) or to amygdalapredominant disease (29%). Amygdala-predominant aSyn pathology was therefore present in 60% of a DLB/Braak stage-conforming pattern. The remaining 20% did not fit into either system of aSyn progression -a previously unreported population group. A group in which aSyn pathology was predominantly neocortical, with minimal amygdala involvement and absent transentorhinal/cingulate brains (8%), corresponds to the diffuse cortical form of DLB [START_REF] Kosaka | Diffuse Lewy body disease[END_REF][START_REF] Yamamoto | Non-uniformity in the regional pattern of Lewy pathology in brains of dementia with Lewy bodies[END_REF]. The MRC CFAS study confirmes that aSyn frequently coexists with AD-type pathology, but suggests that there is no consistent hierarchy in the progression of the two disease processes [START_REF] Parkkinen | Regional distribution of a-synuclein pathology in unimpaired aging and Alzheimer disease[END_REF]. In the hitherto largest autopsy series of 1720 individuals aged at death > 40 years from Kuopio, Finland, the total frequency of aSyn lesions was 14% [START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF]. 83% of them showed a distribution pattern of aSyn lesion comparable to the two current staging systems of PD and DLB, but 55% of subjects with widespread aSyn pathology (Braak PD stages 5-6) lacked clinical signs of dementia (MMSE >26) or EPS. Similarly, among those subjects that fulfilled the McKeith criteria for DLB and displayed only mild concomitant ADrelated pathology, only 48% were demented and 54% displayed EPS. When only demented subjects were included in the analysis, the correlation between aSyn pathology and dementia was 85%. 17% of all cases showed deviations from the suggested caudo-rostral disease progression. One subject with EPS was found already in Braak PD stage 2, whereas none of the cases in stage 4 displayed EPS, and more importantly, no EPS had been reported in 55%

of subjects who exhibited widespread aSyn pathology (Braak PD stages 5-6), as compared to the 14% previously reported [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF]. These and other results suggest that the risk for EPS increases with disease progression though not to the same extent as previously believed. On the other hand, in large autopsy samples between 30 to 55% of elderly subjects with widespread aSyn pathology were neuropsychiatrically intact lacking clinical symptoms or were not classifiable [START_REF] Jellinger | Lewy body-related a-synucleinopathy in the aged human brain[END_REF][START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF][START_REF] Leverenz | Empiric refinement of the pathologic assessment of Lewy-related pathology in the dementia patient[END_REF].

Initial decline in cognition was postulated to occur already during stages 3 and 4, i.e.

around the same time when initial manifestations of somatosensory and motor dysfunction appear. Among 88 subjects, dementia was observed in stage 3 (36%), 4 (67%), stage 5 (94%)

A C C E P T E D M A N U S C R I P T

and in 100% in stage 6, indicating that increasing cognitive decline (decreasing MMSE scores) correlated with increasing aSyn stages [START_REF] Braak | Cognitive status correlates with neuropathologic stage in Parkinson disease[END_REF], which was not confirmed by others [START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF][START_REF] Colosimo | Lewy body cortical involvement may not always predict dementia in Parkinson's disease[END_REF][START_REF] Weisman | In dementia with Lewy bodies, Braak stage determines phenotype, not Lewy body distribution[END_REF][START_REF] Jellinger | Re: In dementia with Lewy bodies, Braak stage determines phenotype, not Lewy body distribution[END_REF]. In the hitherto largest autopsy study of aSyn pathology in humans,the percentage of demented persons increased from 0 to 50% between LB stages 3 and 6.

However, when subjects with either EPS or dementia were included, 91 and 94%, respectively, were assigned to PD stages 5-6. This indicated that only when subjects with clinical symptoms were included in the analysis, the correlation between stage/severity of aSyn pathology and EPS and/or dementia was excellent in line with previous reports [START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF]. In an autopsy series of 330 elderly patiens with clinical parkinsonism (37.6% of which with dementia), only 1.6% of the demented patients (MMSE < 20) showed LB Braak stages 3-5, which were found in the majority of non-demented PD cases, while 35.5% of parkinsondementia (PDD) cases revealed LB stages 4 or 5 with superimposed severe Alzheimer pathology (neuritic Braak stages V and VI). More than half of them showed a strong relationship between aSyn and tau pathologies, particularly in the limbic system. DLB with low or high-grade Alzheimer lesions were seen in 40% of PDD patients, but almost one-third of diffuse DLB cases, i.e. those with mild AD lesions restricted to amyloid plaques or tau pathology in the limbic system, did not show considerable cognitive impairment [START_REF] Jellinger | Morphological substrates of Parkinsonism with and without dementia. A retrospective clinico-pathological study[END_REF].

The clinical relevance of cortical aSyn pathology in relation to cognitive impairment is a matter of intense debate. Some authors have emphasized their key causative role [START_REF] Apaydin | Parkinson disease neuropathology: later-developing dementia and loss of the levodopa response[END_REF][START_REF] Kovari | Lewy body densities in the entorhinal and anterior cingulate cortex predict cognitive deficits in Parkinson's disease[END_REF][START_REF] Hurtig | a-Synuclein cortical Lewy bodies correlate with dementia in Parkinson's disease[END_REF][START_REF] Mattila | a-Synucleinimmunoreactive cortical Lewy bodies are associated with cognitive impairment in Parkinson's disease[END_REF],

whereas others have reported abundant LB cortical lesions in non-demented PD patients [START_REF] Colosimo | Lewy body cortical involvement may not always predict dementia in Parkinson's disease[END_REF] as well as in neuropsychiatrically unimpaired elderly subjects [START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF][START_REF] Parkkinen | Widespread and abundant asynuclein pathology in a neurologically unimpaired subject[END_REF]. These data clearly indicate that detailed regional assessment of aSyn pathology, the inter-and intrarater reliability of which has recently been reviewed [START_REF] Alafuzoff | Assessment of a-synuclein pathology: a study of the BrainNet Europe Consortium[END_REF], cannot reliably predict the clinical status observed intra vitam [START_REF] Parkkinen | Alafuzoff, a-Synuclein pathology does not predict extrapyramidal symptoms or dementia[END_REF]. The neuropathology of PD with and without dementia and DLB

shows both similarities and slight differences. Morphology and immunohistochemistry of cortical and subcortical LBs and the ascending spreading pattern of aSyn pathology do not significantly differ between both subtypes, the late cortical stages 5 and 6 of LB pathology suggesting overlap or transition between PD and DLB [START_REF] Jellinger | Lewy body disorders[END_REF][START_REF] Jellinger | a-Synuclein pathology in Parkinson and Alzheimer disease brain: incidence and topographic distribution -a pilot study[END_REF][START_REF] Jellinger | Lewy body-related a-synucleinopathy in the aged human brain[END_REF][START_REF] Saito | Accumulation of phosphorylated a-synuclein in aging human brain[END_REF], although DLB often has higher density of cortical LBs and AD-lesions than PDD [START_REF] Algom | Dementia with Lewy bodies, Parkinson's disease dementia and Alzheimer dementia: a retrospective clinical comparison and quantitative neuropathologic study[END_REF]. The SN and other subcortical nuclei in DLB show variable neuronal loss often indistinguishable from sporadic PD, except for an occasionally more severe loss in the ventro(dorso)lateral tier compared to predominant cell loss in the medioventral parts of SNc in PD and more frequent involvement by aSyn deposits of the limbic system, in particular the CA 2/3 subarea of the cornu Ammonis in DLB than in PD/PD (79 vs 36%) [START_REF] Jellinger | Lewy body disorders[END_REF]. A major morphologic difference is the significantly more

A C C E P T E D M A N U S C R I P T

frequent and severe load with diffuse amyloid plaques in the striatum in DLB, irrespective of the severity of cortical AD-type lesions, while non-demented PD cases are virtually free of Aß pathology as is the globus pallidus [START_REF] Jellinger | Striatal ß-amyloid deposition in Parkinson disease with dementia. Letter to the Editor[END_REF][START_REF] Jellinger | Does striatal pathology distinguish Parkinson disease with dementia and dementia with Lewy bodies?[END_REF][START_REF] Kalaitzakis | Striatal ß-amyloid deposition in Parkinson disease with dementia[END_REF]. There are neither correlations between LB density in any brain area and neuritic Braak AD stages or frequency of neuritic plaques [START_REF] Jellinger | Prevalence and impact of vascular and Alzheimer pathologies in Lewy body disease[END_REF],

nor between LBs in cortex and SN, suggesting that DLB should not be considered a severe form of PD. Whereas LB densities, in general, cannot separate DLB from PD/PDD, the severity and duration of dementia appears to be related to both increased hippocampal LB densities and neuritic plaque grade. A screening algorithm suggesting that LB density thresholds in parahippocampus may distinguish demented from nondemented PD cases independent from other pathologies [START_REF] Harding | Clinical correlates of selective pathology in the amygdala of patients with Parkinson's disease[END_REF] awaits further confirmation. However, individuals can show significant cognitive disturbance with no or minimal cortical LBs and, converesely, widespread cortical LB pathology without essential cognitive decline [START_REF] Braak | Cognitive status correlates with neuropathologic stage in Parkinson disease[END_REF][START_REF] Colosimo | Lewy body cortical involvement may not always predict dementia in Parkinson's disease[END_REF][START_REF] Burn | Cortical Lewy body disease and Parkinson's disease dementia[END_REF]. In the large Finish autopsy cohort 56% of those subjects where limbic/diffuse neocortical aSyn pathology combined with mild to moderate AD-related changes remained cognitively intact.

However, when only demented subjects with severe AD-related pathology (neuritic Braak stages V and VI), were examined, 85% were assigned to a high likelihood category of DLB and all of them were demented [START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF]. Hence, in cognitively impaired subjects there is a good correlation between dementia and particular pathologies.

In conclusion, recent clinico-pathologic studies confirm that the current staging / categorization systems for can readily be applied to most of the subjects when assessing regional distribution and progression patterns of aSyn pathology, although in a certain percentage of cases this caudo-rostral propagation pattern suggested for PD cannot be confirmed. In such cases the pattern of relevant lesions may have been modified by other coexisting pathologies or genetic factors [START_REF] Uchikado | Alzheimer disease with amygdala Lewy bodies: a distinct form of a-synucleinopathy[END_REF][START_REF] Klunk | Amyloid deposition begins in the striatum of presenilin-1 mutation carriers from two unrelated pedigrees[END_REF][START_REF] Lang | The progression of Parkinson disease: a hypothesis[END_REF][START_REF] Lashley | Cortical a-synuclein load is associated with amyloid-ß plaque burden in a subset of Parkinson's disease patients[END_REF]. Various clinical, biochemical and morphological overlaps between PD, DLB and AD including co-localization of tau and aSyn epitopes in LBs suggest that the process of LB formation is triggered, at least in part, by AD pathology [START_REF] Jellinger | Lewy body disorders[END_REF][START_REF] Iseki | Dementia with Lewy bodies from the perspective of tauopathy[END_REF][START_REF] Marui | Progression and staging of Lewy pathology in brains from patients with dementia with Lewy bodies[END_REF]. Deposition of tau can be demonstrated in a proportion of LBs, being greatest in neurons vulnerable to both LB and NFT formation, such as in LC, NBM and amygdala [START_REF] Duda | Concurrence of a-synuclein and tau brain pathology in the Contursi kindred[END_REF][START_REF] Ishizawa | Colocalization of tau and a-synuclein epitopes in Lewy bodies[END_REF]. This suggests that aSyn and tau may be related to several pathologic processes (bystander effect), which explains the frequent overlap between synucleinopathies and tauopathies [START_REF] Galpern | Interface between tauopathies and synucleinopathies: a tale of two proteins[END_REF][START_REF] Lee | More than just two peas in a pod: common amyloidogenic properties of tau and a-synuclein in neurodegenerative diseases[END_REF]. The collision of two or more processes may occur in the same brain region or even in single cells in the human brain, eg., in LRRK2 mutations [START_REF] Giasson | Biochemical and pathological characterization of Lrrk2[END_REF] and in animal models of PD [START_REF] Duka | a-Synuclein induces hyperphosphorylation of tau in the MPTP model of parkinsonism[END_REF], with association of phospo-tau and aSyn in both NFTs and LBs [START_REF] Shao | Atypical protein kinase C in neurodegenerative disease II: PKCiota/lambda in tauopathies and a-synucleinopathies[END_REF] and in vitro promotion of tau aggregation by aSyn and vice versa [START_REF] Giasson | Initiation and synergistic fibrillization of tau and a-synuclein[END_REF]. Others have suggested that amyloid rather than tau enhances aSyn pathology in human brain and transgenic mice [START_REF] Lashley | Cortical a-synuclein load is associated with amyloid-ß plaque burden in a subset of Parkinson's disease patients[END_REF][START_REF] Pletnikova | Aß deposition is associated with enhanced cortical a-synuclein lesions in Lewy body diseases[END_REF]. These interactions highlight the interface between these and other misfolded proteins [START_REF] Lee | More than just two peas in a pod: common amyloidogenic properties of tau and a-synuclein in neurodegenerative diseases[END_REF][START_REF] Soto | Protein misfolding and neurodegeneration[END_REF] and may represent molecular mechanisms in overlapping pathology of PD/DLB and AD [START_REF] Mandal | Interaction between Aß peptide and a synuclein: molecular mechanisms in overlapping pathology of Alzheimer's and Parkinson's in dementia with Lewy body disease[END_REF][START_REF] Obi | Relationship of phosphorylated a-synuclein and tau accumulation to Aß deposition in the cerebral cortex of dementia with Lewy bodies[END_REF], together with recent biochemical data on tau and ßamyloid [START_REF] Deramecourt | Biochemical staging of synucleinopathy and amyloid deposition in dementia with Lewy bodies[END_REF] challenge the view of PD and DLB as distinct entities.

The recent MRC CFAS study found no evidence for preponderance of amygdalapredominant aSyn pathology over the prototypical DLB/Braak hierarchy in terms of an association with AD [START_REF] Zaccai | Patterns and stages of asynucleinopathy: Relevance in a population-based cohort[END_REF]. According to these authors, this does not support the proposed hypothetical link between aSyn in the amygdala and AD pathology [START_REF] Hamilton | Lewy bodies in Alzheimer's disease: a neuropathological review of 145 cases using a-synuclein immunohistochemistry[END_REF][START_REF] Uchikado | Alzheimer disease with amygdala Lewy bodies: a distinct form of a-synucleinopathy[END_REF], and was explained by the fact that the studies from which these hypotheses emerged included only clinically referred, i.e. selected cases.

On the other hand, in retrospective studies of unselected autopsy cohorts up to 50% of cases displaying abundant aSyn pathology had been reported to be clinically intact without neuropsychiatric deficits. It has been suggested that the key lesions may develop a considerable time prior to the appearance of clinical symptoms [START_REF] Fearnley | Pathology of Parkinson's disease[END_REF], but the duration and progression of this "preclinical" phase is still under discussion. According to functional neuroimaging studies this preclinical periods range from 4.6 to 6.6 years, with an annual decline of striatal dopamine uptake of 8-10% and of DAT between 5.7 and 6.4 years or 10 to 13 %. Higher nigrostriatal dopamine loss has been suggested in early than late onset PD [START_REF] Shih | Higher nigrostriatal dopamine neuron loss in early than late onset Parkinson's disease? A [99mTc]-TRODAT-1 SPECT study[END_REF]. Reduction of striatal dopamine by 57-80% [START_REF] Riederer | Relating mode of action to clinical practice: dopaminergic agents in Parkinson's disease[END_REF] and DAT loss of 56% or approximately 50% of nigral dopaminergic neurons cause motor symptoms [START_REF] Nurmi | Rate of progression in Parkinson's disease: a 6-[18F]fluoro-L-dopa PET study[END_REF]. Thus, about 50% of dopaminergic striatal innervation appears to be sufficient for normal motor function [START_REF] Bernheimer | Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations[END_REF]. Striate dopamine release was 60% reduced in PD patients, whereas in the frontal cortex it was within normal range, indicating that it remained preserved even in severe stages of the disease [START_REF] Piccini | New developments of brain imaging for Parkinson's disease and related disorders[END_REF]. Decreased DAT ultimately results in increased dopamine turnover preposing towards the occurrence of motor complications as PD progresses [START_REF] Sossi | Dopamine transporter relation to dopamine turnover in Parkinson's disease: a positron emission tomography study[END_REF]. However, recent studies in both movement disorders and dementing processes demonstrated that some subjects may tolerate substantial amounts of both aSyn and tau pathology without definite clinical manifestation, suggesting considerable compensatory mechanisms of the aging brain [START_REF] Nandhagopal | Invited article: Functional imaging in Parkinson disease[END_REF][START_REF] Davis | Alzheimer neuropathologic alterations in aged cognitively normal subjects[END_REF][START_REF] Knopman | Neuropathology of cognitively normal elderly[END_REF][START_REF] Morris | Cerebral amyloid deposition and diffuse plaques in "normal" aging: Evidence for presymptomatic and very mild Alzheimer's disease[END_REF][START_REF] Price | Tangles and plaques in nondemented aging and "preclinical" Alzheimer's disease[END_REF][START_REF] Price | Neuron number in the entorhinal cortex and CA1 in preclinical Alzheimer disease[END_REF]. Recent FDG-PET studies in PD showed only a short metabolic preclinical period [START_REF] Frigerio | Substantia nigra Lewy bodies, neurofibrillary tangles, and aging[END_REF].

It should be considered, however, that the biological significance and clinical impact of unresolved [START_REF] Carvey | Progressive dopamine neuron loss in Parkinson's disease: the multiple hit hypothesis[END_REF][START_REF] Sulzer | Multiple hit hypotheses for dopamine neuron loss in Parkinson's disease[END_REF][START_REF] Moore | Molecular pathophysiology of Parkinson's disease[END_REF][START_REF] Przedborski | Etiology and pathogenesis of Parkinson's disease[END_REF][START_REF] Schapira | Etiology of Parkinson's disease[END_REF]. Biophysical studies have suggested that rather the protofibrillary than the fibrillary form of aSyn is cytotoxic [START_REF] Caughey | Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders[END_REF][START_REF] Volles | Zeroing in on the pathogenic form of a-synuclein and its mechanism of neurotoxicity in Parkinson's disease[END_REF][START_REF] Tofaris | a-Synuclein dysfunction in Lewy body diseases[END_REF], whereas the LBs and LNs composed of fibrillary aSyn which are typically observed at autopsy, may be the structural manifestation of a cytoprotective response designed to confine and to eliminate cytotoxic proteins or abnormal cytoskeletal elements [START_REF] Outeiro | Protein aggregation disorders[END_REF][START_REF] Kuusisto | Morphogenesis of Lewy bodies: dissimilar incorporation of a-synuclein, ubiquitin, and p62[END_REF][START_REF] Tanaka | Aggresomes formed by a-synuclein and synphilin-1 are cytoprotective[END_REF][START_REF] Pan | The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson's disease[END_REF]. There is no correlation between the density of LB formation and neuronal cell loss [START_REF] Gomez-Tortosa | Clinical and quantitative pathologic correlates of dementia with Lewy bodies[END_REF], and the comparatively low number of neurons containing LBs in any brain region would not be expected to result from altered synaptic function. Nevertheless, significant intracellular protein aggregation, such as LB formation, are pathologic processes, reflecting changes in the cellular environment that may finally contribute to dysfunction of the involved cells. Recent studies showed development of LBs in grafted neurons in subjects with PD, suggesting host-to-graft propagation [START_REF] Li | Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation[END_REF].

Existing definitions and classification systems should be based on an understanding of the underlying pathologic process which, in the case of synucleinopathies is still incomplete.

Therefore, in view of the caveats discussed above and the inter-and intraindividual biological variabilities, the neuropathologic staging procedures are instruments being far from perfect [START_REF] Litvan | The etiopathogenesis of Parkinson disease and suggestions for future research. Part I[END_REF][START_REF] Uchihara | Research into neurodegenerative disease: an entangled web of mice and men[END_REF]. Although recent studies indicate that LB pathology is indeed progressive, the assessment of regional distribution patterns of aSyn pathology may either evaluate a stage of degeneration or conversely monitors the level of functional neuroprotection, concluding that the topographic mapping of aSyn deposition may be of questionable value, because its is unclear whether LB pathology is the primary (or solely relevant) pathology in PD and related disorders [START_REF] Parkkinen | Applicability of current staging/categorization of a-synuclein pathology and their clinical relevance[END_REF]. If it is the synucleinopathy that characterizes and drives idiopathic PD (and DLB) [START_REF] Wakabayashi | The Lewy body in Parkinson's disease: molecules implicated in the formation and degradation of a-synuclein aggregates[END_REF], the question arises about the degree of lesional density that has to be reached to induce clinical symptoms. Although it has not been proved yet, the presence of abnormal filaments or protein aggregations, which have been observed in PD to develop first in axons as LNs [START_REF] Orimo | Cardiac sympathetic denervation precedes neuronal loss in the sympathetic ganglia in Lewy body disease[END_REF][START_REF] Wakabayashi | Immunocytochemical localization of synaptic vesicle-specific protein in Lewy body-containing neurons in Parkinson's disease[END_REF], may finally damage the parent nerve cell by interfering with axonal transport [START_REF] Braak | Pathology associated with sporadic Parkinson's disease -where does it end?[END_REF][START_REF] Duda | Concurrence of a-synuclein and tau brain pathology in the Contursi kindred[END_REF][START_REF] Duda | Pathology and neurotransmitter abnormalities of dementia with Lewy bodies[END_REF][START_REF] Katsuse | Developmental stages of cortical Lewy bodies and their relation to axonal transport blockage in brains of patients with dementia with Lewy bodies[END_REF][START_REF] Saha | Parkinson's disease a-synuclein mutations exhibit defective axonal transport in cultured neurons[END_REF][START_REF] Del Tredici | A not entirely benign procedure: progression of Parkinson's disease[END_REF]. Keeping all these pros and cons in mind, one has to conclude that if robust correlations between clinical course and morphologic changes will be confirmed by future prospective clinico-pathologic studies, the neuropathologic staging/classification systems will need to be revised accordingly.

  abnormal aSyn aggregations are not yet clear. Like other fibrillary proteinaceous inclusions, such as NFTs or Pick bodies, they may represent end products of reactions to higherto unknown neuronal degenerative processes [149]. The question whether LBs and other aSyn A C C E P T E D M A N U S C R I P T aggregates are cytotoxic and lead to neuronal death due to production of oxidative stress, mitochondrial energy deficit and other noxious factors or are cytoprotective still remains

  medullary nuclei[START_REF] Jellinger | Lewy body-related a-synucleinopathy in the aged human brain[END_REF]. In another autopsy series of 60 autopsy-confirmed cases of PD(29 PD with dementia /PDD/ and 31 without dementia, mean age at death 82.5 years), some early PD symptoms were shown to occur already in rare cases with LB stage 2 (2.3%), eg., autonomic and bladder dysfunctions, sleeping disorders, constipation, orthostatic hypotension, and depression, and more often in stage 3 (15.0%), in which most patients clinically revealed

	stiffness, asymmetric rigidity, and mild hypomimia but no tremor. Stage 4 (50.0%) and stage
	5 (31.7%) showed typical PD-related motor features [3,67]. Thirty-nine brains (65%) showed
	almost equal aSyn load in SN, NBM, LC and both DMV and gigantocellular reticular
	formation, while 11 brains (18.3%) revealed considerable aSyn lesions in SN, LC and NBM,
	but only mild involvement of DMV. In this material, the most frequently involved CNS
	regions were SN (96.7%), NBM (98%), and LC (96.7%), whereas the DMV was involved in
	68-year-old woman with late-onset, dopa-
	responsive parkinsonism of almost 13 years duration, autopsy revealed severe neuronal loss
	with many LBs in DMV but only moderate neuron depletion (60%) in SNc without any LBs
	and moderate cell loss with diffuse aSyn cytoplasmic staining in LC, suggesting unusual

manifestation of LB disease in clinically definite PD

[START_REF] Wakabayashi | Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism[END_REF]

. In an autopsy series of 260 elderly subjects including 71 cases of autopsy proven PD,

[START_REF] Braak | Cognitive status correlates with neuropathologic stage in Parkinson disease[END_REF]

of DLB, 116 AD and 26 age-matched controls (with positive aSyn pathology in 51% of AD and 31% of controls), 30% of AD cases with multifocal aSyn pathology but without clinical EPS showed no involvement of the A C C E P T E D M A N U S C R I P T 91.7%. Five brains (8.3%), despite definite involvement of SN, LC and NBM in four and additional cingulate gyrus and limbic cortex involvement in one each, showed no aSyn lesions and almost no neuronal loss in DMV. The latter cases clinically presented with rigidakinetic and L-dopa responsive PD, with rest tremor in three and dementia in two cases (one each with frontal and limbic cortical involvement). Less frequently affected brain regions

FIGURE 1 .

 1 Progress and distribution pattern of PD-related neuronal pathology. ab accessory basal nucleus of amygdala, ac accessory cortical nucleus of amygdala, ad anterodorsal nucleus of amygdala, am anteromedial nucleus of thalamus, an abducens motor nucleus, ba basal nucleus of amygdala, bn basal nucleus of Meynert, ca1 first Ammon's horn sector, ca2 second Ammon's horn sector, ca caudate nucleus, cc corpus callosum, ce central nuclei of amygdala, cg central grey of mesencephalon, cl claustrum, co cortical nuclei of amygdala, cr central nucleus of raphe, db nucleus of the diagonal band, dm dorsomedial hypothalamic nucleus, dr dorsal nucleus of raphe, ds decussation of superior cerebellar peduncles, dv dorsal nuclear complex of vagal nerve, en entorhinal region, fn facial motor nucleus, fo fornix, gi gigantocellular reticular nucleus, gr granular nucleus of amygdala, hn hypoglossal motor nucleus, in infundibular nucleus, ir intermediate reticular zone, lc locus coeruleus, ld laterodorsal nucleus of the thalamus, lg lateral geniculate body, li nucleus limitans thalami, lt lateral nuclei of the thalamus, md mediodorsal nuclei of thalamus, me medial nuclei of amygdala, mf medial longitudinal fasciculus, mg medial geniculate body, ml medial lemniscus, mm medial mamillary nucleus, ms medial septal nucleus, mt mamillothalamic tract, mv dorsal motor nucleus of vagal nerve, oi oliva inferior, os oliva superior, ot optic tract, pe external pallidum, pf parafascicular nucleus, ph posterior hypothalamic nucleus, pi internal pallidum, po pontine grey, pr praepositus nucleus, pu putamen, pv paraventricular nucleus, re reticular nucleus of the thalamus, rm nucleus raphes magnus, ru nucleus ruber, sb subiculum, sc superior cerebellar peduncle, sf solitary fascicle, so supraoptic nucleus, sn substantia nigra, sp subpeduncular nucleus, st nucleus of the stria terminalis, sn subthalamic nucleus, te transentorhinal region, tl lateral tuberal nucleus, tm tuberomamillary nucleus, tp tegmental pedunculopontine nucleus, vl ventro-lateral nuclei of thalamus, vm ventromedial hypothalamic nucleus, vn vestibular nuclei, vt dopaminergic nuclei of ventral tegmentum (paranigral nucleus and pigmented parabrachial nucleus), zi zona incerta.

Table 1b :

 1b Neuropathologic diagnosis of DLB

			Alzheimer type pathology
	Braak stage:	0-II	III-IV	V-VI
	LBD
	Brainstem predominant ()	low	low	low
	Limbic (transitional)	high	intermediate	low
	Diffuse (neocortical)	high	high	intermediate

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

A C C E P T E D M A N U S C R I P T