R. K. B. Pearce

Whole genome expression profiling of the medial and lateral substantia nigra in Parkinson’s disease

We have used brain tissue from clinically well-documented and neuropathologically confirmed cases of sporadic Parkinson’s disease to establish the transcriptomic expression profile of the medial and lateral substantia nigra. In addition, the superior frontal cortex was analyzed in a subset of the same cases. DNA oligonucleotide microarrays were employed, which provide whole human genome coverage. A total of 570 genes were found to be differentially regulated at a high level of significance. A large number of differentially regulated expressed sequence tags were also identified. Levels of mRNA sequences encoded by genes of key interest were validated by means of quantitative real-time polymerase chain reaction (PCR). Comparing three different normalization procedures, results based on the recently published GeneChip Robust Multi Array algorithm were found to be the most accurate predictor of real-time PCR results. Several new candidate genes which map to PARK loci are reported. In addition, the DNAJ family of chaperones is discussed in the context of Parkinson’s disease pathogenesis.

The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease: a critical analysis of α-synuclein staging

Aims: It has been proposed that alpha‐synuclein (αSyn) pathology in Parkinsons disease (PD) spreads in a predictable caudo‐rostral way with the earliest changes seen in the dorsal motor nucleus of the vagus nerve (DMV). However, the reliability of this stereotypical spread of αSyn pathology has been questioned. In addition, the comparative occurrence of αSyn pathology in the spinal cord and brain has not been closely studied. Methods: In order to address these issues, we have examined 71 cases of PD from the UK Parkinsons Disease Society Tissue Bank at Imperial College, London. The incidence and topographic distribution of αSyn pathology in several brain regions and the spinal cord were assessed. Results: The most affected regions were the substantia nigra (SN; in 100% of cases) followed by the Nucleus Basalis of Meynert (NBM) in 98.5%. Fifty‐three per cent of cases showed a distribution pattern of αSyn compatible with a caudo‐rostral spread of αSyn through the PD brain. However, 47% of the cases did not fit the predicted spread of αSyn pathology and in 7% the DMV was not affected even though αSyn inclusions were found in SN and cortical regions. We also observed a high incidence of αSyn in the spinal cord with concomitant affection of the DMV and in a few cases in the absence of DMV involvement. Conclusions: Our results demonstrate a predominant involvement of the SN and NBM in PD but do not support the existence of a medullary induction site of αSyn pathology in all PD brains.

Striatal β-Amyloid Deposition in Parkinson Disease With Dementia

Dementia is common in Parkinson disease (PD), although its anatomic and pathologic substrates remain undefined. Recently, striatal abnormalities in Lewy body diseases have been described, but their clinical relevance is not clear. Thirty PD cases from the United Kingdom Parkinsons Disease Society Tissue Bank were grouped as demented (PDD; n = 16) and nondemented (PD; n = 14) based on a review of clinical records. The extent of &agr;-synuclein, tau, and amyloid &bgr; peptide (A&bgr;) deposition in the caudate nucleus, putamen, and nucleus accumbens was assessed. All cases showed severe dopaminergic striatal terminal denervation based on tyrosine hydroxylase immunohistochemistry. &agr;-synuclein and tau deposition in the striatum were rare in both groups, but the A&bgr; burden was significantly greater in the striatum of PD cases with dementia than present in the nondemented PD group. Striatal A&bgr; deposition was type-independent of Alzheimer disease changes in the cortex and was minimal in nondemented PD cases. We conclude that A&bgr; deposition in the striatum strongly correlates with dementia in PD.

Dementia and visual hallucinations associated with limbic pathology in Parkinson's disease

The pathological basis of dementia and visual hallucinations in Parkinsons disease (PD) is not yet fully understood. To investigate this further we have conducted a clinico-pathological study based on 30 post-mortem PD brains. PD cases were stratified into groups according to clinical characteristics as follows: (1) cognitively intact (n=9); (2) cases with severe dementia and visual hallucinations (n=12); (3) cases with severe dementia and no visual hallucinations (n=4); and (4) cases with severe visual hallucinations and no dementia (n=5). The extent of alpha-synuclein (alphaSyn), tau and amyloid beta peptide (Abeta) deposition was then examined in the CA2 sector of the hippocampus and in neocortical and subcortical areas known to subserve cognitive function. We find that dementia in PD is significantly associated with alphaSyn in the anterior cingulate gyrus, superior frontal gyrus, temporal cortex, entorhinal cortex, amygdaloid complex and CA2 sector of the hippocampus. Abeta in the anterior cingulate gyrus, entorhinal cortex, amygdaloid complex and nucleus basalis of Meynert is also associated with dementia as is tau in the CA2 sector of the hippocampus. alphaSyn burden in the amygdala is strongly related to the presence of visual hallucinations but only in those PD cases with concomitant dementia. Statistical analysis revealed that alphaSyn burden in the anterior cingulate gyrus could differentiate demented from non-demented PD cases with high sensitivity and specificity. We conclude that alphaSyn in limbic regions is related to dementia in PD as well as to visual hallucinations when there is an underlying dementia.

Transcriptome analysis reveals link between proteasomal and mitochondrial pathways in Parkinson’s disease

There is growing evidence that dysfunction of the mitochondrial respiratory chain and failure of the cellular protein degradation machinery, specifically the ubiquitin–proteasome system, play an important role in the pathogenesis of Parkinson’s disease. We now show that the corresponding pathways of these two systems are linked at the transcriptomic level in Parkinsonian substantia nigra. We examined gene expression in medial and lateral substantia nigra (SN) as well as in frontal cortex using whole genome DNA oligonucleotide microarrays. In this study, we use a hypothesis-driven approach in analysing microarray data to describe the expression of mitochondrial and ubiquitin–proteasomal system (UPS) genes in Parkinson’s disease (PD). Although a number of genes showed up-regulation, we found an overall decrease in expression affecting the majority of mitochondrial and UPS sequences. The down-regulated genes include genes that encode subunits of complex I and the Parkinson’s-disease-linked UCHL1. The observed changes in expression were very similar for both medial and lateral SN and also affected the PD cerebral cortex. As revealed by “gene shaving” clustering analysis, there was a very significant correlation between the transcriptomic profiles of both systems including in control brains. Therefore, the mitochondria and the proteasome form a higher-order gene regulatory network that is severely perturbed in Parkinson’s disease. Our quantitative results also suggest that Parkinson’s disease is a disease of more than one cell class, i.e. that it goes beyond the catecholaminergic neuron and involves glia as well.

Nucleus basalis of Meynert revisited: anatomy, history and differential involvement in Alzheimer’s and Parkinson’s disease

It has been well established that neuronal loss within the cholinergic nucleus basalis of Meynert (nbM) correlates with cognitive decline in dementing disorders such as Alzheimer’s disease (AD). Friedrich Lewy first observed his eponymous inclusion bodies in the nbM of postmortem brain tissue from patients with Parkinson’s disease (PD) and cell loss in this area can be at least as extensive as that seen in AD. There has been confusion with regard to the terminology and exact localisation of the nbM within the human basal forebrain for decades due to the diffuse and broad structure of this “nucleus”. Also, while topographical projections from the nbM have been mapped out in subhuman primates, no direct clinicopathological correlations between subregional nbM and cortical pathology and specific cognitive profile decline have been performed in human tissue. Here, we review the evolution of the term nbM and the importance of standardised nbM sampling for neuropathological studies. Extensive review of the literature suggests that there is a caudorostral pattern of neuronal loss within the nbM in AD brains. However, the findings in PD are less clear due to the limited number of studies performed. Given the differing neuropsychiatric and cognitive deficits in Lewy body-associated dementias (PD dementia and dementia with Lewy bodies) as compared to AD, we hypothesise that a different pattern of neuronal loss will be found in the nbM of Lewy body disease brains. Understanding the functional significance of the subregions of the nbM could prove important in elucidating the pathogenesis of dementia in PD.

The morbid anatomy of dementia in Parkinson's disease.

Dementia in Parkinson’s disease (PD/PDD) is a common complication with a prevalence of up to 50%, but the specific changes underlying the cognitive decline remain undefined. Neuronal degeneration resulting in the dysfunction of multiple subcortical neurochemical projection systems has been described along with Lewy body-type pathology in cortical and limbic regions. Advanced alpha-synuclein (αSyn) pathology is not necessarily sufficient for producing dementia and concomitant Alzheimer’s disease (AD) change has also been proposed as a possible substrate of PDD. A lack of consensus in the extant literature likely stems from clinical heterogeneity and variable reliability in clinical characterisation as well as other historical and methodological issues. The concurrent presence of abnormally deposited αSyn, amyloid-β and tau proteins in the PDD brain and the interaction of these molecules in a linked pathological cascade of AD and PD-related mechanisms may prove important in determining the underlying pathological process for the development of dementia in PD and this concept of combined pathologies awaits further investigation.

Controversies over the staging of α-synuclein pathology in Parkinson’s disease

Parkinson’s disease (PD) is a complex disorder the aetiopathogenesis of which has remained enigmatic almost 200 years after its initial description by James Parkinson. It is now accepted that the neuropathological changes deWning PD are by no means conWned to the substantia nigra pars compacta but that other central and peripheral nervous tissues show degenerative changes as well, which result in dysfunction in a variety of neurotransmitter systems. The papers by Polymeropoulos et al. (15) and Kruger et al. (11), demonstrating that -synuclein ( Syn) gene mutations can cause PD, and the discovery that the encoded protein, which is natively unfolded and still of unknown physiological function, constitutes a major component of Lewy bodies (LBs) and Lewy neurites (LNs) by Spillantini et al. [16], provided the cornerstone for a molecular deWnition of the disease and placed Syn at the centre of PD research interests. Although molecular work is in progress to unravel the mechanisms by which Syn is abnormally deposited in the PD brain, an important issue explored recently and attracting much attention is the natural history of Syn deposition, that is, how Syn pathology progresses within the PD brain [2]. Aiming at a precise patho-architectonic analysis, Braak et al. in 2003 devised a staging system according to which Syn pathology progresses in a systematic fashion and proposed six distinct stages of PD. According to this design, the earliest pathology is to be observed in the dorsal motor nucleus of the vagus nerve (DMV) (stage 1) from where Syn pathology is thought to proceed in a rostral direction via the pons (stage 2) to the midbrain (stage 3) and thence to the basal forebrain and mesocortex (stage 4), Wnally spreading to/involving the neocortex (stages 5–6). However, a certain percentage of incidental and PD cases from diVerent research centres in Europe and the US have been shown/found not to comply with/follow this predicted caudo-rostal pattern [1, 10, 13, 14]. While the observations as such which are documented in the Braak et al., 2003 publication raise little objection, principal methodological considerations make it very unlikely that they provide a reliable and accurate picture of the progression of PD pathology from its earliest stages. Certainly, highlighting the multiple-system and widespread nature of neurodegeneration throughout the central nervous system in PD is important and F. Lewy’s classical work may be cited in this context. The complexity and variety of clinical symptoms found in PD are the reXection of an anatomically wide-ranging disease process. This view is also in agreement with animal studies, which have demonstrated that lesions restricted to the substantia nigra or dopamine pathways alone do not accurately model PD. The review by Meredith et al. in this journal supports this view [12]. Regarding the PD-staging system proposed by Braak, some critical issues need to be considered:

DnaJB6 Is Present in the Core of Lewy Bodies and Is Highly Up-Regulated in Parkinsonian Astrocytes

DnaJ/Hsp40 chaperones determine the activity of Hsp70s by stabilizing their interaction with substrate proteins. We have predicted, based on the in silico analysis of a brain‐derived whole‐genome transcriptome data set, an increased expression of DnaJ/Hsp40 homologue, subfamily B, member 6 (DnaJB6) in Parkinsons disease (PD; Moran et al. [ 2006 ] Neurogenetics 7:1–11). We now show that DnaJB6 is a novel component of Lewy bodies (LBs) in both PD substantia nigra and PD cortex and that it is strongly up‐regulated in parkinsonian astrocytes. The presence of DnaJB6 in the center of LBs suggests an early and direct involvement of this chaperone in the neuronal disease process associated with PD. The strong concomitant expression of DnaJB6 in astrocytes emphasizes the involvement of glial cells in PD and could indicate a route for therapeutic intervention. Extracellular alpha‐synuclein originating from intravesicular alpha‐synuclein is prone to aggregation and the potential source of extracellular aggregates (Lee [ 2008 ] J. Mol. Neurosci. 34:17–22). The observed strong expression of DnaJB6 by astrocytes could reflect a protective reaction, so reducing the neuronal release of toxic alpha‐synuclein and supporting the astrocyte response in PD might limit the progression of the disease process.

Comparative study of commercially available anti-alpha-synuclein antibodies.

Immunohistochemistry for alpha‐synuclein has become the histological technique of choice for the diagnosis for Parkinsons disease, Dementia with Lewy bodies and Multiple System Atrophy (http://www.ICDNS.org). Nevertheless, no standardised protocol has been proposed. We have reviewed 242 of the 270 studies published until June 2005 that mentioned immunohistochemistry for anti‐alpha synuclein on human tissue and we found that only 75 (31%) used commercial antibodies. We also noted that protocols, particularly dilution and antigen unmasking, varied between studies, even when the same antibody was employed. In order to establish a standardised protocol for alpha‐synuclein immunohistochemistry, which can be applied in diagnostic neuropathology we tested seven commercial monoclonal antibodies in brains of subjects with Parkinsons disease, dementia with Lewy bodies, multiple system atrophy, multiple sclerosis with incidental Lewy bodies and aged‐matched normal brain and determined for each antibody the best suited protocol for antigen unmasking. We evaluated the intensity of immunolabelling in Lewy bodies, neuropil threads, dendrites, pre‐synaptic terminals, granular cytoplasmic positivity, peri‐axonal positivity, glial inclusions and non‐specific immunolabelling. Although our results showed that all the antibodies detected alpha‐synuclein inclusions, differences were noted between antibodies, particularly with regard to the detection of glial inclusions. From our study, the best antibodies of the seven tested appeared to be those directed against amino acids 116–131 and 15–123 and we suggest them to be used in routine diagnostic practice for alpha‐synucleinopathies.