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 Parkinson's 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.

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

The pathological basis of dementia and visual hallucinations in Parkinson's 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.

Homocysteine-induced endoplasmic reticulum protein (herp) is up-regulated in parkinsonian substantia nigra and present in the core of Lewy bodies.

BACKGROUND: Recent studies highlight the role of endoplasmic reticulum (ER) stress and aberrant protein degradation in the pathogenesis of neurodegenerative disorders. Herp which is encoded by the HERPUD 1 (homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1) gene is a stress-response protein localized in the ER membrane of neurons and other cell types. Herp has been suggested to improve ER-folding, decrease ER protein load, and participate in ER-associated degradation (ERAD) of proteins. METHODS: Based on microarray expression profiling results we have predicted an increased expression of HERPUD1 in the substantia nigra of Parkinson's disease (PD) patients. We have now used brain tissue of some of the same and additional cases of sporadic PD to localize Herp mRNA and protein in individual cell types. RESULTS: We found expression of Herp in neurons and in glial cells including astrocytes. These findings were corroborated by in situ hybridization. Accumulation of Herp protein was also detected in the core of Lewy bodies suggesting a role in their formation. Hierarchical clustering analysis identified TWINKLE (PEO1) as the gene whose expression profile was most similar to that of Herp across the PD cohort. CONCLUSIONS: The nigral glial cells that expressed Herp at a high level resembled TUNEL-positive glia. While some of these cells likely undergo degeneration, the strong up-regulation of Herp in glia could help to explain the inflammation-like changes observed in PD ("neuroinflammation") as it has been shown that the unfolded protein response serves as an important regulator of inflammatory genes in other organs.

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

AIMS: It has been proposed that alpha-synuclein (alpha Syn) pathology in Parkinson's 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 alpha Syn pathology has been questioned. In addition, the comparative occurrence of alpha Syn 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 Parkinson's Disease Society Tissue Bank at Imperial College, London. The incidence and topographic distribution of alpha Syn 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 alpha Syn compatible with a caudo-rostral spread of alpha Syn through the PD brain. However, 47% of the cases did not fit the predicted spread of alpha Syn pathology and in 7% the DMV was not affected even though alpha Syn inclusions were found in SN and cortical regions. We also observed a high incidence of alpha Syn 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 alpha Syn pathology in all PD brains.

Electron microscopy of tissue adherent to explanted electrodes in dystonia and Parkinson's disease.

Deep brain stimulation (DBS) is used to treat a variety of severe medically intractable movement disorders, including Parkinson's disease, tremor and dystonia. There have been few studies examining the effect of chronic DBS on the brains of Parkinson's disease patients. Most of these post mortem studies concluded that chronic DBS caused mild gliosis around the lead track and did not damage brain tissue. There have been no similar histopathological studies on brains from dystonic patients who have undergone DBS. In this study, our objective was to discover whether tissue would be attached to DBS electrodes removed from patients for routine clinical reasons. We hoped that by examining explanted DBS electrodes using scanning (SEM) and/or transmission (TEM) electron microscopy we might visualize any attached tissue and thus understand the electrode-human brain tissue interaction more accurately. Initially, SEM was performed on one control DBS electrode that had not been implanted. Then 21 (one subthalamic nucleus and 20 globus pallidus internus) explanted DBS electrodes were prepared, after fixation in 3% glutaraldehyde, for SEM (n = 9) or TEM (n = 10), or both (n = 2), according to departmental protocol. The electrodes were sourced from two patients with Parkinson's disease, one with myoclonic dystonia, two with cervical dystonia and five with primary generalized dystonia, and had been in situ for 11 and 31 months (Parkinson's disease), 16 months (myoclonic dystonia), 14 and 24 months (cervical dystonia) and 3-24 months (primary generalized dystonia). Our results showed that a foreign body multinucleate giant cell-type reaction was present in all TEM samples and in SEM samples, prewashed to remove surface blood and fibrin, regardless of the diagnosis. Some of the giant cells were >100 microm in diameter and might have originated from either fusion of parenchymal microglia, resident perivascular macrophage precursors and/or monocytes/macrophages invading from the blood stream. The presence of mononuclear macrophages containing lysosomes and sometimes having conspicuous filopodia was detected by TEM. Both types of cell contained highly electron-dense inclusions, which probably represent phagocytosed material. Similar material, the exact nature of which is unknown, was also seen in the vicinity of these cells. This reaction was present irrespective of the duration of implantation and may be a response to the polyurethane component of the electrodes' surface coat. These findings may be relevant to our understanding of the time course of the clinical response to DBS in Parkinson's disease and various forms of dystonia, as well as contributing to the design characteristics of future DBS electrodes.

Neurogenetic diseases: molecular diagnosis and therapeutic approaches.

A neurogenetic disorder is defined as a clinical disease caused by a defect in one or more genes which affect the differentiation and function of the neuroectoderm and its derivatives. Genetic findings in various neurogenetic disorders are discussed. Huntington disease, spinobulbar muscular atrophy, and the autosomal dominant cerebellar ataxias are examples of autosomal dominant disorders caused by the expansion of trinucleotides (CAG) within disease genes. The CAG expansions appear to result in a gain of gene function. Prenatal, presymptomatic, and differential diagnostic tests are based on the detection of the repeat expansions. Point mutations within disease genes result in many additional neurogenetic disorders. An autosomal dominant form of amyotrophic lateral sclerosis and various types of craniosynostotic syndromes are described. The mutations in the disease genes also appear to result in a gain of gene function. Molecular diagnosis in these disorders is based on the direct examination of the mutated gene by methods such as single-strand conformation polymorphism analysis, denaturing gradient gel electrophoresis, and direct DNA sequencing. In many neurogenetic disorders the disease gene has not yet been identified. Here molecular diagnosis relies on indirect approaches based on methods such as the analysis of linkage and of allelic association. Hereditary forms of dystonia are presented as examples. Common sporadic neurological disorders such as Alzheimer and Parkinson diseases frequently have multifactorial causes. Investigations into the molecular basis and the development of diagnostic tests in these two important diseases are discussed. At present no curative therapies exist in neurogenetic disorders. Gene therapeutic approaches, however, provide promise for a cure in at least some of these diseases. Basic principles of gene therapy are explained and attempts at gene therapy in Alzheimer and Parkinson diseases are described. Finally, some of the many obstacles are summarized that must be overcome before gene therapy becomes feasible in most monogenic neurological diseases.

Ultrastructural location of major histocompatibility complex (MHC) class II positive perivascular cells in histologically normal human brain.

The expression of major histocompatibility complex (MHC) class I and II antigens was studied in surgical and postmortem brain biopsy tissue using light and electron microscopic immunocytochemistry. In addition, monoclonal antibodies directed against human macrophages (EBM11) and alpha-smooth muscle actin were applied. It is shown that blood vessel-associated MHC class II immunoreactivity in histologically normal human brain can be localized to a distinct class of cells, termed perivascular cells, which share macrophage but not smooth muscle cell antigen. This immunophenotype, the location in the perivascular space as well as the morphology, frequency and tissue distribution distinguish perivascular cells from pericytes and intraparenchymal microglia. It is suggested that MHC class II positive perivascular cells are a normal constituent of the human cerebral microvasculature. The potential role of these cells in immunological reactions occurring at the blood-brain interface is discussed.

Microglia and the development of spongiform change in Creutzfeldt-Jakob disease.

Recent in vitro experiments suggest that neurotoxicity of the prion protein is dependent on the presence of microglia. We have studied 11 cases of Creutzfeldt-Jakob disease (CJD) using immunocytochemistry in combination with computerized image analysis to clarify the relationship between spongiform change and microglial activation. MHC class II-positive microglia were almost exclusively confined to cortical gray matter where the neuropil area occupied by these cells exceeded that of controls more than 350-fold. In cortical regions with a bimodal distribution of spongiform degeneration, the presence of class II-positive microglia correlated well with the presence of vacuolation in layer V, but significantly less with spongiform change in layers II and III. In areas where spongiform degeneration affected the entire depth of the cortex, activated microglia were predominantly located in the inner one-half of the cortex or were evenly distributed throughout all cortical laminae. Here, microglia exhibited atypical, tortuous cell processes and occasionally intracytoplasmic vacuoles, suggesting that microglia themselves may become a disease target. Taken together, our results provide indirect evidence against an early causative involvement of microglia in the development of spongiform change. At later stages, however, diseased microglia could produce harmful factors which mediate both astrogliosis and neuronal injury.

Oligodendrogliomas: reproducibility and prognostic value of histologic diagnosis and grading.

Prognostic value of histological grading of oligodendroglial tumors is controversial and interobserver reproducibility in grading of these tumors is unknown. Seven neuropathologists and 6 surgical pathologists experienced in brain tumor-diagnosis assessed 124 oligodendroglial tumors operated at the Mayo Clinic (1960-1990). Among histologic parameters upon which current oligodendroglioma grading systems are based, only high cellularity, presence of mitoses, microcalcifications, endothelial hypertrophy, endothelial proliferation, and necrosis appeared to be reproducible. Reproducible histologic features, based on consensus ratings among neuropathologists (defined as > 60%), were evaluated for the association with cause-specific survival by fitting Cox regression models. By univariate analysis, a significant association with survival was found for age, high cellularity, presence of mitoses, endothelial hypertrophy and proliferation and necrosis. On multivariable analysis with a stepwise variable selection method, only age and presence of endothelial proliferation were found to be independently associated with survival with a discriminatory index of the model of 0.68. Mitotic index was significantly associated with survival based on the grading from each separate neuropathologist, but it was not based on consensus, most likely because this was classified as indeterminate in 54% of cases. Alternatively, "models fit" considering the assessment of single neuropathologists, identified a model based on age and on mitotic index with similar discriminatory indices of 0.69-0.7. Our study found few factors independently associated with cause specific-survival among morphological parameters. These findings are consistent with the present WHO stratification of oligodendrogliomas into low- and high-grade variants.

No man alone: the rediscovery of Alois Alzheimer's original cases.

In 1992 and 1997, respectively, the histological slides of Alois Alzheimer's original cases were rediscovered in Munich. This material, which has survived two world wars, was not originally kept at the Institute of Neuropathology of the University of Munich where it was found. Parviz Mehraein, head of the institute, saved anonymous neuropathological material given away by the Psychiatric Clinic of the University of Munich and the Max-Planck-Institute of Psychiatry. Yet it was not until Kohshiro Fujisawa [corrected] of the Tokyo Metropolitan Institute of Neuroscience wrote a letter that the search leading to the rediscovery of Alzheimer's cases was initiated. Henry deF. Webster [corrected] of the National Institutes of Health in Bethesda, Maryland, had mediated his contact to Munich. Histological and molecular genetic findings obtained on the tissue sections have been reported previously (Neurogenetics 1997, 1:73-80; 1998, 1:223-228). The present article summarizes the unusual history of this rediscovery and at the same time illustrates the great value of international exchange in science.

Non-radioactive direct sequencing of PCR products amplified from neuropathological specimens.

We have developed a simple, rapid and relatively inexpensive protocol for direct non-isotopic cycle sequencing of DNA amplified using the polymerase chain reaction (PCR). PCR is performed on routine and archival neuropathological tissue. For sequencing, a 5'-digoxigenin end-labelled oligonucleotide primer is annealed and extended during thermal cycling, sequencing reactions are separated on a standard sequencing gel and the gel is contact-blotted to a nylon membrane. Sequenced DNA is visualized using immunological detection of digoxigenin.

The X-linked dystonia-parkinsonism syndrome (XDP): clinical and molecular genetic analysis.

Dystonia and parkinsonism are two major representatives of movement disorders. The X-linked dystonia-parkinsonism syndrome (XDP) serves as a model system for the study of both dystonia and parkinsonism since both symptom complexes occur together and are inherited as Mendelian traits with very high penetrance. XDP, which is endemic to the Philippine island of Panay, originated by a single mutation ("genetic founder effect"), thus assuring homogeneity of the disorder at the molecular level. The disease locus, DYT3, has been assigned to the proximal long arm (Xq12-21.1) of the human X chromosome. A strategy is described to isolate this gene by positional cloning. The rationale of this strategy, the major methods involved and technical terms are explained.

Microglia: immune network in the CNS.

In recent years much progress has been made toward a better understanding of the nature and function of microglial cells. This review summarizes new developments and attempts to provide a perspective for future avenues to take in microglial research. Microglia are considered to play an active role in a variety of neurological diseases. Their function in forming a network of immune competent cells within the CNS is discussed.

Microglial activation in Alzheimer disease: Association with APOE genotype.

Microglial cells are considered to play an important role in the pathogenesis of Alzheimer disease. Apart from producing the Alzheimer amyloid precursor (APP) as an acute phase protein, microglial cells seem to be involved in the deposition of its amyloidogenic cleavage product, the amyloid-beta peptide (Abeta). Abeta is bound by apolipoprotein E (APOE) in an isoform-specific manner, and it has been demonstrated that inheritance of the AD susceptibility allele, APOE epsilon4, is associated with increased deposition of Abeta in the cerebral cortex. However, the relationship between APOE epsilon4 gene dose and microglial activation is unknown. Using microglial expression of major histocompatibility complex class II molecules as a marker, we have performed a quantitative genotype-phenotype analysis on microglial activation in frontal and temporal cortices of 20 APOE genotyped AD brains. The number of activated microglia and the tissue area occupied by these cells increased significantly with APOE epsilon4 gene dose. When a model of multiple linear regression was used to compare the relative influence of APOE genotype, sex, disease duration, age at death, diffuse and neuritic plaques as well as neurofibrillary tangles on microglial activation, only APOE genotype was found to have a significant effect. Thus, the APOE gene product represents an important determinant of microglial activity in AD. Since microglial activation by APP has been shown to be modulated by apoE in vitro, a direct role of microglia in AD pathogenesis is conceivable.

Neuronal FasL induces cell death of encephalitogenic T lymphocytes.

Apoptosis of inflammatory cells plays a crucial role in the recovery from autoimmune CNS disease. However, the underlying mechanisms of apoptosis induction are as yet ill-defined. Here we report on the neuronal expression of FasL and its potential function in inducing T-cell apoptosis. Using a combination of facial nerve axotomy and passive transfer encephalomyelitis, the fate of CD4+ encephalitogenic T cells engineered to express the gene for green fluorescent protein was followed. FasL gene transcripts and FasL protein were detected in neurons by in sit-hybridization and immunohistochemistry. T cells infiltrating preferentially the injured brain parenchyma were found in the immediate vicinity of FasL expressing neurons and even inside their perikarya. In contrast to neurons, T cells rapidly underwent apoptosis. In co-cultures of hippocampal nerve cells and CD4 T lymphocytes, we confirmed expression of FasL in neurons and concomitant induction of T-cell death. Antibodies blocking neuronal FasL were shown to have a protective effect on T-cell survival. Thus, FasL expression by neurons in neuroinflammatory diseases may constitute a pivotal mechanism underlying apoptosis of encephalitogenic T cells.

Neuronal MCP-1 expression in response to remote nerve injury.

Direct injury of the brain is followed by inflammatory responses regulated by cytokines and chemoattractants secreted from resident glia and invading cells of the peripheral immune system. In contrast, after remote lesion of the central nervous system, exemplified here by peripheral transection or crush of the facial and hypoglossal nerve, the locally observed inflammatory activation is most likely triggered by the damaged cells themselves, that is, the injured neurons. The authors investigated the expression of the chemoattractants monocyte chemoattractant protein MCP-1, regulation on activation normal T-cell expressed and secreted (RANTES), and interferon-gamma inducible protein IP10 after peripheral nerve lesion of the facial and hypoglossal nuclei. In situ hybridization and immunohistochemistry revealed an induction of neuronal MCP-1 expression within 6 hours postoperation, reaching a peak at 3 days and remaining up-regulated for up to 6 weeks. MCP-1 expression was almost exclusively confined to neurons but was also present on a few scattered glial cells. The authors found no alterations in the level of expression and cellular distribution of RANTES or IP10, which were both confined to neurons. Protein expression of the MCP-1 receptor CCR2 did not change. MCP-1, expressed by astrocytes and activated microglia, has been shown to be crucial for monocytic, or T-cell chemoattraction, or both. Accordingly, expression of MCP-1 by neurons and its corresponding receptor in microglia suggests that this chemokine is involved in neuron and microglia interaction.

Transferrin Receptor Expression and Iron Uptake in the Injured and Regenerating Rat Sciatic Nerve.

Iron-saturated transferrin is a ubiquitous growth factor that plays a critical role in cellular iron uptake, growth and proliferation. Here we have studied the expression and distribution of transferrin receptors and iron uptake following injury of the rat sciatic nerve. Axotomy led to a massive but transient increase (days 2 - 9, maximum day 4) in [125I]transferrin binding at the site of the injury and in the distal, denervated part of the crushed or resected sciatic nerve, shortly preceding the time course of cellular proliferation (Friede and Johnstone, Acta Neuropathol, 7, 218 - 231, 1967; Jurecka et al., Acta Neuropathol, 32, 299 - 312, 1975). An additional, transient increase in specific binding was observed during reinnervation after reconnection of the resected sciatic nerve. Immunocytochemistry using the Ox-26 monoclonal antibody revealed strong and simultaneous expression of the transferrin receptor protein on two different cell types: on a subpopulation of blood-borne macrophages invading the injured peripheral nerve and on Schwann cells reacting to denervation and reinnervation. In addition, studies using intravenously injected radioactive iron (59Fe3+) showed a massive increase in endoneural iron uptake confined to the lesion site and to the distal part of the axotomised sciatic nerve, parallel to the time course of reactive transferrin receptor expression. Since iron is an essential cofactor of a number of key enzymes needed in energy metabolism and DNA synthesis, these data suggest that the induction of transferrin receptor expression may play an important role in the regulation of cellular growth and proliferation during peripheral nerve regeneration.

Apolipoprotein E epsilon 4 allele, cognitive decline, and deterioration of everyday performance in Alzheimer's disease.

Many studies have demonstrated an association between the apolipoprotein E (apoE) epsilon 4 allele and Alzheimer's disease (AD). The present study is concerned with the relationship between the apoE epsilon 4 allele and the progression of symptoms in AD. We determined rates of cognitive decline and deterioration in everyday performance prospectively over 3 years in 64 patients with clinically diagnosed AD using the Cambridge Cognitive Examination (CAMCOG), the Mini-Mental State Examination (MMSE), and the Dementia Scale (DS) included in the Cambridge Mental Disorders of the Elderly Examination (CAMDEX). Carriers and noncarriers of the epsilon 4 allele did not significantly differ in cognitive functioning and everyday performance at baseline measurements. The time that had elapsed since the estimated onset of symptoms was also not different between the two groups. This suggested that the clinical progression of AD was not associated with the epsilon 4 status before the patients entered the study. On prospective observation, the rate of cognitive decline assessed with the CAMCOG and the MMSE and the rate of deterioration in everyday performance rated with the DS were also not different between carriers and noncarriers of the epsilon 4 allele. We conclude that the clinical course of AD is independent of the apoE epsilon 4 allele.

Peripheral nerve lesion produces increased levels of major histocompatibility complex antigens in the central nervous system.

Proliferation of central nervous system (CNS) glia in response to peripheral nerve injury occurs without apparent participation of cells of the immune system. It is shown here that following transection of the rat facial nerve there is strongly elevated expression of class I, and to a lesser extent, class II antigens of the major histocompatibility complex (MHC) in the facial nucleus. It is demonstrated by double-immunofluorescence studies that the cells responsible for increased levels of MHC class I antigens are endogenous brain microglia. These findings emphasize the thought that microglia are immunocompetent cells, but, at the same time, raise the possibility for a non-immunological function of MHC antigens under conditions of neural regeneration.

New expression of myelomonocytic antigens by microglia and perivascular cells following lethal motor neuron injury.

The results of the present study demonstrate that following lethal motor neuron injury microglia and perivascular cells, as well as brain macrophages derived from the latter two cell types, newly express antigens of the myelomonocytic lineage as recognized by the monoclonal antibodies ED1 and ED3. It is suggested that differences in the immunophenotype of resident brain macrophage precursor cells, i.e. microglia and perivascular cells, and macrophages occurring outside the central nervous system (CNS) may be explained by differences in local macrophage antigen expression rather than by a different embryological lineage. The new appearance of antigens common to peripheral macrophages on neural phagocytes in CNS lesions may therefore not necessarily imply that most or all of these cells are of recent blood origin.