Up-regulation of matrix metallopeptidase 12 in motor neurons undergoing synaptic stripping.

Axotomy of the rodent facial nerve represents a well-established model of synaptic plasticity. Post-traumatic "synaptic stripping" was originally discovered in this system. We report upregulation of matrix metalloproteinase MMP12 in regenerating motor neurons of the mouse and rat facial nucleus. Matrix metalloproteinases (matrix metallopeptidases, MMPs) are zinc-binding proteases capable of degrading components of the extracellular matrix and of regulating extracellular signaling networks including within synapses. MMP12 protein expression in facial motor neurons was enhanced following axotomy and peaked at day 3 after the operation. The peak of neuronal MMP12 expression preceded the peak of experimentally induced synaptic plasticity. At the same time, MMP12 redistributed intracellularly and became predominantly localized beneath the neuronal somatic cytoplasmic membrane. Both findings point to a role of MMP12 in the neuronal initiation of the synaptic stripping process. MMP12 is the first candidate molecule for such a trigger function and has potential as a therapeutic target. Moreover, since statins have been shown to increase the expression of MMP12, interference with synaptic stability may represent one mechanism by which these widely used drugs exert their side effects on higher CNS functions.

The potential for genetically altered microglia to influence glioma treatment.

Diffuse and unstoppable infiltration of brain and spinal cord tissue by neoplastic glial cells is the single most important therapeutic problem posed by the common glioma group of tumors: astrocytoma, oligoastrocytoma, oligodendroglioma, their malignant variants and glioblastoma. These neoplasms account for more than two thirds of all malignant central nervous system tumors. However, most glioma research focuses on an examination of the tumor cells rather than on host-specific, tumor micro-environmental cells and factors. This can explain why existing diffuse glioma therapies fail and why these tumors have remained incurable. Thus, there is a great need for innovation. We describe a novel strategy for the development of a more effective treatment of diffuse glioma. Our approach centers on gaining control over the behavior of the microglia, the defense cells of the CNS, which are manipulated by malignant glioma and support its growth. Armoring microglia against the influences from glioma is one of our research goals. We further discuss how microglia precursors may be genetically enhanced to track down infiltrating glioma cells.

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.

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.

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.

Analysis of alpha-synuclein, dopamine and parkin pathways in neuropathologically confirmed parkinsonian nigra.

The identification of mutations that cause familial Parkinson's disease (PD) provides a framework for studies into pathways that may be perturbed also in the far more common, non-familial form of the disorder. Following this hypothesis, we have examined the gene regulatory network that links alpha-synuclein and parkin pathways with dopamine metabolism in neuropathologically verified cases of sporadic PD. By means of an in silico approach using a database of eukaryotic molecular interactions and a whole genome transcriptome dataset validated by qRT-PCR and histological methods, we found parkin and functionally associated genes to be up-regulated in the lateral substantia nigra (SN). In contrast, alpha-synuclein and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene expression levels were significantly reduced in both the lateral and medial SN in PD. Gene expression for Septin 4, a member of the GTP-binding protein family involved in alpha-synuclein metabolism was elevated in the lateral parkinsonian SN. Additionally, catalase and mitogen-activated protein kinase 8 and poly(ADP-ribose) polymerase family member 1 (PARP1) known to function in DNA repair and cell death induction, all members of the dopamine synthesis pathway, were up-regulated in the lateral SN. In contrast, two additional PD-linked genes, glucocerebrosidase and nuclear receptor subfamily 4, group A, member 2 (NR4A2) showed reduced expression. We show that in sporadic PD, parkin, alpha-synuclein and dopamine pathways are co-deregulated. Alpha-synuclein is a member of all three gene regulatory networks. Our analysis results support the view that alpha-synuclein has a central role in the familial as well as the non-familial form of the disease and provide steps towards a pathway definition of PD.

The medial and lateral substantia nigra in Parkinson's disease: mRNA profiles associated with higher brain tissue vulnerability.

Sporadic Parkinson's disease (PD) is characterized by progressive death of dopaminergic neurons within the substantia nigra. However, pathological cell death within this nucleus is not uniform. In PD, the lateral tier of the substantia nigra (SNl) degenerates earlier and more severely than the more medial nigral component (SNm). The cause of this brain regional vulnerability remains unknown. We have used DNA oligonucleotide microarrays to compare gene expression profiles from the SNl to those of the SNm in both PD and control cases. Genes expressed more highly in the PD SNl included the cell death gene, p53 effector related to PMP22, the tumour necrosis factor (TNF) receptor gene, TNF receptor superfamily, member 21, and the mitochondrial complex I gene, NADH dehydrogenase (ubiquinone) 1beta subcomplex, 3, 12 kDa (NDUFbeta3). Genes that were more highly expressed in PD SNm included the dopamine cell signalling gene, cyclic adenosine monophosphate-regulated phosphoprotein, 21 kDa, the activated macrophage gene, stabilin 1, and two glutathione peroxidase (GPX) genes, GPX1 and GPX3. Thus, there is increased expression of genes encoding pro-inflammatory cytokines and subunits of the mitochondrial electron transport chain, and there is a decreased expression of several glutathione-related genes in the SNl suggesting a molecular basis for pathoclisis. Importantly, some of the genes that are differentially regulated in the SNl are known to be expressed highly or predominantely in glial cells. These findings support the view that glial cells can be primarily affected in PD emphasizing the importance of using a whole tissue approach when investigating degenerative CNS disease.

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.

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

Immunohistochemistry for alpha-synuclein has become the histological technique of choice for the diagnosis for Parkinson's 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 Parkinson's 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.

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.

Microglia in culture: what genes do they express?

The cell culture model utilized in this study represents one of the most widely used paradigms of microglia in vitro. After 14 days, microglia harvested from the neonatal rat brain are considered 'mature'. However, it is clear that this represents a somewhat arbitrary definition. In this paper, we provide a transcriptome definition of such microglial cells. More than 7,000 known genes and 1,000 expressed sequence tag clusters were analysed. 'Microglia genes' were defined as sequences consistently expressed in all microglia samples tested. Accordingly, 388 genes were identified as microglia genes. Another 1,440 sequences were detected in a subset of the cultures. Genes consistently expressed by microglia included genes known to be involved in the cellular immune response, brain tissue surveillance, microglial migration as well as proliferation. The expression profile reported here provides a baseline against which changes of microglia in vitro can be examined. Importantly, expression profiling of normal microglia will help to provide the presently purely operational definition of 'microglial activation' with a molecular biological correlate. Furthermore, the data reported here add to our understanding of microglia biology and allow projections as to what functions microglia may exert in vivo, as well as in vitro.

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.

Towards a transcriptome definition of microglial cells.

This study provides an expression signature of interferon-gamma (IFN-gamma)-activated microglia. Microglia are macrophage precursor cells residing in the brain and spinal cord. The microglial phenotype is highly plastic and changes in response to numerous pathological stimuli. IFN-gamma has been established as a strong immunological activator of microglial cells both in vitro and in vivo. Affymetrix RG_U34A microarrays were used to determine the effect of IFN-gamma stimulation on migroglia cells isolated from newborn Lewis rat brains. More than 8,000 gene sequences were examined, i.e., 7,000 known genes and 1,000 expressed sequence tag (EST) clusters. Under baseline conditions, microglia expressed 326 of 8,000 genes examined (approximately 4% of all genes, 182 known and 144 ESTs). Transcription of only 34 of 7,000 known genes and 8 of 1,000 ESTs was induced by IFN-gamma stimulation. The majority of the newly expressed genes encode pro-inflammatory cytokines and components of the MHC-mediated antigen presentation pathway. The expression of 60 of 182 identified genes and of 9 of 144 ESTs was increased by IFN-gamma, whereas 29 of 182 known genes and 7 of 144 ESTs were down-regulated or undetectable in IFN-gamma-stimulated cultures. Overall, the activating effect of IFN-gamma on the microglial transcriptome showed restriction to pathways involved in antigen presentation, protein degradation, actin binding, cell adhesion, apoptosis, and cell signaling. In comparison, down-regulatory effects of IFN-gamma stimulation appeared to be confined to pathways of growth regulation, remodeling of the extracellular matrix, lipid metabolism, and lysosomal processing. In addition, transcriptomic profiling revealed previously unknown microglial genes that were de novo expressed, such as calponin 3, or indicated differential regulatory responses, such as down-regulation of cathepsins that are up-regulated in response to other microglia stimulators.

Nuclear hormone and orphan receptors: their role in neuronal differentiation and cytoprotection and in the pathogenesis of Parkinson's disease.

Human nuclear hormone receptors (NHR) and orphan receptors (NOR) act as transcription factors in response to a wide range of circulating hormones and unknown ligands. A role for NHR and NOR in disorders of the subcortical dopaminergic pathways such as Parkinson's disease (PD) is suggested by a wealth of recent data including experimental observations. Both classes of receptors promote the formation of specific neuronal identities, tissue patterning during embryonic development and the maturation of vulnerable monoaminergic and cholinergic neurons. NHR and NOR are also known to exert a neuroprotective function on adult neurons. The scope of this review is to revisit the functional profile of these receptors with particular reference to their activity in the development of selected neuronal populations relevant to the pathophysiology of PD and to discuss how they may relate to the neuropathological and clinical expression of the disease.

Transformation of donor-derived bone marrow precursors into host microglia during autoimmune CNS inflammation and during the retrograde response to axotomy.

Macrophages in the brain can have a triple source. They may originate from recently blood-derived precursors, from the largely resident perivascular cell population (perivascular macrophages and related cells), and from intrinsic parenchymal as well as perivascular microglia. Although continuous exchange of part of the perivascular cell population with bone marrow-derived precursors is now accepted, the turnover of adult parenchymal microglia has remained enigmatic. Using bone-marrow chimeras carrying an unexpressed marker gene and carbon labeling of peripheral monocyte/macrophages in a combined model of facial nerve axotomy and transfer experimental autoimmune encephalitis, we demonstrate for the first time that there is an easy to induce exchange between parenchymal central nervous system (CNS) microglia and the macrophage precursor cell pool of the bone marrow. Furthermore, very low level infiltration of the CNS parenchyma by recently bone marrow-derived microglia could be observed after simple peripheral nerve axotomy that is followed by neuronal regeneration. Thus, microglial cells can be considered wanderers between the peripheral immune system and the CNS where they may act as a "Trojan horse" in infections. The fact that recently bone marrow-derived parenchymal microglia fully integrate into a regenerating brain nucleus' architecture encourages entirely new approaches for delivering genes into the adult CNS.

Inter-laboratory comparison of DNA preservation in archival paraffin-embedded human brain tissue from participating centres on four continents.

DNA extracted from formalin-fixed and paraffin-embedded brain tissue is known to contain as yet ill-characterized inhibitors of the PCR process. As part of a project that aims to clarify the role of mitochondrial DNA sequence variation in human neurodegenerative diseases using DNA from various ethnic backgrounds, we have investigated factors that influence the preservation of archival DNA and its suitability for PCR. In this study, neuropathological tissue samples were analysed that had been routinely processed in 18 international centres on four continents. Following DNA extraction, PCR amplification of mitochondrial and nuclear DNA sequences was performed with and without additional purification of the template DNA. In addition, the DNA used for PCR was analysed by HPLC. Phosphate-buffered formalin proved to be a superior fixative compared with unbuffered aldehyde: DNA extraction resulted in greater yields, the molecular weight of the isolated DNA was higher and PCR was more successful. PCR inhibitors were identified as (1) high concentrations of small (<300 bp) DNA fragments that competitively compete with template DNA and (2) contaminants of the DNA template solution including denatured protein that cannot be completely removed by phenolic extraction. HPLC analysis did not reveal significant qualitative differences between DNA isolated from fresh-frozen tissue samples and DNA recovered from formalin-fixed, paraffin-embedded brain tissue. The fact that DNA could be amplified from the majority of tissue specimens in this study suggests that rare diseases and diseases where ethnic background plays an important role can be sampled for genetic polymorphism analysis on a global scale using archival neuropathological collections.

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.