Hideaki Yokoo

Neuropathologic diagnostic and nosologic criteria for frontotemporal lobar degeneration: consensus of the Consortium for Frontotemporal Lobar Degeneration

The aim of this study was to improve the neuropathologic recognition and provide criteria for the pathological diagnosis in the neurodegenerative diseases grouped as frontotemporal lobar degeneration (FTLD); revised criteria are proposed. Recent advances in molecular genetics, biochemistry, and neuropathology of FTLD prompted the Midwest Consortium for Frontotemporal Lobar Degeneration and experts at other centers to review and revise the existing neuropathologic diagnostic criteria for FTLD. The proposed criteria for FTLD are based on existing criteria, which include the tauopathies [FTLD with Pick bodies, corticobasal degeneration, progressive supranuclear palsy, sporadic multiple system tauopathy with dementia, argyrophilic grain disease, neurofibrillary tangle dementia, and FTD with microtubule-associated tau (MAPT) gene mutation, also called FTD with parkinsonism linked to chromosome 17 (FTDP-17)]. The proposed criteria take into account new disease entities and include the novel molecular pathology, TDP-43 proteinopathy, now recognized to be the most frequent histological finding in FTLD. TDP-43 is a major component of the pathologic inclusions of most sporadic and familial cases of FTLD with ubiquitin-positive, tau-negative inclusions (FTLD-U) with or without motor neuron disease (MND). Molecular genetic studies of familial cases of FTLD-U have shown that mutations in the progranulin (PGRN) gene are a major genetic cause of FTLD-U. Mutations in valosin-containing protein (VCP) gene are present in rare familial forms of FTD, and some families with FTD and/or MND have been linked to chromosome 9p, and both are types of FTLD-U. Thus, familial TDP-43 proteinopathy is associated with defects in multiple genes, and molecular genetics is required in these cases to correctly identify the causative gene defect. In addition to genetic heterogeneity amongst the TDP-43 proteinopathies, there is also neuropathologic heterogeneity and there is a close relationship between genotype and FTLD-U subtype. In addition to these recent significant advances in the neuropathology of FTLD-U, novel FTLD entities have been further characterized, including neuronal intermediate filament inclusion disease. The proposed criteria incorporate up-to-date neuropathology of FTLD in the light of recent immunohistochemical, biochemical, and genetic advances. These criteria will be of value to the practicing neuropathologist and provide a foundation for clinical, clinico-pathologic, mechanistic studies and in vivo models of pathogenesis of FTLD.

Clinical and neuropathologic variation in neuronal intermediate filament inclusion disease.

Background: Recently described neuronal intermediate filament inclusion disease (NIFID) shows considerable clinical heterogeneity. Objective: To assess the spectrum of the clinical and neuropathological features in 10 NIFID cases. Methods: Retrospective chart and comprehensive neuropathological review of these NIFID cases was conducted. Results: The mean age at onset was 40.8 (range 23 to 56) years, mean disease duration was 4.5 (range 2.7 to 13) years, and mean age at death was 45.3 (range 28 to 61) years. The most common presenting symptoms were behavioral and personality changes in 7 of 10 cases and, less often, memory loss, cognitive impairment, language deficits, and motor weakness. Extrapyramidal features were present in 8 of 10 patients. Language impairment, perseveration, executive dysfunction, hyperreflexia, and primitive reflexes were frequent signs, whereas a minority had buccofacial apraxia, supranuclear ophthalmoplegia, upper motor neuron disease (MND), and limb dystonia. Frontotemporal and caudate atrophy were common. Histologic changes were extensive in many cortical areas, deep gray matter, cerebellum, and spinal cord. The hallmark lesions of NIFID were unique neuronal IF inclusions detected most robustly by antibodies to neurofilament triplet proteins and α-internexin. Conclusion: NIFID is a neuropathologically distinct, clinically heterogeneous variant of frontotemporal dementia (FTD) that may include parkinsonism or MND. Neuronal IF inclusions are the neuropathological signatures of NIFID that distinguish it from all other FTD variants including FTD with MND and FTD tauopathies.

α-Internexin Is Present in the Pathological Inclusions of Neuronal Intermediate Filament Inclusion Disease

Neuronal intermediate filament (IF) inclusion disease (NIFID) is a novel neurological disease of early onset with a variable clinical phenotype including frontotemporal dementia, pyramidal, and extrapyramidal signs. Pathologically, in affected areas, there is neuronal loss, astrocytosis, and neuronal intracytoplasmic aggregates of abnormal neuronal IFs that contain neither tau nor alpha-synuclein. Thus, to characterize the neuronal IF protein profile of inclusions in NIFID, immunohistochemistry (IHC) was performed on 10 cases of NIFID, four normal aged controls (NL), and two cases of Alzheimers disease (AD) using a panel of anti-neuronal IF proteins. Immunoelectron microscopy was performed on selected cases and frozen tissue from the frontal lobe of four cases was used for biochemical studies including sequential extractions and Western blotting. Based on these studies, we report here for the first time that alpha-internexin, a neuronal IF protein, is present within the inclusions of NIFID as are all three neurofilament subunits: heavy, medium, and light. Thus, all class IV neuronal IF proteins are present within the pathological inclusions of this disease. Biochemistry revealed that IF aggregates were soluble in sodium dodecyl sulfate (SDS) and no post-translational modification was detected when compared with Alzheimers disease or aged control brains. Hence, we conclude that NIFID is characterized by the pathological cytoplasmic aggregation of all class IV neuronal IF proteins in brain. The discovery of alpha-internexin in the cytoplasmic inclusions implicates novel mechanisms of pathogenesis in NIFID and other neurological diseases with pathological accumulations of IFs.

Anti-Human Olig2 Antibody as a Useful Immunohistochemical Marker of Normal Oligodendrocytes and Gliomas

Olig2 is a recently identified transcription factor involved in the phenotype definition of cells in the oligodendroglial lineage. The expression of Olig2 transcript has been demonstrated in human oligodendroglial tumors, although the protein expression has not been studied extensively. We developed a polyclonal antibody to human Olig2 and analyzed it immunohistochemically. The antibody depicted a single distinct band of predicted molecular weight by Western blotting, and did not cross-react with human Olig1. In normal human brain tissue, the nuclei of oligodendrocytes of interfascicular, perivascular, and perineuronal disposition were clearly labeled by the antibody. Similarly, the nuclei of oligodendroglial tumors were labeled. There was no apparent correlation between the staining intensity and histological grade. Astrocytic components within the tumors were generally less or not stained. Astrocytic tumors were also positive with the Olig2 antiserum to a lesser extent, and the difference between oligodendroglial and astrocytic tumors was demonstrated by a statistical analysis. Olig2 and glial fibrillary acidic protein were expressed in a mutually exclusive manner, and Olig2 expression was cell-cycle related. Neither central neurocytoma nor schwannoma cases were stained. Our antibody was demonstrated to be useful in recognizing normal oligodendrocytes on paraffin sections, and applicable in diagnosis of some brain tumors.

Haploinsufficiency of CSF-1R and clinicopathologic characterization in patients with HDLS

Objective: To clarify the genetic, clinicopathologic, and neuroimaging characteristics of patients with hereditary diffuse leukoencephalopathy with spheroids (HDLS) with the colony stimulating factor 1 receptor (CSF-1R) mutation. Methods: We performed molecular genetic analysis of CSF-1R in patients with HDLS. Detailed clinical and neuroimaging findings were retrospectively investigated. Five patients were examined neuropathologically. Results: We found 6 different CSF-1R mutations in 7 index patients from unrelated Japanese families. The CSF-1R mutations included 3 novel mutations and 1 known missense mutation at evolutionarily conserved amino acids, and 1 novel splice-site mutation. We identified a novel frameshift mutation. Reverse transcription PCR analysis revealed that the frameshift mutation causes nonsense-mediated mRNA decay by generating a premature stop codon, suggesting that haploinsufficiency of CSF-1R is sufficient to cause HDLS. Western blot analysis revealed that the expression level of CSF-1R in the brain from the patients was lower than from control subjects. The characteristic MRI findings were the involvement of the white matter and thinning of the corpus callosum with signal alteration, and sequential analysis revealed that the white matter lesions and cerebral atrophy relentlessly progressed with disease duration. Spotty calcifications in the white matter were frequently observed by CT. Neuropathologic analysis revealed that microglia in the brains of the patients demonstrated distinct morphology and distribution. Conclusions: These findings suggest that patients with HDLS, irrespective of mutation type in CSF-1R, show characteristic clinical and neuroimaging features, and that perturbation of CSF-1R signaling by haploinsufficiency may play a role in microglial dysfunction leading to the pathogenesis of HDLS.

Systematic immunohistochemical profiling of 378 brain tumors with 37 antibodies using tissue microarray technology

We performed a systematic immunohistochemical study on 378 brain tumors using 37 antibodies and tissue microarray (TMA) technology. The aim of this study was to find new diagnostic biomarkers using antibodies established in our laboratory. Our TMA consisted of a grid of 1.5-mm cores that were extracted from individual donor blocks. Staining for each antibody was scored using a three-point system. We used hierarchical clustering analysis to interpret these data, which resulted in separation of all the brain tumors into seven groups. Although there were some exceptions, cases with the same histological diagnosis were generally grouped together. We then carried out statistical analyses to find the most useful antibodies for grouping of brain tumors. Ten antibodies [glial fibrillary acidic protein (GFAP), Olig2, vimentin, epithelial membrane antigen (EMA), cytokeratin (AE1/AE3), alpha-internexin, nestin, pinealocytes PP5, aquaporin-4 (AQP4) M13d and AQP4M13e] discriminated between astrocytomas and oligodendroglial tumors. Six antibodies [EMA, AE1/AE3, TUJ1, nestin, neurofilament protein-MH (NF-MH) and perivascular cells GP-1] showed significant differences between high-grade and low-grade gliomas. Our data have revealed new antibodies with potential diagnostic utility (Olig2, PP5, GP-1) and demonstrate that TMA technology is highly useful for evaluating newly established antibodies in brain-tumor research.

α-Internexin aggregates are abundant in neuronal intermediate filament inclusion disease (NIFID) but rare in other neurodegenerative diseases

Abnormal neuronal aggregates of α-internexin and the three neurofilament (NF) subunits, NF-L, NF-M, and NF-H have recently been identified as the pathological hallmarks of neuronal intermediate filament (IF) inclusion disease (NIFID), a novel neurological disease of early onset with a variable clinical phenotype including frontotemporal dementia, pyramidal and extrapyramidal signs. α-Internexin, a class IV IF protein, a major component of inclusions in NIFID, has not previously been identified as a component of the pathological protein aggregates of any other neurodegenerative disease. Therefore, to determine the specificity of this protein, α-internexin immunohistochemistry was undertaken on cases of NIFID, non-tau frontotemporal dementias, motor neuron disease, α-synucleinopathies, tauopathies, and normal aged control brains. Our results indicate that class IV IF proteins are present within the pleomorphic inclusions of all cases of NIFID. Small subsets of abnormal neuronal inclusions in Alzheimer’s disease, Lewy body diseases, and motor neuron disease also contain epitopes of α-internexin. Thus, α-internexin is a major component of the neuronal inclusions in NIFID and a relatively minor component of inclusions in other neurodegenerative diseases. The discovery of α-internexin in neuronal cytoplasmic inclusions implicates novel mechanisms of pathogenesis in NIFID and other neurological diseases with pathological filamentous neuronal inclusions.

Gene expression profiling and subgroup identification of oligodendrogliomas

The histological diagnosis of low-grade astrocytomas and oligodendrogliomas (WHO grade II) is often challenging, particularly in cases that show both astrocytic and oligodendroglial differentiation. We carried out gene expression profiling on 17 oligodendrogliomas (93% with LOH 1p and/or 19q) and 15 low-grade astrocytomas (71% with a TP53 mutation), using a cDNA array containing 1176 cancer-related genes. In oligodendrogliomas, 40 genes showed on average higher expression (at least a two-fold increase) than in astrocytomas, including DES, TDGF1, TGF-β, GABA-BR1A, Histone H4, CDKN1A, PCDH43, Rho7 and Jun-D, while 39 genes were expressed at lower levels (at least a two-fold decrease), including JNK2, ITGB4, JNK3A2, RhoC, IFI-56K, AAD14 and EGFR. Immunohistochemistry revealed nuclear staining of Jun-D in oligodendrogliomas, in contrast to the immunoreactivity of cytoplasm and cell processes in low-grade astrocytomas. Partial least-squares analysis of the 79 genes at least two-fold differentially expressed between oligodendrogliomas and low-grade astrocytomas demonstrated perfect separation of oligodendrogliomas from low-grade astrocytomas and normal cerebral white matter. Clustering analysis based on the entire gene set divided the 17 subjects with oligodendrogliomas into two subgroups with significantly different survival (log-rank test, P=0.0305; survival to 5-years, 80 vs 0%, P=0.048). These results demonstrate that oligodendrogliomas and low-grade astrocytomas differ in their gene expression profiles, and that there are subgroups of oligodendroglioma with distinct expression profiles related to clinical outcome.

A distinct pattern of Olig2-positive cellular distribution in papillary glioneuronal tumors: a manifestation of the oligodendroglial phenotype?

Mixed neuronal-glial tumors of the central nervous system display a wide spectrum of differentiation. Among them, the papillary glioneuronal tumor (PGNT) is characterized by pseudopapillary structures composed of astroglial cells covering hyalinized vessels, and by neurocytic, ganglioid and ganglion cells. In addition, a “nonspecific” cell type, not similar to either astrocytes or neurocytes, has been recognized since the initial reports. Recently, minigemistocytic cells and a population immunostained by anti-Olig2 antibody have also been recognized in PGNT. Olig2 is a transcription factor that is specific for the cellular phenotype of oligodendrocytes. The aim of this study was to further investigate the histological diversity of PGNT. We examined six cases of PGNT, each of which showed Olig2 immunopositivity. Minigemistocytes were encountered in three cases at close proximity to the Olig2-positive area. Olig2-positive cells were negative for glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen by double immunostaining, and mainly occupied the interpapillary area laterally adjacent to the GFAP-positive cells. They had relatively small, round and vesicular nuclei, and were formerly regarded as neurocytic cells or nonspecific cellular elements. Fluorescence in situ hybridization targeting chromosome 1p failed to demonstrate any deletion. This study disclosed an additional cellular component of PGNT that is characterized by Olig2 positivity, suggestive of oligodendroglial phenotype, and the results also encourage us to investigate oligodendroglial participation in various glioneuronal tumors.

An unusual variant of ependymoma with extensive tumor cell vacuolization

Abstract We report a case of ependymoma with unusual vacuolar features arising in the left occipital lobe of a 2-year-old child. The tumor was composed of cells with single or multiple cytoplasmic vacuoles and clear cells. Some cells showed a signet ring-like configuration. Clear cells were compactly arranged and showed an oligodendroglioma-like appearance. In addition, there were cellular ependymoma-like areas including perivascular pseudorosettes. On immunohistochemistry, glial fibrillary acidic protein and vimentin were mainly detected in cytoplasmic processes, and epithelial membrane antigen (EMA) staining showed granular and small vesicular reactivity. Ultrastructural investigation demonstrated intercellular microrosettes with or without cilia and long zonula adherens-type junctions that are typical of ependymoma. Furthermore, many intracytoplasmic lumina (ICL) were observed. Some ICL had microvilli and some did not. The latter varied in size, and may have fused with each other to develop giant ICL which could correspond to the signet ring-like configuration. Small ICL without microvilli had an appearance similar to that of distended endoplasmic reticula. Serial semithin and ultrathin sections revealed that EMA-positive structures were consistent with ICL containing microvilli and intercellular microrosettes. To determine the presence of unusual vacuolated ependymoma, electron microscopical examination was required. However, light microscopy was useful for detecting EMA-positive microvesicular and granular structures.