K. Miyazu

Cytoskeletal protein abnormalities in patients with olivopontocerebellar atrophy — an immunocytochemical and Gallyas silver impregnation study

A highly sensitive silver technique for glial cytoplasmic inclusions (GCI) in olivopontocerebellar atrophy (OPCA) was applied to tissues from 15 patients with neurodegenerative disorders inluding OPCA, Joseph disease, Alzheimers disease (AD), Huntingtons chorea, Pick disease and three control non‐neurological subjects. Brain tissue from both OPCA and AD impregnated positively. Neurons, astroglia and oligodendroglia in the putamen, pontine nucleus and inferior olivary nucleus all impregnated in addition to white matter oligodendroglia. Neuronal inclusions in the pontine nucleus appeared as compact or fibrillary masses, and GCI‐bearing oligodendroglia and astrocytes showed homogeneously impregnated somata. The myelinated pontocerebellar tract and the white matter surrounding the inferior olivary nucleus contained a small number of impregnated nerve fibres with a hollow structure, which resembled the myelin sheath. Immunocytochemical studies to clarify these argyrophilic structures in the OPCA subjects employed paired helical filament (PHF), microtubule associated proteins (MAPS), MAPl, MAPZ, MAPS, tau, ubiquitin, neurofilament (200 or 70 kilodaltons) and myelin basic protein (MBP) antisera. GCI‐bearing white matter oligodendroglia expressed PHF, tau, MAP5 and ubiquitin immunoreactives and non‐argyrophilic astroglia were positive for MAP5 antiserum alone. In the putamen, pontine nuclei and inferior olivary nuclei, impregnated neurons as well as the GCI‐bearing oligodendroglia immunostained with PHF, tau, MAP5 and ubiquitin antisera and impregnated astroglia were also immunoreactive to these antisera except for being tau negative in the putamen. Silver impregnated nerve fibres showed only MBP immunoreactivity. These findings indicate that the argyrophilia in the OPCA subjects closely correlates with PHF and tau immunoreactivities.

Borrelia burgdorferi-seropositive chronic encephalomyelopathy: Lyme neuroborreliosis? An autopsied report.

A 36-year-old Japanese woman presented with progressive cerebellar signs and mental deterioration of subacute course after her return from the USA. Her serum antibody to spirochete Borrelia burgdorferi was significantly elevated. A necropsy 4 years after her initial neurological signs revealed multifocal inflammatory change in the cerebral cortex, thalamus, superior colliculus, dentate nucleus, inferior olivary nucleus and spinal cord. The lesions showed spongiform change, neuronal cell loss, astrocytosis and proliferation of activated microglial cells. The internal capsule was partially vacuolated and the spinal cord, notably at the thoracic level, was demyelinated and cavitated in the lateral funiculus. Microglial cells aggregated within and around the spongiform lesions and microglial nodules were present in the medulla oblongata. Use of Warthin-Starry stain demonstrated silver-impregnated organisms strongly suggesting B. burgdorferi in the central nervous tissues. The dentate nucleus and inferior olivary nucleus showed the most advanced lesions with profound fibrillary gliosis. Occlusive vascular change was relatively mild, and fibrous thickening of the leptomeninges with lymphocyte infiltrates was localized in the basal midbrain. The ataxic symptoms were due to the dentate and olivary nucleus lesions and mental deterioration was attributable to the cortical and thalamic lesions. Spongiform change, neuronal cell loss, and microglial activation are characteristic pathological features in the present case. The cerebellar ataxia and subsequent mental deterioration are unusual clinical features of Lyme neuroborreliosis. Spirochete B. burgdorferi can cause focal inflammatory parenchymal change in the central nervous tissues and the present case may be an encephalitic form of Lyme neuroborreliosis.

KP-1 Is a Marker for Extraneuronal Neurofibrillary Tangles and Senile Plaques in Alzheimer Diseased Brains

KP-1 immunostaining with microwave pretreatment in formalin-fixed, paraffin-embedded sections enhanced its immunoreactivity revealing extraneuronal neurofibrillary tangles (NFTs) called ghost tangles, senile plaques (SPs) and perivascular deposits as well as microglial labelling in Alzheimer-diseased brains. KP-1 stained cored and uncored SPs, granules within the SPs, perivascular β-amyloid protein (βAP) and star-like βAP deposits in cortical layer I, which was confirmed in comparison to silver-impregnated structures in the Reusche-stained or Gallyas-Schiff-stained sections. On double immunostaining with KP-1 and ubiquitin, ghost tangles were labelled by KP-1 and intraneuronal NFTs were positive for ubiquitin. A few KP-1-positive granules deposits different from amyloid core were found within the SPs and the outer margin of amyloid cores of SPs were stained by KP-1. KP-1-positive microglia were attached to the ubiquitin-positive intraneuronal NFTs. Microglia were more numerously labelled by CR3/43 than by KP-1, and CR3/43-positive microglia were found to be preferentially attached to SPs. As KP-1 recognizes lysosome-associated antigen CD68, similarities between KP-1 positivity and Reusche-stained structures suggested that lysosomal activity was associated with βAP deposits and ghost tangles were involved in lysosome-associated processes. It is speculated that lysosomes play a role in the process of ghost tangle formation and in βAP deposits leading to SP formation.

Another phenotype of frontotemporal dementia and parkinsonism linked to chromosome-17 (FTDP-17) with a missense mutation of S305N closely resembling Pick’s disease

Sirs: Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is an inherited neurodegeneration caused by mutation of the tau gene and clinically shows personality change, parkinsonism and dementia [17]. The frontotemporal lobe, substantia nigra and basal ganglia are affected with neuronal and glial tau-positive inclusions. FTDP-17 sometimes assumes a different phenotype for the same mutation [13]. Pick’s disease (PD)-like syndrome has been reported in FTDP-17 patients with mutations at G389R [3, 10, 12], K257T [12, 14] and K369I [11], which were characterized by Pick bodies (PB)-like inclusions. The first patient was reported by Iijima et al. [6] with missense mutation of S305N featured ring-like neurofibrillary tangles (NFT) and tau-positive glial inclusions but lacked circumscribed cortical atrophy and parkinsonism. In this study, we report the two brothers with S305N mutation [9] of whom the elder brother was previously reported as PD [8]. A 47-year-old man, a younger brother of a previously reported patient [8], presented with inertia and forgetfulness in August 2001 [9]. The brothers’ paternal grandmother had been known to be eccentric and the brothers’ father had shown curious and aggressive behavior and died at age of 44 years in a mental hospital. He scored 23 on MMSE and 71 on full-scale IQ. MRI showed atrophy of the right temporal lobe where the middle and inferior temporal gyrus, medial and lateral occipitotemporal gyri and hippocampal formation were selectively atrophied with a knife-edge appearance. After informed consent approved by the ethical committee of Kanazawa University, genomic DNA was extracted from paraffinembedded tissue from the brain of his elder brother and from the peripheral blood sample from the patient and his mother. The regions encoding exon 9–13 of the tau gene were amplified by PCR using unique primer sets, and the PCR products were directly sequenced [16]. Since mutation of S305N in exon 10 of the tau gene generates BsrDI recognition site, restriction fragment length polymorphism analysis using BsrDI was employed to confirm the mutation. DNA sequence analysis of exon 10 of the tau gene identified AGT to AAT transition in circulating leucocytes from the patient and a normal sequence in his mother, thus indicating a heterozygous mutation of S305N. The restriction fragment length polymorphism method analysis using BsrDI identified the same mutation in the paraffin section of the brain of the elder brother and leucocytes from the younger brother because this mutation generates the recognition site for BsrDI, which recognizes GCAATG. Immunolabeling with a polyclonal antibody to phosphorylated serine262 (pSer262), serine422 (pSer422) (Sigma-Aldrich) and AT8 was performed to discriminate PB-like inclusions from PB. PB-like inclusions intensely expressed AT8 and pSer422 but pSer262 expression was found only in a limited number of them (Fig. 1). Astrocytic inclusions in the hippocampus, amygdala, basal ganglia and thalamus were also labeled with AT8 and pSer422 but unlabeled with pSer262. The pSer262 expression of PB-like inclusions and astrocytic inclusions are different from tau expressions of PB [7]. Electron microscopic study with a JEM-1210 of 0.5-micrometer thick sections of small cubic specimens taken from the hippocampus and cerebral cortex of the older brother disclosed that PB-like inclusions consisted of straight, randomly orientated fibrils measuring approximately 10–20 nm in width and 60–600 nm in length with some of the fibrils showing constriction (Fig. 2). FTDP-17 cases mimicking Pick’s disease in five studies [3, 10–12, 14] showed tau-positive PB-like inclusions. So missense mutations can produce filamentous structure. Ultrastructurally PB-like inclusions in our case were similar to those seen in PB [7, 19] and in FTDP-17 patients in the five studies. Three patients with S305S mutation FTDP-17 [15] mimicked progressive supranuclear palsy, in which pathological tau aggregation was similar to that of Alzheimer type NFT. Iijima’s case [6] had no lobar atrophy but had NFT with twisted tubules close to Alzheimer’s disease. The codon 305 is located near the stem-loop and forms part of a stem-loop structure at the 5’ splice donor site. This codon mutation causes stem-loop instability or alterations in the consensus seLETTER TO THE EDITORS

Regional Analysis of Differently Phosphorylated Tau Proteins in Brains from Patients with Alzheimer's Disease

Neurofibrillary tangles (NFT) in Alzheimer’s disease (AD) are composed of abnormally phosphorylated tau proteins. Many phosphorylation sites have been reported in the AD brain, and NFT distribution was now roughly classified into 3 stages by Braak stage; this classification is based on pathological studies using the specific silver impregnation technique. The aim of our study was to examine the regional distribution of differently phosphorylated tau proteins with 5 site-specific monoclonal antibodies against the tau proteins, AT8, AT180, HT7, Tau2 and Tau5. We then compared our findings with those obtained from silver-stained NFT in an attempt to clarify the relationship between abnormal phosphorylation sites of the tau protein and NFT development. AT180 and AT8 labeled the highest and Tau2 the lowest density of NFT in any regions, while Tau5 and HT7 showed inconsistent distribution. In the limbic cortex, cornu ammonis, entorhinal cortex and cingulate cortex, silver-stained NFT density significantly correlated with density of NFT labeled with the 5 anti-tau antibodies, but cerebral isocortices showed heterogenous patterns of tau-positive NFT. Quantification of tau-positive regional NFT density showed that the AD-associated phosphorylation process progresses from the C-terminal to the N-terminal of the amino acid sequence, and correlation of Gallyas-stained NFT density with tau-labeled NFT density was more significant in the limbic cortices than the cerebral isocortices, which implies that stereotypical phosphorylation occurs in the limbic structures.

Membranous lipodystrophy (Nasu-Hakola disease) with thalamic degeneration: report of an autopsied case

SummaryAn autopsied case of membranous lipodystrophy (Nasu-Hakola disease, NHD) with thalamic degeneration was reported. A 34-year-old Japanese man was diagnosed as having NHD by bone biopsy prior to the onset of clinical symptoms. His maternal grandfather and paternal grandmother are cousins, but this family history is negative for NHD. He developed frontal lobe syndrome at the age of 35 with progressive dementia, and died of acute renal failure at the age of 46. Gross inspection of the brain detected atrophy and softening of the cerebral white matter, predominantly in the frontal lobe. Microscopically, numerous spheroids, predominant fibrillary gliosis with less prominent demyelination “dissociation glio-myélinique” and scanty sudanophilic lipid droplets were observed, indicating the sclerosing type of NHD. An unusual pathological finding in this case was selective involvement of the thalamic nuclei with preservation of the other gray matter except for focal cortical necrosis. The topography of the affected thalamic nuclei is similar to that of systemic thalamus degeneration. An association with thalamic degeneration in NHD has not been previously reported. The present case suggests that NHD also affects the thalamus.

Non-familial olivopontocerebellar atrophy combined with late onset Alzheimer's disease: a clinico-pathological case report.

A 76-year-old woman with olivopontocerebellar atrophy (OPCA) presented with progressive intellectual deterioration. She showed cerebellar ataxia and muscle atrophy and weakness, and gradually developed generalized dementia with visuospatial disturbance. An autopsy revealed numerous senile plaques (SPs), neurofibrillary tangles (NFTs) and neuropil threads particularly in the CA1, subiculum and entorhinal cortex and to a lesser degree in the cerebral neocortex shown by immunostaining and specific silver impregnation techniques. The nucleus basalis of Meynert had numerous NFTs with fibrillary gliosis and neuronal cell loss. The basis pontis was markedly atrophied and the pontine nucleus had severe neuronal depopulation and gliosis. The pontine transverse fibers were demyelinated with their axons being fragmented. The cerebellar white matter was also severely degenerated. The striatum, Onufs and intermediolateral nuclei of the spinal cord remained unchanged. Ubiquitin immunohistochemistry and Gallyas silver impregnation technique revealed oligodendroglial inclusions in the pontine nucleus, corticopontine tract, cerebral and cerebellar white matter. On double immunostaining of KP1 and ubiquitin, globular neurite SPs encircled by KP1-positive fibrous structures were found in the hippocampus and cerebral neocortex. The curly neurite SPs contained KP1-positive granules. The KP1-positive microglial cells were distributed widely in the cerebral white matter and HLA-DR-positive ones were found around the SPs. The present case showed generalized dementia compatible with Alzheimers disease (AD) and had a pathologically limbic type of late onset AD. This is the first case where AD affected non-familial OPCA.

Morphometric Study on the CH4 of the Nucleus Basalis of Meynert in Alzheimer's Disease

Ch4 neurons were studied morphometrically in three normal controls and three patients with Alzheimers disease (AD). Two series of preparations stained with cresyl violet and with the indirect immunoperoxidase method using a monoclonal antibody to acetylcholinesterase (AChE) counterstained with cresyl violet were morphometrically analyzed. The cholinergic neurons were identified as positive reaction products of AChE in the perikarya. The cross-sectional area of all the Ch4 neurons with clearly visible nucleoli in one preparation was measured using a computer imaging system. Evaluation of these data provided a mean value of the cross-sectional neuronal area (CNA), number of neurons, standard deviation of the CNA and coefficient of variation of the cholinergic and noncholinergic neurons. The cholinergic neurons were decreased in number but showed hypertrophy in the early stage of AD, and they gradually became atrophic in the advanced stage. The noncholinergic neurons showed only atrophy without definite neuronal cell depletion. These findings indicate that cholinergic neurons have a plasticity and remodeling property against the AD process and suggest that hypertrophy of the cholinergic neurons in the Ch4 is the result of the effect of neurotrophic factors.

Striatonigral Degeneration Combined with Olivopontocerebellar Atrophy with Subcortical Dementia and Hallucinatory State

We present an autopsied case of striatonigral degeneration (SND) combined with olivopontocerebellar atrophy (OPCA) with subcortical dementia and hallucinatory state. A Japanese woman without a remarkable family history showed hand tremor at the age of 35 years, followed by bradykinesia, muscle rigidity, orthostatic hypotension, neurogenic bladder and pyramidal signs. No obvious cerebellar symptoms were found. Various antiparkinsonian drugs were administered, but were not markedly effective for the parkinsonism. She developed a mild dementia characterized by mild memory disturbance with preservation of orientation, slowing of thought processes, emotional lability toward sadness, impaired ability to manipulate acquired knowledge and poor calculating, and by the absence of aphasia, apraxia and agnosia. The features in this patient were consistent with those seen in subcortical dementia. She also had auditory hallucinations. MRI revealed hypointense T2 signals in the putamina and substantia nigra. T1-weighted MRI demonstrated atrophy of both the pons and cerebellum in addition to atrophy of the putamina and substantia nigra. EEG showed slowing of background activity. She died of cardiac failure at the age of 47. Autopsy disclosed brain stem tegmental atrophy, SND, OPCA and many glial cytoplasmic inclusions in the central nervous system, but well-preserved cerebrum. We discuss the relationship between the psychiatric symptoms and pathologic findings of brain stem tegmentum.

Are there sequential morphometrical changes in the nucleus basalis in Alzheimer's disease?

Degenerated neurons of the nucleus basalis of Meynert (nbM) were quantitatively analyzed in 3 normal and 3 Alzheimers disease (AD) subjects. In this study, the Ch4 of the nbM was examined using the indirect immunoperoxidase method with a monoclonal antibody to acetylcholinesterase (AChE) counterstained with cresyl violet. AChE-rich neurons were designated as the cholinergic neurons. The cross-sectional area of all the Ch4 neurons with clearly visible nucleoli in one preparation was measured using a computer image analyzing system. Furthermore, we compared these data with the numbers of neurofibrillary tangles (NFTs) and neuritic plaques (NPs) in the temporal cortex by Gallyas silver stain. The cholinergic neurons decreased in number and size according to the length of the disease duration but the surviving cholinergic neurons in the size range from 800 to 1,000 micron 2 in a case with short clinical duration were increased in number. The non-cholinergic neurons showed only atrophy without definite neuronal cell depletion. The 400- to 1,000-microns 2-sized non-cholinergic neurons were markedly decreased in number, and the number of 300-microns 2-sized non-cholinergic neurons remained unchanged. Although there was an inverse correlation between the degree of atrophy and depletion of the cholinergic neurons with the number of NFTs and NPs in 2 AD cases with 3 and 6 years of disease duration, this correlation was not found in an AD case with 12 years of disease duration, probably due to extensive and profound grey matter degeneration.