Aberrant accumulation of ErbB4 in progressive supranuclear palsy.

AIMS: The human epidermal growth factor receptor family consists of four members that belong to the ErbB lineage of proteins (ErbB1-4). Neuregulin-1 (NRG1)/ErbB signalling regulates brain development and function. Abnormalities in this signalling have been implicated in the aetiology or development of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. So, we aimed at investigating whether the expression of NRG1 or ErbB proteins are altered in progressive supranuclear palsy (PSP). METHODS: The brains of 10 PSP and six control patients were investigated by immunohistochemical analysis. RESULTS: Whereas C-terminal ErbB4 immunoreacitivity was partially but distinctly present in the cytoplasm and/or in the nucleus of neurons in control patients, it was rarely observed in the neuronal nuclei in PSP patients. In contrast, neurofibrillary tangles, coiled bodies and threads were robustly immunoreactive for C-terminal ErbB4 in PSP. Double immunofluorescence for C-terminal ErbB4 and phospho-tau revealed co-localization of these proteins within neuronal and glial inclusions. To the contrary, there was no difference in the subcellular localization of NRG1, ErbB1, ErbB2, and N-terminal ErbB4 between control and PSP patients. These proteins were localized in the cytoplasm of neurons. CONCLUSIONS: Our present results suggest that NRG1/ErbB4 signalling could be an important event in the pathogenesis of PSP.

Regionally different immunoreactivity for Smurf2 and pSmad2/3 in TDP-43-positive inclusions of amyotrophic lateral sclerosis.

AIMS: Smad ubiquitination regulatory factor-2 (Smurf2), an E3 ubiquitin ligase, can interact with Smad proteins and promote their ubiquitin-dependent degradation, thereby controlling the cellular levels of these signalling mediators. We previously reported that phosphorylated Smad2/3 (pSmad2/3) was sequestered in transactive response DNA-binding protein-43 (TDP-43) inclusions in the spinal cord of patients with amyotrophic lateral sclerosis (ALS). Recent biochemical and immunohistochemical studies on spinal cord and brain of ALS patients demonstrated that the composition of the TDP-43 inclusions is regionally distinct, suggesting different underlying pathogenic processes. We aimed to elucidate regional differences in pathomechanisms and composition of TDP-43 inclusions in relation to pSmad2/3 and Smurf2. METHODS: The spinal cord and brain tissues of 13 sporadic ALS (SALS) patients were investigated using immunohistochemical analysis. RESULTS: TDP-43-positive inclusions in lower motor neurones of SALS patients were immunopositive for Smurf2 and pSmad2/3. Multiple immunofluorescence staining for Smurf2, pSmad2/3, TDP-43 and ubiquitin revealed co-localization of these four proteins within the inclusions in lower motor neurones of SALS patients. Furthermore, the loss of nuclear pSmad2/3 immunoreactivity was observed in cells bearing TDP-43 inclusions. In contrast, TDP-43-positive inclusions in the extramotor neurones in the brain of SALS patients were noticeably negative for Smurf2 and pSmad2/3. In addition, pSmad2/3 immunoreactivity was preserved in the nuclei of inclusion-bearing cells. CONCLUSIONS: This regional difference in the expression of Smurf2 and pSmad2/3 within TDP-43-positive inclusions might be one of the pathomechanisms underlying the loss of lower motor neurones and comparatively spared cortical neurones seen in ALS.

Levodopa challenge test and (123) I-metaiodobenzylguanidine scintigraphy for diagnosing Parkinson's disease.

OBJECTIVES: To explore the possibility of a generally applicable tool for the immediate diagnosis of Parkinson's disease (PD) in its early stage, we compared the sensitivity and specificity of an acute levodopa challenge test with that of (123) I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy. MATERIALS AND METHODS: A consecutive series of 45 patients with extrapyramidal symptoms were recruited to the acute levodopa challenge and evaluated for improvement by use of the Unified Parkinson's Disease Rating Scale motor scores. Of these patients, 32 of them were also examined by MIBG scintigraphy. The patients were followed up for at least 24 months, and 22 patients were diagnosed as having clinically definite PD. RESULTS: The sensitivity and specificity of the acute levodopa challenge test to predict clinical diagnosis of PD were 81.8% and 81.8%, respectively, which were better than those obtained by MIBG scintigraphy (62.5% and 62.5%). In both early- and middle-stages of PD, the test gave better sensitivity than MIBG scintigraphy. CONCLUSIONS: Considering that the well-established and frequently referred clinical diagnostic criteria require longitudinal observation for at least 24 months, the acute levodopa challenge test can be used as an immediate diagnostic tool for PD with sensitivity and specificity comparable to those of MIBG.

Smad ubiquitination regulatory factor-2 in progressive supranuclear palsy.

AIMS: Smad ubiquitination regulatory factor-2 (Smurf2) is an E3 ligase that belongs to the HECT domain ubiquitin ligase family. Smurf2 can interact with Smad proteins and promote their ubiquitin-dependent degradation, thereby controlling the cellular levels of these signalling mediators. Phosphorylated Smad2/3 (pSmad2/3) was recently identified in phosphorylated tau (phospho-tau) inclusions in patients with progressive supranuclear palsy (PSP). As Smurf2 is the E3 ligase of pSmad2, we aimed at investigating the relationship among Smurf2, pSmad2/3 and phospho-tau in this study. METHODS: The brains of six PSP and three control patients without neurological disorder were investigated by immunohistochemical analysis. RESULTS: In the control subjects, Smurf2 immunoreactivity was not demonstrable in the neurones and glial cells, and that for pSmad2/3 was observed exclusively in neuronal and glial nuclei. In PSP patients, the pathognomonic neuronal and glial phospho-tau inclusions were immunopositive for both Smurf2 and pSmad2/3. The intensity of pSmad2/3 immunosignals of neuronal and glial nuclei containing phospho-tau inclusions was less than that for the cells without the inclusions. Triple immunofluorescence staining for Smurf2, pSmad2/3 and phospho-tau revealed co-localization of these proteins within the neuronal and glial inclusions; and in some globose neurofibrillary tangles, the Smurf2 immunoreactivity appeared more centrally distributed than that of pSmad2/3 and phospho-tau. CONCLUSIONS: This is the first demonstration of the presence of Smurf2 immunoreactivity in the phospho-tau inclusions in PSP. These findings suggest that Smurf2 plays a significant role in the pathomechanism of PSP by causing abnormal redistribution of neuronal nuclear pSmad2/3 to the cytoplasm.

HtrA2/Omi-immunoreactive intraneuronal inclusions in the anterior horn of patients with sporadic and Cu/Zn superoxide dismutase (SOD1) mutant amyotrophic lateral sclerosis.

AIMS: HtrA2/Omi is a mitochondrial serine protease that promotes the apoptotic processes, but the relationship between HtrA2/Omi and amyotrophic lateral sclerosis (ALS) is still unknown. The purpose of the present study was to determine whether abnormal expression of HtrA2/Omi occurs in patients with ALS. METHODS: We prepared autopsied spinal cord tissues from 7 control subjects, 11 patients with sporadic ALS (SALS) and 4 patients with Cu/Zn superoxide dismutase (SOD1)-related familial ALS (FALS). We then performed immunohistochemical studies on HtrA2/Omi using formalin-fixed, paraffin-embedded sections from all of the cases. RESULTS: In the control subjects, the anterior horn cells were mildly to moderately immunostained with HtrA2/Omi. In the patients with SALS, strong HtrA2/Omi immunoreactivity was found in some skein-like inclusions and round hyaline inclusions as well as many spheroids, but Bunina bodies were immunonegative for HtrA2/Omi. In the patients with SOD1-related FALS, Lewy body-like hyaline inclusions were observed in three cases and conglomerate inclusions were observed in the remaining case, and both types of inclusions were intensely immunopositive for HtrA2/Omi. CONCLUSIONS: These results suggest that abnormal accumulations of HtrA2/Omi may occur in several types of motor neuronal inclusions in the anterior horn from SALS and SOD1-linked FALS cases, and that HtrA2/Omi may be associated with the pathogenesis of both types of ALS.

Thoracoamniotic shunting with double-basket catheters for fetal chylothorax in the second trimester.

The progress of a fetal severe pleural effusion at mid-trimester is extremely poor. We encountered a fetus that developed a severe left pleural effusion at 21 weeks of gestation. The pleural effusion was removed by thoracocentesis at 22 weeks. Cytology revealed abundant lymphocytes, suggesting chylothorax. However, a reaccumulation of pleural effusion with hydrops was subsequently noted, and a thoracoamniotic shunt with double-basket catheters was installed at 23 weeks. The pleural effusion decreased after 24 weeks and completely disappeared at 26 weeks. At 40 weeks of gestation, a female infant was born by vaginal delivery, with no evidence of pleural effusion. We would like to stress that thoracoamniotic shunt with double-basket catheters in the second trimester is effective for pleural effusion with hydrops.

Fine structure of anterior horns in patients without amyotrophic lateral sclerosis.

Light and electron microscopic study of the anterior horns in nine patients without amyotrophic lateral sclerosis (ALS) disclosed several features usually described in ALS. Central chromatolysis and spheroids with fine structure identical to that seen in ALS were observed in three cases. Rare honeycomb-like structures, dense filamentous aggregates, dense granular deposits, stubby mitochondria and membrane-bound aggregates of spherical and tubular particles were also observed. No typical Bunina bodies or focal argyrophilia of the chromatolytic neurons were found.

Basal lamina formation by astrocytes in organotypic cultures of mouse spinal cord tissue.

The relationship between astrocytes, basal lamina and mesenchymal tissue was analyzed ultrastructurally in myelinated organotypic cultures of mouse spinal cord tissue grown in combination with its pia-arachnoid membrane. A discontinuous, well-developed basal lamina covered flat astrocytic processes which formed the basal layer of the explant opposing the pia-arachnoid membrane. Some astrocytic processes on the surface facing the pia-arachnoid membrane lacked basal lamina, had an irregular surface with microvillus-like protrusions but within the explant they formed intercellular chambers which were surrounded by basal lamina. Even in the presence of mesenchymal tissue which appeared to facilitate the formation of basal lamina in this system, the absence of basal lamina on some areas of the astrocytic plasma membrane suggests regional differences of the astrocytic processes and might reflect the epithelial nature of the astrocyte.

Striatal efferent involvement and its correlation to levodopa efficacy in patients with multiple system atrophy.

We report an immunohistochemical investigation of the striatal efferents in the striatum, globus pallidus, and substantia nigra of five patients with multiple system atrophy (MSA): olivopontocerebellar atrophy (2), striatonigral degeneration (2), and Shy-Drager syndrome (1). All patients manifested parkinsonism during the clinical course of their illness. The administration of levodopa improved the symptoms of two patients, but not of the other three. Brain tissues from five age-matched neurologically normal subjects served as controls. Immunohistochemical assays were carried with antibodies against met-enkephalin, substance P, and calbindin-D28k. Irrespective of the clinical form of multiple system atrophy, the immunoreactivity with the antibodies was reduced at the dorsolateral portion of the striatum and the ventrolateral portions of the globus pallidus and of the substantia nigra. The woolly fiber arrangement of reaction product deposits seen in both segments of the globus pallidus of normal individuals was totally absent in the ventrolateral portions of the three patients who did not have a response to levodopa. By contrast, there were positively stained woolly fibers in globus pallidum segments of the two levodopa-responsive patients, even though their number and size were decreased in comparison with controls. These results indicate that the three clinical forms of multiple system atrophy share common topographic alterations of the striatal efferent system and that the severity of the involvement correlates with the clinically observed effect of levodopa on the parkinsonism.

alpha-Tocopherol transfer protein gene: exon skipping of all transcripts causes ataxia.

This report concerns the characterization of the alpha-tocopherol transfer protein (alpha-TTP) gene in a Japanese family affected by ataxia with isolated vitamin E deficiency (AVED). The sequence analysis revealed a G-to-A transition at the 3' end of exon 3 in both alleles, which predicts outsplicing of this exon from premessenger RNA and the concomitant frame shift in the ataxic patient. We used reverse transcriptase-polymerase chain reaction to analyze alpha-TTP gene transcripts. All transcripts in peripheral blood lymphocytes of the AVED patient, who was treated with large doses of vitamin E, lacked exon 3. The deduced truncated protein shares only 43% of the normal alpha-TTP. Normal control tissues and cells contained normal transcripts and, unexpectedly, also the same mutant transcripts as those of the patient, although with different transcription levels. Treatment of normal fibroblasts with clinically relevant concentrations of vitamin E increased production of transcripts in a dose-dependent manner. We propose that exon skipping of all transcripts through the complete inactivation of the splice site accounts for the clinical onset of AVED and for the clinical resistance to vitamin E in our patient.

The problem of de novo colorectal carcinoma.

From April 1985 to March 1995, colonoscopy was carried out at our institution in 24,059 patients, 31,800 times in symptomatic and/or asymptomatic average risk persons. 184 submucosal invasive carcinomas were detected. Unlike protruding-type lesion, the depressed-type invades the submucosal layer, even though the size is within 10 mm. The depressed type of invasive carcinoma accounted for 20 lesions, and represented 10.9% (20 of 184) of all the invasive carcinomas. The pit pattern of depressed-type lesions shows a small round pit (type IIIs pit pattern) and that of carcinoma lesions shows the irregular pit and non-structure (type V pit pattern).

A new amyloidogenic transthyretin variant, [D38A], detected by electrospray ionization/mass spectrometry.

A new variant of transthyretin (TTR) was detected by mass spectrometry (MS) in a 63-year-old Japanese female patient suffering from amyloidosis. TTR was analyzed by 2-dimensional liquid chromatography coupled with electrospray ionization MS. Variant TTR showed extra peaks in addition to normal TTR peaks. The extra peaks were about 44 Da smaller than normal TTR peaks, and the abundance of variant peaks showed about 80% of the corresponding normal free and adduct peaks. Direct genomic DNA sequencing of TTR exon 2 showed both adenine and cytosine in the position corresponding to the second base of codon 38. This codes for a variant alanine (GCT) as well as the normal aspartic acid (GAT), indicating that the case is heterozygous for the substitution, [D38A].

Colorectal tumours and pit pattern.

AIMS: To investigate the morphological and histopathological associations between an individual pit seen on stereomicroscopy or magnifying colonoscopy and an individual crypt seen in histological sections; and to examine these associations in colorectal tumours. METHODS: Fourteen thousand and twenty three cases were examined by colonoscope at Akita Red Cross Hospital. The surface mucosal pits of the lesions were observed using a magnifying endoscope in vivo and the pits of the extracted specimens were observed in vitro using a stereo microscope. Histological diagnoses were determined by light microscopy: the pit patterns in 100 glands were analysed. RESULTS: Pit pattern was classified into seven principal types: (1) normal round pit; (2) small round pit; (3) small asteroid pit; (4) large asteroid pit; (5) oval pit; (6) gyrus-like pit; and (7) non-pit. There was a correlation between pit pattern and the structure of the underlying crypt or gland. Furthermore, there was an association between pit pattern and the histology of the cells in the gland. Macroscopically, types 3, 4, 5, and 6 were common in protruding lesions. Type 2 was common in depressed lesions. The non-pit pattern was recognised in both. The depressed lesions had invaded the deeper layers more rapidly than protruding lesions. CONCLUSIONS: There were associations between individual pits and crypts. The branching carcinoma gland is thought to be the result of malignant transformation of the adenoma gland. The straight carcinoma gland is thought to result from the normal gland becoming malignant. The gland of the small round pit is thought to change from normal to the straight carcinoma gland via malignant transformation.

Tracheal dimensions at full inflation and deflation in adolescent twins.

Tracheal dimensions at total lung capacity (TLC) and residual volume (RV) were analyzed roentgenographically in 17 pairs of male adolescent twins (mean age 16.3 yr; 12 monozygotic pairs and 5 dizygotic pairs). Genetic factors dominated environmental traits in intra- as well as extrathoracic tracheal width at RV. Extrathoracic tracheal width at TLC was also governed by genetic components. Intrathoracic tracheal depth (anteroposterior diameter), length, and cross-sectional area did not seem to be genetically controlled at TLC and RV. Intrathoracic tracheal cross-sectional area increased by 14.4% and became more elliptical from RV to TLC, owing mainly to an increase in tracheal depth (16.7%). Increments from RV to TLC in tracheal depth but not width correlated with increases in lung width, depth, and height. Intrathoracic trachea was elongated 14% in association with increase in lung height from RV to TLC. At TLC, extrathoracic tracheal width was larger than intrathoracic tracheal width, but this dimension did not differ at RV. These results indicate that genetic factors influence, at least at RV, the tracheal rings more strongly than membranous parts. Intrathoracic tracheal depth but not width increases during inspiration in accordance with increase in lung volume. Extrathoracic tracheal width widens more than intrathoracic trachea from RV to TLC.

Trachea and lung dimensions in nonsmoking twins: morphological and functional studies.

To compare genetic and environmental factors that determine lung function and dimensions, chest radiographs and pulmonary function were measured in 17 pairs of nonsmoking twin adolescent boys (12 monozygotic pairs and 5 dizygotic pairs). Genetic factors dominated in tracheal width and lung dimensions (height, width, and apicofissural and fissurodiaphragmatic distances) at residual volume. Genetic factors also affected forced vital capacity, functional residual capacity, forced expiratory volume in 1 s, maximum expiratory flow at 25% vital capacity, and maximum flow at 50% vital capacity-to-forced vital capacity ratio. Peak expiratory flow correlated with tracheal width at residual volume. Age correlated with lung dimensions (width and depth) but not with tracheal width. These results indicate that genetic factors determine the dimensions and function of central airways, peripheral airways, and lung parenchyma in adolescent males. The effects of genetic factors on some functional measurements (airway resistance, closing volume-to-vital capacity ratio, and phase III in single-breath N2 washout) may be masked because of poor reproducibility of the tests.

Production mechanism of crackles in excised normal canine lungs.

Lung crackles may be produced by the opening of small airways or by the sudden expansion of alveoli. We studied the generation of crackles in excised canine lobes ventilated in an airtight box. Total airflow, transairway pressure (Pta), transpulmonary pressure (Ptp), and crackles were recorded simultaneously. Crackles were produced only during inflation and had high-peak frequencies (738 +/- 194 Hz, mean +/- SD). During inflation, crackles were produced from 111 +/- 83 ms (mean +/- SD) prior to the negative peak of Pta, presumably when small airways began to open. When end-expiratory Ptp was set constant between 15 and 20 cmH2O and end-expiratory Ptp was gradually reduced from 5 cmH2O to -15 or -20 cmH2O in a breath-by-breath manner, crackles were produced in the cycles in which end-expiratory Ptp fell below -1 to 1 cmH2O. This pressure was consistent with previously known airway closing pressures. When end-expiratory Ptp was set constant at -10 cmH2O and end inspiratory Ptp was gradually increased from -5 to 15 or 20 cmH2O, crackles were produced in inspiratory phase in which end-inspiratory Ptp exceeded 4-6 cmH2O. This pressure was consistent with previously known airway opening pressures. These results indicate that crackles in excised normal dog lungs are produced by opening of peripheral airways and are not generated by the sudden inflation of groups of alveoli.

Ataxic variant of adrenoleukodystrophy: MRI and CT findings.

A 28-year-old man developed slowly progressive dysarthria and gait disturbance over 7 years. Neurological examination revealed marked ataxia of articulation and gait, mild ataxia and spasticity of all four limbs without intellectual, visual, auditory, sensory or sphincter dysfunction. No physical signs of adrenal hypofunction were found. However, analysis of fatty acid of plasma sphingomyelin showed an increase in very long chain fatty acids, compatible with the diagnosis of adrenoleukodystrophy. Computed tomography disclosed marked atrophy of the cerebellum and pons, and bilateral low-density lesions in the deep while matter of the cerebellum. Magnetic resonance imaging showed these lesions more clearly, as well as other lesions in the middle and superior cerebellar peduncles, despite the absence of cerebral white matter involvement at the time of presentation. This is a rare case of adrenoleukodystrophy presenting as spinocerebellar degeneration and showing marked atrophy and several parenchymatous lesions of the cerebellum and brain stem.

Ubiquitin-positive intraneuronal inclusions in the extramotor cortices of presenile dementia patients with motor neuron disease.

Ubiquitin-positive intraneuronal inclusions were found in the extramotor cortices of ten presenile dementia patients with motor neuron disease. There were inclusions in the hippocampal granular cells and in the small neurons of the superficial layers of the temporal and frontal cortices. Bunina bodies were present in the anterior horn cells in all cases. These results suggest that ubiquitin-related cytoskeletal abnormalities are common in cerebral non-motor small neurons in these patients.

Fine structure of astrocytic processes during serum-induced demyelination in vitro.

Astrocytic changes were examined ultrastructurally during serum-induced demyelination in organotypic cultures of mouse spinal cord tissue. The myelin sheaths of most myelinated fibers showed myelin breakdown. Sheet-like astrocytic processes completely and closely surrounded the demyelinating fibers and frequently engulfed myelin fragments. These processes were virtually devoid of glial filaments or microtubules and contained flocculogranular material, round and elongated cisterns and glycogen granules. Penetration of the myelin sheath by astrocytic processes was only rarely found. The cell body of the astrocyte was never insinuated between the myelin sheath and the axon. No endocytosis of myelin droplets via coated pits on the surface of astrocytes was observed. In comparison with phagocytic mononuclear cells in vivo, astrocytes in vitro dispose of myelin debris less actively. Astrocytes in serum-induced demyelination probably play a nonspecific but fundamental role during degeneration in segregating damaged nerve fibers from surrounding neuronal elements.

Synaptic pathology of spinal anterior horn cells in amyotrophic lateral sclerosis: an immunohistochemical study.

We have applied immunohistochemical techniques to study synaptic alterations of the spinal anterior horn in amyotrophic lateral sclerosis (ALS), and other disorders involving upper or lower motor neurons. A monoclonal antibody to synaptophysin was used. Spinal cord tissues from normal individuals served as controls. As compared to these, a decrease in synaptophysin immunoreactivity was evident in the neuropil in the spinal anterior horn of ALS patients. However, synaptophysin expression in the perikarya and dendrites of remaining normal-appearing neurons in these patients was not decreased and occasionally it was even higher than in control neurons. Similar results were obtained with specimens from patients with lower motor neuron disease. Synaptophysin immunoreactivity in the neuropil and perikarya of the cases with focal spinal cord lesions with bilateral descending tract degeneration was similar to normal controls. Our data suggest that the alterations in synaptophysin expression occurring in ALS are mainly associated with the loss of lower motor neurons, and that the occasional increased perikaryal expression may be due to the neuronal atrophy, compensatory accumulation or abnormal synaptic vesicle degradation.