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 alpha-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.

Age-dependent synuclein pathology following traumatic brain injury in mice.

Synucleins (Syn), a family of synaptic proteins, includes alpha-Syn, which plays a pivotal role in Parkinson's disease and related neurodegenerative diseases (synucleinopathies) by forming distinct brain pathologies (Lewy bodies and neurites). Since traumatic brain injury (TBI) is a poorly understood risk factor for Parkinson's disease, we examined the effects of TBI in the young and aged mouse brain on alpha-, beta-, and gamma-Syn. Immunohistochemical analysis showed that brains from sham-injured young and aged mice had normal alpha- and beta-Syn immunoreactivity (IR) in neuropil of cortex, striatum, and hippocampus with little or no gamma-Syn IR. At 1 week post TBI, the aged mouse brain showed a transient increase of alpha- and beta-Syn IR in the neuropil as well as an induction of gamma-Syn IR in subcortical axons. This was associated with strong labeling of striatal axon bundles by antibodies to altered or nitrated epitopes in alpha-Syn as well as by antibodies to inducible nitric oxide synthase. However, these TBI-induced changes disappeared by 16 weeks post TBI, and altered Syn IR was not seen in young mice subjected to TBI nor in alpha-Syn knockout mice while Western blots confirmed that TBI induced transient alterations of alpha-Syn in the mouse brains. This model of age-dependent TBI-induced transient alterations in alpha-Syn provides an opportunity to examine possible links between TBI and mechanisms of disease in synucleinopathies.

Ultrastructural evidence for differential axonal sprouting in the striatum after thermocoagulatory and aspiration lesions of the cerebral cortex in adult rats.

Thermocoagulation of pial blood vessels overlying the cerebral cortex induces an ischemic degeneration of the cortex. We have previously shown with anatomical tracing techniques that thermocoagulatory lesions of the sensorimotor cortex trigger a robust axonal sprouting of contralateral cortical neurons into the denervated striatum. Similar sprouting was not observed after acute aspiration lesions of the same cortical region. We have now examined immunostaining for the growth-associated protein (GAP)-43 at the ultrastructural level after both types of lesions. A modest increase in growth cone-like structures was observed just below the corpus callosum after both lesions. However, GAP-43-positive growth cone-like structures were markedly increased in the denervated dorsolateral striatum only after thermocoagulatory lesions. In contrast, no significant increase in growth cone immunostaining was found in the dorsolateral striatum after aspiration lesions, confirming the absence of axonal sprouting in the dorsolateral striatum in this condition. Corticostriatal inputs make asymmetric synapses with dendritic spines of striatal neurons. As expected, the density of asymmetric synapses was markedly decreased in the dorsolateral striatum after aspiration lesions. However, it was not different from control after thermocoagulatory lesions that removed the same cortical area. The density of symmetric synapses was decreased after both types of lesions at 16 but not 42 days post-surgery. These data reveal that robust axonal and synaptic remodeling can occur in the dorsolateral striatum of adult rats after ischemic lesions of the cerebral cortex and further demonstrate marked differences in the degree of anatomical plasticity induced by two different types of cortical lesions.

Increased lipid peroxidation precedes amyloid plaque formation in an animal model of Alzheimer amyloidosis.

Oxidative stress is a key feature in the Alzheimer's disease (AD) brain and manifests as lipid peroxidation (LPO). Isoprostanes (iPs) are specific and sensitive markers of in vivo LPO. To determine whether amyloid beta (Abeta) deposition in vivo is associated with increased LPO, we examined iP levels in a transgenic mouse model (Tg2576) of AD amyloidosis. Urine, plasma, and brain tissues were collected from Tg2576 and littermate wild-type (WT) animals at different time points starting at 4 months of age and continuing until 18 months of age. Levels of urinary 8,12-iso-iPF(2alpha)-VI were higher in Tg2576 than in WT animals as early as 8 months of age and remained this high for the rest of the study. A similar pattern was observed for plasma levels of 8,12-iso-iPF(2alpha)-VI. Homogenates from the cerebral cortex and hippocampus of Tg2576 mice had higher levels of 8,12-iso-iPF(2alpha)-VI than those from WT mice starting at 8 months of age. In contrast, a surge of Abeta 1-40 and 1-42 levels as well as Abeta deposits in Tg2576 mouse brains occurred later, at 12 months of age. A direct correlation was observed between brain 8,12-iso-iPF(2alpha)-VI and Abeta 1-40 and 1-42. Because LPO precedes amyloid plaque formation in Tg2576 mice, this suggests that brain oxidative damage contributes to AD pathogenesis before Abeta accumulation in the AD brain.

Frontotemporal dementia with novel tau pathology and a Glu342Val tau mutation.

It is unclear how tau gene mutations cause frontotemporal dementia (FTD) with parkinsonism linked to chromosome 17 (FTDP-17), but those in exon 10 (E10) or the following intron may be pathogenic by altering E10 splicing, perturbing the normal 1:1 ratio of four versus three microtubule-binding repeat tau (4R:3R tau ratio) and forming tau inclusions. We report on a 55-year old woman with frontotemporal dementia and a family history of FTDP-17 in whom we found a novel E12 (Glu342Val) tau gene mutation, prominent frontotemporal neuron loss, intracytoplasmic tau aggregates, paired helical tau filaments, increased 4R tau messenger RNA, increased 4R tau without E2 or E3 inserts, decreased 4R tau with these inserts, and a 4R:3R tau ratio greater than 1 in gray and white matter. Thus, this novel Glu342Val mutation may cause FTDP-17 by unprecedented mechanisms that alter splicing of E2, E3, and E10 to preferentially increase 4R tau without amino terminal inserts and promote aggregation of tau filaments into cytopathic inclusions.

Inhibition by neuromuscular blocking drugs of norepinephrine transporter in cultured bovine adrenal medullary cells.

UNLABELLED: Pancuronium stimulates the cardiovascular system, whereas vecuronium, a derivative of pancuronium, has far fewer effects. The inhibition of norepinephrine transporter (NET) in the sympathetic nervous system may partly account for the stimulatory actions of pancuronium. To investigate the mechanism of action of pancuronium on NET, we examined the effects of pancuronium on NET activity by using cultured bovine adrenal medullary cells and compared pancuronium with other neuromuscular blocking drugs. Pancuronium (1-300 microM) inhibited desipramine-sensitive [(3)H]norepinephrine (NE) uptake in a concentration-dependent manner. Vecuronium (100-300 microM) and d-tubocurarine (300 microM) also decreased [(3)H]NE uptake but were less potent than pancuronium at clinical concentrations. Succinylcholine had little effect on [(3)H]NE uptake. Saturation analysis showed that pancuronium and vecuronium reduced an apparent maximum velocity (V(max)) of [(3)H]NE uptake without altering Michaelis-Menten constant, indicating noncompetitive inhibition. Pancuronium did not inhibit the specific binding of [(3)H]desipramine to plasma membranes isolated from bovine adrenal medulla. A protein kinase C inhibitor, GF109203X, did not affect the inhibition of [(3)H]NE uptake by pancuronium. Pancuronium enhanced the inhibition of NET induced by ketamine. These results suggest that pancuronium, with clinically relevant concentrations, inhibits NET activity by interacting with a site distinct from the recognition site for NE and the desipramine binding site on the transporter. IMPLICATIONS: In this study, pancuronium inhibited norepinephrine uptake and was the most potent of the neuromuscular blocking drugs we tested, including pancuronium, vecuronium, d-tubocurarine, and succinylcholine. Pancuronium may affect the sympathetic nervous system by inhibiting the activity of the presynaptic norepinephrine transporter at clinically relevant concentrations.

Activation of extracellular signal-regulated protein kinases is associated with a sensitized locomotor response to D(2) dopamine receptor stimulation in unilateral 6-hydroxydopamine-lesioned rats.

Evidence indicates that mitogen-activated protein kinase (MAPK) pathways play a crucial role in the neurobiology of the nervous system. In the present study, dopamine receptor-mediated regulation of extracellular signal-regulated kinases (ERKs) was examined in rats in which the nigrostriatal dopaminergic pathway was unilaterally lesioned by 6-hydroxydopamine (6-OHDA). Subcutaneous injections of the D(2) receptor agonist quinpirole significantly increased tyrosine-phosphorylated ERK1/2 in lesioned striatum, whereas the D(1) receptor agonist SKF38393 failed to activate ERKs. Quinpirole-induced phosphorylation of ERK1/2 was seen as early as 3 min and peaked at 15 min after the challenge. In parallel, striatal ERK kinase activity, measured by the in vitro kinase assay, was increased 2.5-fold on the lesioned side after the administration of quinpirole. Immunohistochemical examination of brain sections after quinpirole administration revealed significant increases in ERK1/2 immunostaining in perinuclear and intranuclear areas of striatal neurons. This increase was much more pronounced on the lesioned than the intact side. Furthermore, quinpirole-induced contralateral rotation was decreased by 48.7 and 50.7%, respectively, when the striatal ERK pathway was selectively inhibited by a single intrastriatal injection of the MAPK/ERK kinase inhibitor PD098059 or after a continuous 7 d intrastriatal infusion of ERK1/2 antisense oligodeoxynucleotide. The results demonstrate, for the first time, that the ERK signaling pathway is activated in denervated striatum in response to stimulation of D(2) dopamine receptors and that the resulting imbalance in striatal ERK activity contributes, at least in part, to neuronal plasticity that underlies D(2) dopamine receptor-mediated contralateral rotation in unilateral 6-OHDA denervated rats.

Axon pathology in Parkinson's disease and Lewy body dementia hippocampus contains alpha-, beta-, and gamma-synuclein.

Pathogenic alpha-synuclein (alphaS) gene mutations occur in rare familial Parkinson's disease (PD) kindreds, and wild-type alphaS is a major component of Lewy bodies (LBs) in sporadic PD, dementia with LBs (DLB), and the LB variant of Alzheimer's disease, but beta-synuclein (betaS) and gamma-synuclein (gammaS) have not yet been implicated in neurological disorders. Here we show that in PD and DLB, but not normal brains, antibodies to alphaS and betaS reveal novel presynaptic axon terminal pathology in the hippocampal dentate, hilar, and CA2/3 regions, whereas antibodies to gammaS detect previously unrecognized axonal spheroid-like lesions in the hippocampal dentate molecular layer. The aggregation of other synaptic proteins and synaptic vesicle-like structures in the alphaS- and betaS-labeled hilar dystrophic neurites suggests that synaptic dysfunction may result from these lesions. Our findings broaden the concept of neurodegenerative "synucleinopathies" by implicating betaS and gammaS, in addition to alphaS, in the onset/progression of PD and DLB.

N-methyl-D-aspartate receptor blockade affects polysialylated neural cell adhesion molecule expression and synaptic density during striatal development.

Glutamatergic neurons innervate the striatum and form asymmetric synapses with the dendritic spines of striatal efferent neurons. The role of glutamate in striatal development, however, remains largely unknown. Previous studies have shown a dramatic increase in the density of asymmetric synapses in the rat striatum during the third postnatal week, followed by a decrease to adult levels by postnatal day 25. At the same time, the highly polysialylated form of the neural cell adhesion molecule becomes progressively restricted to synaptic regions and then disappears. We have now examined the effects of antagonists of the N-methyl-D-aspartate subtype of glutamatergic receptors on the expression of the polysialylated form of the neural cell adhesion molecule and on synaptic density during this late period of striatal development. Peripheral administration of the N-methyl-D-aspartate receptor antagonist dizocilpine maleate markedly decreased immunoreactivity for the highly polysialylated form of the neural cell adhesion molecule in the dorsolateral striatum and cerebral cortex when drug treatment included postnatal day 20, but not earlier in development. This effect was regionally specific and loss of the polysialylated neural cell adhesion molecule in the striatum was reproduced by the local administration of dizocilpine maleate, DL-2-amino-5-phosphonovalerate or ketamine on postnatal day 20. Quantitative ultrastructural studies of synaptic density with the physical disector method performed after one of the regimens inducing loss of the polysialylated neural cell adhesion molecule (postnatal days 18-20) revealed a 30% decrease in asymmetric synapses in the dorsolateral striatum of treated rats. Symmetric synapses, which presumably do not use glutamate, were not affected. The data indicate that N-methyl-D-aspartate receptors play a role in the late stages of synaptogenesis in the striatum and suggest that a subset of synapses expressing immunoreactivity for the highly polysialylated form of the neural cell adhesion molecule may be dependent on N-methyl-D-aspartate receptor stimulation during a critical period of striatal development.

Mutant and wild type human alpha-synucleins assemble into elongated filaments with distinct morphologies in vitro.

alpha-Synuclein is a soluble presynaptic protein which is pathologically redistributed within intracellular lesions characteristic of several neurodegenerative diseases. Here we demonstrate that wild type and two mutant forms of alpha-synuclein linked to familial Parkinson's disease (Ala30 --> Pro and Ala53 --> Thr) self-aggregate and assemble into 10-19-nm-wide filaments with distinct morphologies under defined in vitro conditions. Immunogold labeling demonstrates that the central region of all these filaments are more robustly labeled than the N-terminal or C-terminal regions, suggesting that the latter regions are buried within the filaments. Since in vitro generated alpha-synuclein filaments resemble the major ultrastructural elements of authentic Lewy bodies that are hallmark lesions of Parkinson's disease, we propose that self-aggregating alpha-synuclein is the major subunit protein of these filamentous lesions.

Synaptogenesis and ultrastructural localization of the polysialylated neural cell adhesion molecule in the developing striatum.

The polysialylated neural cell adhesion molecule (PSA-NCAM) plays a role in axonal development and synaptic plasticity. Its pattern of expression is regulated temporally and topographically in the brain during development. However, it is unclear whether or not its subcellular location also changes. We have examined PSA-NCAM expression in relation to synapse formation in the developing rat striatum with immunohistochemistry and electron microscopy. Early in development, PSA-NCAM was present along the cytoplasmic membranes of neurons and in growth cones. PSA-NCAM expression became progressively confined to pre- and postsynaptic elements as neurons matured morphologically. Confirming previous results, a marked increase in the density of asymmetric synapses determined by using the physical dissector method was observed in the dorsolateral striatum between postnatal day 14 (P14) and P18. It was followed by a reduction between P18 and P25, when asymmetric synapse density reached adult levels. In contrast, the density of symmetric synapses had surpassed adult levels by P14. In the dorsomedial striatum, the density of asymmetric and symmetric synapses was similar at P18, at P25, and in adults. PSA-NCAM was associated with most asymmetric and symmetric synapses at P14 and P18 and was expressed in both pre- and postsynaptic elements of a majority (P14) or approximately half (P18) of the synapses. Most synapses lost PSA-NCAM expression between P18 and P25 in the dorsolateral striatum and between P25 and adult in the dorsomedial striatum. The data indicate that PSA-NCAM expression becomes restricted topographically during neuronal maturation but remains strategically associated with developing synapses during late postnatal development in the striatum.

Age-associated impairment in brain MAPK signal pathways and the effect of caloric restriction in Fischer 344 rats.

Mitogen-activated protein kinases (MAPKs) play important roles in cell proliferation, differentiation, and apoptosis. Important functional roles for MAPKs in postmitotic cells have recently been suggested. In the present study, we investigated the effect of aging on the brain ERK (extracellular signal-regulated kinase) and p38 MAPK signaling pathways of Fischer 344 rats. The results show that basal tyrosine-phosphorylated ERK1/ERK2 in cortex of 24-month-old rats was reduced by 36%-59%, compared to 6- and 12-month-old rats (p<.05, 24- vs. 12- or 6-month-old rats). Similarly, the phosphotransferase activities of ERK and p38 MAPK, measured by in vitro immunocomplex kinase assays using myelin basic protein (MBP) as substrate, were shown to be reduced approximately 50% and 59% respectively, in the cerebrocortex of 24-month-old rats (p<.01, 24- vs. 12- or 6-month-old rats). The reductions in basal ERK and p38 MAPK activities are not due to altered protein levels of these kinases as assessed by Western analysis. Immunohistochemically, no age-related differences in ERK expression and cellular distribution were observed However, cytosolic ERK tended to aggregate in brain neurons of aged rats. In contrast brain tyrosine-phosphorylated PLCgamma1 did not change with age. Activation of ERK in response to EGF or PMA was also reduced in cortical brain slices of 24-month-old rats. These results demonstrate an age-associated selective impairment in the MAPK signaling pathways. Moreover, lifelong caloric restriction completely prevented the age-related decrease in basal brain ERK activity and diminished the age-related reduction of p38 MAPK activity. Taken together, these data indicate that ERK and p38 MAPK signaling pathways are impaired in the aged brain and that lifelong caloric restriction modulates these defects in brain intracellular signaling pathways.

D1 dopamine receptor agonists mediate activation of p38 mitogen-activated protein kinase and c-Jun amino-terminal kinase by a protein kinase A-dependent mechanism in SK-N-MC human neuroblastoma cells.

We investigated the effects of D1 dopamine receptor stimulation on the activation of mitogen-activated protein kinases (MAPKs) in SK-N-MC human neuroblastoma cells. We found that the D1 dopamine receptor agonist SKF38393 induced similar time- and dose-related activation of p38 MAPK and c-Jun amino-terminal kinase (JNK), whereas extracellular signal-regulated kinase activity was not affected by D1 dopamine receptor stimulation. Maximal stimulation of p38 MAPK and JNK was observed after a 15-min incubation with 100 microM SKF38393. In contrast, 10 microM quinpirole, a D2 dopamine receptor agonist, did not activate p38 MAPK or JNK. Treatment of cells with 10 muM SCH23390, a D1 dopamine receptor antagonist, significantly inhibited the activation of both kinases by SKF38393. These results indicate that activation of the p38 MAPK and JNK signaling pathways is mediated by dopamine D1 receptors in SK-N-MC neuroblastoma cells. Furthermore, dibutyryl-cAMP mimicked SKF38393-mediated stimulation of p38 MAPK and JNK. Inhibition of protein kinase A by 1 microM H-89 or 10 microM adenosine 3', 5'-cyclic monophosphothioate (Rp-isomer, triethylammonium salt) markedly attenuated the activation of p38 MAPK and JNK. Conversely, the selective protein kinase C inhibitor calphostin C did not block D1 dopamine receptor-stimulated activation of p38 MAPK and JNK. These results demonstrate, for the first time, that the Gs-coupled D1 dopamine receptor activates the p38 MAPK and JNK signaling pathways by a protein kinase A-dependent mechanism.

A role for N-methyl-D-aspartate receptors in the regulation of synaptogenesis and expression of the polysialylated form of the neural cell adhesion molecule in the developing striatum.

Striatal development proceeds during a protracted postnatal period in rats. In the dorsolateral striatum, the number of asymmetric synapses, formed mostly by glutamatergic afferents innervating the dendritic spines of medium-sized striatal neurons, increases during the 3rd postnatal week and then rapidly declines before reaching adult levels. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM), which is widely expressed along neuronal membranes early in development, becomes progressively localized to synapses, and is no longer detectable in remaining synapses after synaptic pruning has occurred. Administration of MK-801, an antagonist of N-methyl-D-aspartate receptors, on day 20, either peripherally or locally into the striatum, decreases asymmetric synapse number by 30% and totally abolishes immunolabelling for PSA-NCAM in the dorsolateral striatum.

Pattern of expression of the serotonin2C receptor messenger RNA in the basal ganglia of adult rats.

The distribution of the serotonin (5-HT) receptor 5-HT2C mRNA was examined at the single-cell level with in situ hybridization histochemistry and emulsion autoradiography in the basal ganglia and mesolimbic system of adult rats, with focus on the pallidum and the substantia nigra, which receive striatal inputs and play a critical role in basal ganglia function. 5-HT2C receptor mRNA expression was always restricted to a subpopulation of neurons in the regions examined. In the neostriatum, labeled neurons were more numerous in the rostral nucleus accumbens than in the caudal nucleus accumbens and were more numerous in the ventral and ventrolateral caudate-putamen than in the dorsal caudate-putamen, where labeled neurons were restricted to isolated clusters. In striatal target areas, dense labeling in the entopeduncular nucleus (internal pallidum, direct striatal output pathway) contrasted with an absence of labeling in the globus pallidus (external pallidum, indirect striatal output pathway). Double-label in situ hybridization in the substantia nigra revealed coexpression of 5-HT2C receptor mRNA with glutamic acid decarboxylase but not with tyrosine hydroxylase mRNA, indicating that it was restricted to gamma-aminobutyric acid (GABA)ergic neurons. In this region, dense labeling for 5-HT2C mRNA was found in half of the neurons at middle and caudal levels of both the pars compacta and the pars reticulata, with little labeling rostrally. The data suggest that drugs acting on the 5-HT2C receptor could selectively affect discrete neuronal populations in the basal ganglia and mesolimbic systems and indicate a new level of neurochemical heterogeneity among GABAergic neurons of the substantia nigra.

Relationship between TA01 and TA02 polypeptides associated with lung adenocarcinoma and histocytological features.

TA01 (molecular weight 35.0 kDa, isoelectric point 5.45) and TA02 (molecular weight 35.0 kDa, isoelectric point 5.29) polypeptides were detected using two-dimensional polyacrylamide gel electrophoresis (2-DE). A previous study has shown that these polypeptides are distributed in primary adenocarcinomas and some large-cell carcinomas of the lung. However, various expression levels of TA01 and TA02 polypeptides were demonstrated in adenocarcinoma, while large-cell carcinoma expressed low levels. To evaluate the relationship between the expression of TA01 and TA02 polypeptides and the histocytological features of primary adenocarcinoma of the lung, these two polypeptides were analysed by 2-DE combined with a non-enzymatic sample preparation technique, and their expression levels were compared with the histocytological features of primary lung adenocarcinoma. Out of 57 primary lung adenocarcinoma cases, 46 cases (80.7%) and 52 cases (91.2%) expressed TA01 and TA02 polypeptides respectively. Furthermore, the expression levels of TA01 and TA02 polypeptides correlated with the degree of cellular atypia, structural atypia and histocytological differentiation of primary lung adenocarcinoma. On the other hand, these two polypeptides were not detected in adenocarcinoma of the lung, metastatic from the colon and mammary glands. High expression of TA01 and TA02 polypeptides reflected the differentiation of primary adenocarcinoma in the lung. These two polypeptides are valuable in determining the histocytological differentiation of primary lung adenocarcinoma as well as in distinguishing between primary and metastatic adenocarcinoma of the lung.

Regulation of the polysialylated form of the neural cell adhesion molecule in the developing striatum: effects of cortical lesions.

Early in development, the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is expressed by growth cones, neuronal processes, and neuronal cell bodies. In rat striatum, PSA-NCAM expression becomes progressively restricted to pre- and postsynaptic membranes and is undetectable by postnatal day 25 (P25), i.e., after corticostriatal synaptogenesis. This study examined the effects of cortical lesions performed on P14, when the corticostriatal projection is already primarily unilateral and cortical inputs have not yet formed asymmetric synapses on striatal neurons. Rats were killed on P25, and PSA-NCAM expression was examined by immunoblotting and immunohistochemistry with light and electron microscopy. In contrast to the case in controls, PSA-NCAM expression was maintained in the striatum of lesioned pups. Ultrastructural studies showed that PSA-NCAM was present 1) in growth cone-like structures and neuronal processes and 2) in striatal neurons. Together with the presence of growth cones, the observation that the number of asymmetric synapses was unchanged in the denervated striatum suggests that axonal sprouting occurred in response to the lesion. This was confirmed by axonal labeling in the denervated striatum after injection of Fluoro-Ruby in the contralateral cortex. The data indicate that P14 cortical lesions affect PSA-NCAM expression in the developing striatum 1) by inducing a robust axonal plasticity resulting in the presence of immature presynaptic elements that contain PSA-NCAM and 2) by delaying the loss of PSA-NCAM expression in striatal neurons, suggesting that the lesion affects the time course of striatal maturation.

Assessment of homogeneity in polypeptide expression in breast carcinomas shows widely variable expression in highly malignant tumors.

We describe the results from a protein-based approach to the study of heterogeneity in gene expression between human tumors. Cell preparations from 5 benign breast lesions, 5 potentially weakly malignant and 4 potentially highly malignant invasive ductal breast carcinomas were examined by 2-dimensional gel electrophoresis (2-DE) gels. Qualitative and quantitative differences were recorded by computerized analysis. Analysis of samples from different areas of the same tumor showed a high degree of similarity in the pattern of polypeptide expression. Analysis of 2 tumors and their metastases revealed similar 2-DE profiles. In contrast, variations between different lesions with comparable histological characteristics were considerable. Greater differences in polypeptide expression were observed between potentially highly malignant carcinomas compared with comparisons of less malignant lesions. Our results show that malignant human breast carcinomas may be highly heterogeneous in their patterns of gene expression.

Expression of tropomyosin isoforms in benign and malignant human breast lesions.

High molecular weight tropomyosins (tms) are commonly down-regulated in fibroblasts transformed by oncogenes. Previous studies have also demonstrated that specific tm isoforms are down-regulated in human breast carcinoma cell lines. We examined tropomyosin isoforms in cells prepared from non-cancerous breast lesions and primary human breast carcinomas. The average level of expression of all three high molecular weight tm isoforms (tm 1-3) in carcinomas was generally found to be less than 25% of that observed in non-cancerous breast lesions. Interestingly, the expression of tm 1 was found to be 1.7-fold higher in primary tumours with metastatic spread to axillary lymph nodes compared with primary tumours with no evidence of metastasis (p<0.05). Similarly, tm 1 expression was higher in two 12V-H-ras transformed fibroblast cell lines capable of experimental metastasis compared with three weakly metastatic cell lines. We conclude from these studies that expression of high molecular weight tm isoforms is low in primary breast carcinomas, and that metastatic tumours express relatively high levels of tm 1.

Detection of polypeptides associated with the histopathological differentiation of primary lung carcinoma.

Two-dimensional polyacrylamide gel electrophoresis combined with a non-enzymatic sample preparation technique is useful for analysing clinical tumour material. Using these techniques, we analysed the relationship between the histopathological findings in primary lung malignancies and the expression of a number of unidentified polypeptides that were detected in the molecular weight region 20-35 kDa. In this study 45 cases of primary lung cancer (PLC) (21 cases of adenocarcinoma, ten cases of squamous cell carcinoma, five cases of large-cell carcinoma, one case of adenosquamous cell carcinoma, five cases of small-cell carcinoma and three cases of carcinoid tumour) were examined. For reference, a human diploid fibroblast cell line (W138) and normal peripheral lymphocytes were used. Sixteen polypeptides were judged to be associated with histopathological features. These polypeptides seem to be valuable as differentiation markers. The simultaneous evaluation of these polypeptides and some other proliferation markers (e.g. PCNA, PCNA 'satellite', Numatin/protein B23 and lamin B) seems to clarify the characteristics of each case of PLC. Furthermore, it is possible to classify PLC based on the two-dimensional electrophoresis findings, and this classification of PLC is suggested to reflect the biological features of the tumour more precisely than that based only on morphology.