Neuroprotective effects of gelsolin during murine stroke.

Increased Ca2+ influx through activated N-methyl-D-aspartate (NMDA) receptors and voltage-dependent Ca2+ channels (VDCC) is a major determinant of cell injury following brain ischemia. The activity of these channels is modulated by dynamic changes in the actin cytoskeleton, which may occur, in part, through the actions of the actin filament-severing protein gelsolin. We show that gelsolin-null neurons have enhanced cell death and rapid, sustained elevation of Ca2+ levels following glucose/oxygen deprivation, as well as augmented cytosolic Ca2+ levels in nerve terminals following depolarization in vitro. Moreover, major increases in infarct size are seen in gelsolin-null mice after reversible middle cerebral artery occlusion, compared with controls. In addition, treatment with cytochalasin D, a fungal toxin that depolymerizes actin filaments, reduced the infarct size of both gelsolin-null and control mice to the same final volume. Hence, enhancement or mimicry of gelsolin activity may be neuroprotective during stroke.

Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer disease.

Prostate apoptosis response-4 (Par-4) is a protein containing both a leucine zipper and a death domain that was isolated by differential screening for genes upregulated in prostate cancer cells undergoing apoptosis. Par-4 is expressed in the nervous system, where its function is unknown. In Alzheimer disease (AD), neurons may die by apoptosis, and amyloid beta-protein (A beta) may play a role in this. We report here that Par-4 expression is increased in vulnerable neurons in AD brain and is induced in cultured neurons undergoing apoptosis. Blockade of Par-4 expression or function prevented neuronal apoptosis induced by Ab and trophic factor withdrawal. Par-4 expression was enhanced, and mitochondrial dysfunction and apoptosis exacerbated, in cells expressing presenilin-1 mutations associated with early-onset inherited AD.

Perikaryal accumulation and proteolysis of neurofilament proteins in the post-mortem rat brain.

Investigations of neurofilament alterations in neurodegenerative disorders utilize postmortem human tissues obtained at autopsy. To determine if alterations in the levels or distribution of neurofilament proteins might occur during the interval between death and autopsy, the postmortem cooling curve of the human brain was modeled in Sprague-Dawley rats and neurofilament proteins were examined by immunocytochemistry and immunoblots. One hour after death, enhanced perikaryal immunostaining of NF-M and both phosphorylated and nonphosphorylated NF-H epitopes was observed throughout the hippocampal formation. A greater number of neurons exhibited increased somatic immunostaining 4-h postmortem. In addition, loss of neurofilament protein immunostaining was observed in the neuropil, particularly in the molecular layer of the dentate gyrus. This corresponded with, but lagged behind, the pattern of calpain activation determined using an antibody against calpain-cleaved alpha-spectrin. Immunoblots confirmed the postmortem loss of neurofilament proteins in both triton-soluble and insoluble fractions. These results demonstrate that the levels and localization of neurofilament proteins observed in tissues obtained at autopsy even with short postmortem intervals may not accurately reflect the premortem condition.

Postmortem changes in the levels and localization of microtubule-associated proteins (tau, MAP2 and MAP1B) in the rat and human hippocampus.

The neuronal cytoskeleton is disrupted in neurodegenerative disorders such as Alzheimer's disease. Due to the lack of suitable animal models, studies examining the events involved in the neurodegeneration have relied on postmortem human brain tissue obtained from individuals with the disease and from normal controls. However, it is uncertain if the neuronal cytoskeleton is stable during the postmortem interval. Immunohistochemistry and immunoblots were used to examine the microtubule-associated proteins tau, MAP2, and MAP1B in the rat hippocampus at various times after death. Shortly after death, tau immunoreactivity was lost from axons and accumulated in somatodendritic compartments. MAP2 and MAP1B also accumulated in neuronal cell bodies prior to a loss of immunostaining in some regions, notably subiculum. Immunoblots confirmed a loss of MAP2 and MAP1B within a few hours after death. Tau levels remained constant during the 8-hour postmortem interval examined, although the electrophoretic mobility of some tau bands was altered. Human brain tissue obtained at autopsy and at surgery demonstrated similar cytoskeletal alterations in postmortem tissue. These results demonstrate that microtubules and associated proteins are not stable postmortem.

Effects of intrahippocampal colchicine administration on the levels and localization of microtubule-associated proteins, tau and MAP2.

Colchicine, a microtubule disrupting agent, has been used to model several aspects of Alzheimer's disease-related neuropathology. The formation of neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, involves the loss of tau (a low mol. wt. microtubule-associated protein) from axons and accumulation of abnormally phosphorylated tau in somatodendritic compartments. Other cytoskeletal proteins, such as microtubule-associated protein 2 (MAP2), disappear as tau accumulates. The present study was directed at evaluating the effects of colchicine on tau and MAP2, to determine if changes in their levels or distribution might be similar to those which precede the formation of neurofibrillary tangles in Alzheimer's disease. Six hours following intrahippocampal colchicine injection (3.5 micrograms injected into two rostro-caudal locations) tau-1 immunostaining was enhanced in CA1 s. radiatum and decreased in the outer molecular layer of the dentate gyrus. In addition, a shift in the relative abundance of tau isoforms was observed in Western blots. Both the immunocytochemical and immunoblot results are consistent with a dephosphorylation of tau. Loss of MAP2 was evident 3 days postinjection which coincided with a loss of Cresyl violet staining in granule cell, CA3, subicular and entorhinal neurons. Accumulation of tau or MAP2 in neuronal perikarya was not observed at any postinjection time points. Thus, intrahippocampal colchicine administration does not model the shift in tau localization, excessive tau phosphorylation, or other cytoskeletal alterations that are suggested to precede or accompany the formation of neurofibrillary pathology in Alzheimer's disease.

Prognostic significance of proliferation associated nucleolar antigen P120 in human breast carcinoma.

Nucleolar antigen P120 is detected in rapidly proliferating cells but not in normal resting cells or in many benign and slowly growing malignant tumors. The objective of the study was to determine whether the expression of P120 in breast cancer correlated with histopathological or biological properties associated with prognosis. In this retrospective study, 120 primary breast tumors were analyzed for P120; 114 of these tumors were also stained for the erbB-2 protein. Immunopositive staining was correlated with patient survival, nodal status, estrogen receptor levels, and number of mitoses. Sixty-nine % (83 of 120) of the tumors were positive for P120; 25% (28 of 114) stained positively for erbB-2. Of the 28 erbB-2 positive tumors 26 were also positive for the P120 protein. Forty-six % (55 of 120) of the specimens were from patients who later died from recurrent breast cancer; P120 was detected in 89% (49 of 55) of these specimens. In 52% of the survivors the P120 protein was also expressed. P120 negative tumors were highly correlative with survival (P = 0.0001); 84% (32 of 37) of patients with P120 negative tumors survived more than 7 years without evidence of recurrent disease. Multivariate analysis showed that the worst prognosis was for patients who had tumor positive nodes and expressed P120 (P = 0.0001); death occurred in 73% (30 of 41) of these patients. For the node negative patients who did not express P120, 5-year survival was 90% (19 of 21 patients); 5-year survival for the node negative patients who expressed P120 was significantly less (67%; 28 of 42 patients). Patients with P120 negative tumors had a good prognosis, irrespective of their nodal status. In this group, survival of node negative patients was 86% (18 of 21) and for those with positive nodes survival was 82% (13 of 16). A poor prognosis was found for patients with intense erbB-2 stained tumors (5 of 7 patients died). Weak staining of erbB-2 tumors (21 specimens) was not correlated with patient survival. Compared to P120 negative tumors, P120 positive tumors had greater numbers of mitoses (9.06 versus 6.65) and an almost 2-fold increase in the occurrence of positive nodes (one of every 4.67 versus one of every 8.81). The number of P120 positive tumors was greater in estrogen receptor positive tumors (75%) than in estrogen negative tumors (54%).(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation-related nucleolar antigens P145 and P120 associated with separate nucleolar elements and differences in tissue distribution.

Nucleolar antigens p145 and p120 are associated with proliferating cells (Freeman, J.W.; McRorie, D.K.; Busch, R.K.; Gyorkey, P.; Gyorkey, F.; Ross, B.E.; Spohn, W.H.; Busch, H. Cancer Res. 46:3593; 1986 and Freeman, J.W.; Busch, R.K.; Gyorkey, P.; Gyorkey, F.; Ross, B.E.; Busch, H. Cancer Res. 48:1244; 1988) and are not detectable in normal resting cells. Recent immunoelectron microscopic studies (Ochs, R.L.; Reilly, M.T.; Freeman, J.W.; Busch, H. Cancer Res. 48:6523; 1988) suggest that the two antigens have overlapping nucleolar localizations. In this study the nucleolus was physicochemically and biochemically studied to determine whether p145 and p120 were associated with a common nucleolar component. Antigen p145 was associated with 40-80 S ribonucleoprotein particles (RNPs), and the p145 antigen was not detected in HeLa cells following in situ RNAse digestion. P120 was found in a 40-80 S, RNAse resistant complex. Sequential extraction of HeLa nucleoli showed that most of antigen p145 was extractable in 10 mM Tris with 0.2% deoxycholate, whereas p120 was found in a nucleolar residue fraction requiring DNAse and high salt treatment for optimal extraction. Neither antigen p145 nor p120 was detectable in normal resting tissues. Antigen p145 was detected in all proliferating tissues examined, including a variety of malignant tumors (ten of ten), benign tissues including adenomas and hyperplasias (eight of eight), and in normal proliferating cells such as colonic epithelium and spermatogonia of the testes. Antigen p120 was not detected in all tumors, being absent in three of seven lymphomas and in one melanoma examined.(ABSTRACT TRUNCATED AT 250 WORDS)