No abstract

A number of studies have provided evidence that neuronal cell loss after stroke involves programmed cell death or apoptosis. In particular, recent biochemical and immunohistochemical studies have demonstrated the expression and activation of intracellular proteases, notably caspase-3, which act as both initiators and executors of the apoptotic process. To further elucidate the involvement of caspases in neuronal cell death induced by focal stroke we developed a panel of antibodies and investigated the spatial and temporal pattern of both caspase-8 and caspase-3 expression. Our efforts focused on caspase-8 because its "apical" position within the enzymatic cascade of caspases makes it a potentially important therapeutic target. Constitutive expression of procaspase-8 was detectable in most cortical neurons, and proteolytic processing yielding the active form of caspase-8 was found as early as 6 hr after focal stroke induced in rats by permanent middle cerebral artery occlusion. This active form of caspase-8 was predominantly seen in the large pyramidal neurons of lamina V. Active caspase-3 was evident only in neurons located within lamina II/III starting at 24 hr after injury and in microglia throughout the core infarct at all times examined. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, gel electrophoresis of DNA, and neuronal cell quantitation indicated that there was an early nonapoptotic loss of cortical neurons followed by a progressive elimination of neurons with features of apoptosis. These data indicate that the pattern of caspase expression occurring during delayed neuronal cell death after focal stroke will vary depending on the neuronal phenotype.

Background and Purpose-Microglia and astrocytes in the peri-infarct region are activated in response to focal stroke. A critical function of activated glia is formation of a protective barrier that ultimately forms a new glial-limiting membrane. Osteopontin, a provisional matrix protein expressed during wound healing, is induced after focal stroke. The present study was performed to determine the spatial and temporal expression of osteopontin and its integrin receptor ␣ v ␤ 3 during formation of the peri-infarct gliotic barrier and subsequent formation of a new glial-limiting membrane. Methods-Spontaneously hypertensive rats (nϭ19) were subjected to permanent occlusion of the middle cerebral artery and killed 3, 6, and 24 hours and 2, 5, and 15 days after occlusion. The spatial and temporal expression of osteopontin mRNA was determined by in situ hybridization, and that of osteopontin ligand and its integrin receptor ␣ v ␤ 3 was determined by immunohistochemistry. Results-Osteopontin mRNA was expressed de novo in the peri-infarct region from 3 to 48 hours; by 5 days osteopontin mRNA expression was restricted to the infarct. Osteopontin protein was expressed by peri-infarct microglia beginning at 24 hours and by microglia/macrophages at 48 hours in the infarct. Integrin receptor ␣ v ␤ 3 was expressed in peri-infarct astrocytes at 5 and 15 days. Conclusions-Early

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin‐associated protein 1 (HAP‐1), a brain‐enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP‐1 is unknown, but recent evidence supports a role in microtubule‐dependent organelle transport. We examined the subcellular localization of HAP‐1 with an antibody made against the NH2‐terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP‐1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP‐1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP‐1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP‐1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP‐1 localized to membrane‐bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP‐1 was also strongly associated with an unusual large “dense” organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP‐1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP‐1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons. J. Comp. Neurol. 403:421–430, 1999. © 1999 Wiley‐Liss, Inc.

No abstract

In vivo studies of granule cell gene expression during corticocerebellar development and in vitro studies of Purkinje cell neurite outgrowth suggest that neurotrophin-3 may influence growth of Purkinje cell dendrites. To determine whether neurotrophic substances affect the growth of specific neuronal processes (i.e. axons and dendrites) or nonspecifically cause process development by exerting a trophic influence upon neuronal physiology we performed an immunohistochemical examination of trkC protein expression during early postnatal development of the rat cerebellum. Our findings indicate that Purkinje cells begin to synthesize trkC protein coincident with the onset of dendritic outgrowth. Robust immunostaining was evident throughout the entire somatodendritic domain of Purkinje cells during dendritic development but became faint and restricted to the cell body subsequent to the completion of dendritogenesis. These results suggest that growth and maturation of the Purkinje cell dendritic arbor may be influenced by neurotrophin-3 activation of trkC receptors distributed within developing dendrites.

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH2-terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large "dense" organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons.