Vascular endothelial growth factor (VEGF) is an angiogenic protein with therapeutic potential in ischemic disorders, including stroke. VEGF confers neuroprotection and promotes neurogenesis and cerebral angiogenesis, but the manner in which these effects may interact in the ischemic brain is poorly understood. We produced focal cerebral ischemia by middle cerebral artery occlusion for 90 minutes in the adult rat brain and measured infarct size, neurological function, BrdU labeling of neuroproliferative zones, and vWF-immunoreactive vascular profiles, without and with intracerebroventricular administration of VEGF on days 1-3 of reperfusion. VEGF reduced infarct size, improved neurological performance, enhanced the delayed survival of newborn neurons in the dentate gyrus and subventricular zone, and stimulated angiogenesis in the striatal ischemic penumbra, but not the dentate gyrus. We conclude that in the ischemic brain VEGF exerts an acute neuroprotective effect, as well as longer latency effects on survival of new neurons and on angiogenesis, and that these effects appear to operate independently. VEGF may, therefore, improve histological and functional outcome from stroke through multiple mechanisms.

Alterations in Ca 2+ homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose-regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress-induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c-mediated caspase activation in a cell-free system, and expression of GRP78 blocks both caspase activation and caspase-mediated cell death. GRP78 forms a complex with caspase-7 and -12 and prevents release of caspase-12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm to set in motion the cytosolic component of the ER stressinduced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

CorrectionsAGRICULTURAL SCIENCES. For the article ''Characterization of capsaicin synthase and identification of its gene (csy1) for pungency factor capsaicin in pepper (Capsicum sp.),'' by B. C. Narasimha

Vascular endothelial growth factor (VEGF) is an angiogenic protein with therapeutic potential in ischemic disorders, including stroke. VEGF confers neuroprotection and promotes neurogenesis and cerebral angiogenesis, but the manner in which these effects may interact in the ischemic brain is poorly understood. We produced focal cerebral ischemia by middle cerebral artery occlusion for 90 minutes in the adult rat brain and measured infarct size, neurological function, BrdU labeling of neuroproliferative zones, and vWF-immunoreactive vascular profiles, without and with intracerebroventricular administration of VEGF on days 1-3 of reperfusion. VEGF reduced infarct size, improved neurological performance, enhanced the delayed survival of newborn neurons in the dentate gyrus and subventricular zone, and stimulated angiogenesis in the striatal ischemic penumbra, but not the dentate gyrus. We conclude that in the ischemic brain VEGF exerts an acute neuroprotective effect, as well as longer latency effects on survival of new neurons and on angiogenesis, and that these effects appear to operate independently. VEGF may, therefore, improve histological and functional outcome from stroke through multiple mechanisms.

SummaryNeurogenesis, which may contribute to the ability of the adult brain to function normally and adapt to disease, nevertheless declines with advancing age. Adult neurogenesis can be enhanced by administration of growth factors, but whether the aged brain remains responsive to these factors is unknown. We compared the effects of intracerebroventricular fibroblast growth factor (FGF)-2 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) on neurogenesis in the hippocampal dentate subgranular zone (SGZ) and the subventricular zone (SVZ) of young adult (3-month) and aged (20-month) mice. Neurogenesis, measured by labelling with bromodeoxyuridine (BrdU) and by expression of doublecortin, was reduced by ∼ ∼ ∼ ∼ 90% in SGZ and by ∼ ∼ ∼ ∼ 50% in SVZ of aged mice. HB-EGF increased BrdU labelling in SGZ at 3 months by ∼ ∼ ∼ ∼ 60% and at 20 months by ∼ ∼ ∼ ∼ 450%, which increased the number of BrdU-labelled cells in SGZ of aged mice to ∼ ∼ ∼ ∼ 25% of that in young adults. FGF-2 also stimulated BrdU labelling in SGZ, by ∼ ∼ ∼ ∼ 25% at 3 months and by ∼ ∼ ∼ ∼ 250% at 20 months, increasing the number of newborn neurones in older mice to ∼ ∼ ∼ ∼ 20% of that in younger mice. In SVZ, HB-EGF and FGF-2 increased BrdU incorporation by ∼ ∼ ∼ ∼ 140% at 3 months and ∼ ∼ ∼ ∼ 170% at 20 months, so the number of BrdU-labelled cells was comparable in untreated 3-month-old and growth factor-treated 20-month-old mice. These results demonstrate that the aged brain retains the capacity to respond to exogenous growth factors with increased neurogenesis, which may have implications for the therapeutic potential of neurogenesis enhancement in age-associated neurological disorders.

Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of T he biosynthesis of enkephalin opioid peptides as well as numerous peptide neurotransmitters and hormones requires proteolytic processing of respective proprotein precursors within regulated secretory vesicles (1-4). The mature, processed enkephalin peptide is stored within these vesicles and undergoes stimulated secretion to mediate neurotransmission and cell-cell communication in the regulation of analgesia, behavior, and immune-cell functions. Secretory vesicles of neuroendocrine chromaffin cells (also known as chromaffin granules) contain enkephalin and its precursor proenkephalin (PE) (5, 6), with relevant prohormone convertases for converting PE into active enkephalin.The primary PE-cleaving activity in chromaffin granules has been characterized as a cysteine protease complex known as ''prohormone thiol protease'' (PTP) (7-10). The cysteine protease activity cleaves PE and enkephalin-containing peptide substrates at paired basic residues, as well as at certain monobasic residues, to generate appropriate enkephalin-related peptide products. Cellular inhibition of PTP by a cysteine protease inhibitor results in reduced production of enkephalin (11). Molecular identification of the protease component responsible for this cysteine protease activity will facilitate our understanding of multiple proteolytic enzymes that produce active peptides including the opioid [Met]enkephalin (ME) (12,13).In this study the protease responsible for PE-cleaving activity in chromaffin granules was identified by using an activity-based probe for cysteine proteases (14, 15) combined with mass spectrometry (MS) for peptide sequencing. Results identified secretory vesicle cathepsin L as the enzyme responsible for the previously described PTP cysteine protease activity involved in enkephalin and neuropeptide production (7-10). Cathepsin L generated the active peptide ME by cleaving enkephalin-containing peptide substrates at native dibasic and monobasic sites. Notably, cathepsin L colocalized with ME in the regulated secretory pathway of chromaffin cells. In cathepsin L gene knockout (KO) mice (16-1...

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

There is no satisfactory treatment for Huntington's disease (HD), a hereditary neurodegenerative disorder that produces chorea, dementia, and death. One potential treatment strategy involves the replacement of dead neurons by stimulating the proliferation of endogenous neuronal precursors (neurogenesis) and their migration into damaged regions of the brain. Because growth factors are neuroprotective in some settings and can also stimulate neurogenesis, we treated HD transgenic R6͞2 mice from 8 weeks of age until death by s.c. administration of FGF-2. FGF-2 increased the number of proliferating cells in the subventricular zone by Ϸ30% in wild-type mice, and by Ϸ150% in HD transgenic R6͞2 mice. FGF-2 also induced the recruitment of new neurons from the subventricular zone into the neostriatum and cerebral cortex of HD transgenic R6͞2 mice. In the striatum, these neurons were DARPP-32-expressing medium spiny neurons, consistent with the phenotype of neurons lost in HD. FGF-2 was neuroprotective as well, because it blocked cell death induced by mutant expanded Htt in primary striatal cultures. FGF-2 also reduced polyglutamine aggregates, improved motor performance, and extended lifespan by Ϸ20%. We conclude that FGF-2 improves neurological deficits and longevity in a transgenic mouse model of HD, and that its neuroprotective and neuroproliferative effects may contribute to this improvement.trophic factor ͉ polyglutamine