Anesthetic concerns in patients with known cerebrovascular insufficiency.

This review outlines the perioperative anesthesia considerations of patients with vascular diseases of the central nervous system, including occlusive cerebrovascular diseases with ischemic risks and various cerebrovascular malformations with hemorrhagic potential. The discussion emphasizes perioperative management strategies to prevent complications and minimize their effects if they occur. Planning the anesthetic and perioperative management is predicated on understanding the goals of the therapeutic intervention and anticipating potential problems.

Proteolytic cleavage of ataxin-7 by caspase-7 modulates cellular toxicity and transcriptional dysregulation.

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin-7 is cleaved by caspase-7, and we map two putative caspase-7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin-7 protein. Site-directed mutagenesis of these two caspase-7 cleavage sites in the polyQ-expanded form of ataxin-7 produces an ataxin-7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin-7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ length-dependent manner, expression of the non-cleavable D266N/D344N form of polyQ-expanded ataxin-7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase-7 truncation product of ataxin-7-69Q or -92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin-7, showed increased cellular toxicity. We also detected N-terminal polyQ-expanded ataxin-7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase-7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase-7 into the nucleus by polyQ-expanded ataxin-7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin-7 by caspase-7 may contribute to SCA7 disease pathogenesis.

The rat vanilloid receptor splice variant VR.5'sv blocks TRPV1 activation.

The capsaicin receptor (VR1, TRPV1) is a ligand-gated ion channel predominantly expressed in peripheral nociceptors and activated by multiple noxious stimuli including products of inflammation. A 5'-splice variant (VR.5'sv) of TRPV1 has been previously isolated and found to be insensitive to noxious stimuli. We report in this study that coexpression of VR.5'sv with TRPV1 in Xenopus oocytes blocks TRPV1-mediated current responses. Oocytes expressing the inhibitory profile demonstrated colocalization of TRPV1 and VR.5'sv-associated immunostaining in the plasma membrane. TRPV1 protein expression was comparable in all groups. Evidence of endogenous VR.5'-splice variant-like-protein expression was detected in dorsal root ganglion. These results support the idea that coexpression of VR.5'sv or a similar variant could result in inhibitory modulation of TRPV1 activation.

Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664.

The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of beta-amyloid peptides, a set of 39-43 amino acid fragments derived proteolytically from beta-amyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp-664 to generate a second cytotoxic peptide, APP-C31, but whether this C-terminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp-664 caspase cleavage site. Surprisingly, whereas beta-amyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp-664. These results suggest that Asp-664 plays a critical role in the generation of Alzheimer-related pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via protein-protein interactions.

FGF-2 promotes neurogenesis and neuroprotection and prolongs survival in a transgenic mouse model of Huntington's disease.

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 R6/2 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 approximately 30% in wild-type mice, and by approximately 150% in HD transgenic R6/2 mice. FGF-2 also induced the recruitment of new neurons from the subventricular zone into the neostriatum and cerebral cortex of HD transgenic R6/2 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 approximately 20%. 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.

Comparison of ischemia-directed migration of neural precursor cells after intrastriatal, intraventricular, or intravenous transplantation in the rat.

Cell replacement therapy may have the potential to promote brain repair and recovery after stroke. To compare how focal cerebral ischemia affects the entry, migration, and phenotypic features of neural precursor cells transplanted by different routes, we administered neuronal precursors from embryonic cerebral cortex of green fluorescent protein (GFP)-expressing transgenic mice to rats that had undergone middle cerebral artery occlusion (MCAO) by the intrastriatal, intraventricular, and intravenous routes. MCAO increased the entry of GFP-immunoreactive cells, most of which expressed neuroepithelial (nestin) or neuronal (doublecortin) markers, from the ventricles and bloodstream into the brain, and enhanced their migration when delivered by any of these routes. Transplanted neural precursors migrated into the ischemic striatum and cerebral cortex. Thus, transplantation of neural precursors by a variety of routes can deliver cells with the potential to replace injured neurons to ischemic brain regions.

Effect of aging on neuroglobin expression in rodent brain.

Neuroglobin (Ngb), a recently discovered O2-binding heme protein related to hemoglobin and myoglobin, protects neurons from hypoxic-ischemic injury in vitro and in vivo. In immunostained mouse brain sections, we found widespread expression of Ngb protein in neurons, but not astrocytes, of several brain regions that are prominently involved in age-related neurodegenerative disorders. Western blots from young adult (3 month), middle-aged (12 month), and aged (24 month) rats showed an age-related decline in Ngb expression in cerebral neocortex, hippocampus, caudate-putamen, and cerebellum. Loss of this neuroprotective protein may have a role in increasing susceptibility to age-related neurological disorders.

Immunolocalization of TASK-3 (KCNK9) to a subset of cortical neurons in the rat CNS.

Tandem pore domain (2P) K channels constitute the most diverse family of K channels and are responsible for background (leak or baseline) K currents. Of the 15 human 2P K channels, TASK-1, TASK-2, and TASK-3 are uniquely sensitive to physiologic pH changes as well as being inhibited by local anesthetics and activated by volatile anesthetics. In this study polyclonal antibodies selective for TASK-3 have been used to localize its expression in the rat central nervous system (CNS). TASK-3 immunostaining was found in rat cortex, hypothalamus, and hippocampus. Double immunofluorescent studies identified a discrete population of TASK-3 expressing neurons scattered throughout cortex. Using immunogold electron microscopy TASK-3 was identified at the cell surface associated with synapses and within the intracellular synthetic compartments. These results provide a more finely detailed picture of TASK-3 expression and indicate a role for TASK-3 in modulating cerebral synaptic transmission and responses to CNS active drugs.

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter.

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 [Met]enkephalin by cathepsin L occurred by proteolytic processing at dibasic and monobasic prohormone-processing sites. Cellular studies showed the colocalization of cathepsin L with [Met]enkephalin in secretory vesicles of neuroendocrine chromaffin cells by immunofluorescent confocal and immunoelectron microscopy. Functional localization of cathepsin L to the regulated secretory pathway was demonstrated by its cosecretion with [Met]enkephalin. Finally, in cathepsin L gene knockout mice, [Met]enkephalin levels in brain were reduced significantly; this occurred with an increase in the relative amounts of enkephalin precursor. These findings indicate a previously uncharacterized biological role for secretory vesicle cathepsin L in the production of [Met]enkephalin, an endogenous peptide neurotransmitter.

Neurogenesis and aging: FGF-2 and HB-EGF restore neurogenesis in hippocampus and subventricular zone of aged mice.

Neurogenesis, 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 approximately 90% in SGZ and by approximately 50% in SVZ of aged mice. HB-EGF increased BrdU labelling in SGZ at 3 months by approximately 60% and at 20 months by approximately 450%, which increased the number of BrdU-labelled cells in SGZ of aged mice to approximately 25% of that in young adults. FGF-2 also stimulated BrdU labelling in SGZ, by approximately 25% at 3 months and by approximately 250% at 20 months, increasing the number of newborn neurones in older mice to approximately 20% of that in younger mice. In SVZ, HB-EGF and FGF-2 increased BrdU incorporation by approximately 140% at 3 months and approximately 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.

VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia.

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.

Nanotubules formed by highly hydrophobic amphiphilic alpha-helical peptides and natural phospholipids.

We previously reported that the 18-mer amphiphilic alpha-helical peptide, Hel 13-5, consisting of 13 hydrophobic residues and five hydrophilic amino acid residues, can induce neutral liposomes (egg yolk phosphatidylcholine) to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures induces the formation of membrane structures resembling cellular organelles such as the Golgi apparatus. In the present study we focused our attention on the effects of peptide sequence and chain length on the nanotubule formation occurring in mixture systems of Hel 13-5 and various neutral and acidic lipid species by means of turbidity measurements, dynamic light scattering measurements, and electron microscopy. We designed and synthesized two sets of Hel 13-5 related peptides: 1) Five peptides to examine the role of hydrophobic or hydrophilic residues in amphiphilic alpha-helical structures, and 2) Six peptides to examine the role of peptide length, having even number residues from 12 to 24. Conformational, solution, and morphological studies showed that the amphiphilic alpha-helical structure and the peptide chain length (especially 18 amino acid residues) are critical determinants of very long tubular structures. A mixture of alpha-helix and beta-structures determines the tubular shapes and assemblies. However, we found that the charged Lys residues comprising the hydrophilic regions of amphiphilic structures can be replaced by Arg or Glu residues without a loss of tubular structures. This suggests that the mechanism of microtubule formation does not involve the charge interaction. The immersion of the hydrophobic part of the amphiphilic peptides into liposomes initially forms elliptic-like structures due to the fusion of small liposomes, which is followed by a transformation into tubular structures of various sizes and shapes.

Cerebral neurogenesis is induced by intranasal administration of growth factors.

Neurogenesis persists in the adult brain, where it may contribute to repair and recovery after injury, but the lack of methods for noninvasive stimulation of cerebral neurogenesis limits its potential for clinical application. We report that intranasal administration of either fibroblast growth factor-2 or heparin-binding epidermal growth factor-like growth factor increases neurogenesis, measured by the incorporation of bromodeoxyuridine into cells that express the early neuronal marker protein doublecortin in the subventricular zone of mouse brain. These findings indicate that intranasal growth factors may have potential as neurogenesis-promoting therapeutic agents.

Type 1 insulin-like growth factor receptor (IGF-IR) signaling inhibits apoptosis signal-regulating kinase 1 (ASK1).

The type 1 insulin-like growth factor receptor (IGF-IR) is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGF-IR binding to its ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress- and TNFalpha-induced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGF-IR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling inhibited ASK1 irrespective of TNFalpha-induced ASK1 activation and resulted in decreased ASK1-dependent JNK1 stimulation. Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGF-IR signaling suppresses the ASK-1-mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stress-activated arm of the cascade by IGF-IR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGF-I can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings.

The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells.

This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.4 x 10(6) and 1.7 x 10(5) M(-1) s(-1), respectively. Endopin 2 formed SDS-stable complexes with papain and elastase, a characteristic property of serpins. Interactions of the RSL domain of endopin 2 with papain and elastase were indicated by cleavage of endopin 2 near the predicted P1-P1' residues by these proteases. Endopin 2 did not inhibit the cysteine protease cathepsin B, or the serine proteases chymotrypsin, trypsin, plasmin, and furin. Endopin 2 in neuroendocrine chromaffin cells was colocalized with the secretory vesicle component (Met)enkephalin by confocal immunonfluorescence microscopy, and was present in isolated secretory vesicles (chromaffin granules) from chromaffin cells as a glycoprotein of 72-73 kDa. Moreover, regulated secretion of endopin 2 from chromaffin cells was induced by nicotine and KCl depolarization. Overall, these results demonstrate that the serpin endopin 2 possesses dual specificity for inhibiting both papain-like cysteine and elastase-like serine proteases. These findings demonstrate that endopin 2 inhibitory functions may occur in the regulated secretory pathway.

Caspase cleavage of members of the amyloid precursor family of proteins.

The synapse loss and neuronal cell death characteristic of Alzheimer's disease (AD) are believed to result in large part from the neurotoxic effects of beta-amyloid peptide (Abeta), a 40-42 amino acid peptide(s) derived proteolytically from beta-amyloid precursor protein (APP). However, APP is also cleaved intracellularly to generate a second cytotoxic peptide, C31, and this cleavage event occurs in vivo as well as in vitro and preferentially in the brains of AD patients (Lu et al. 2000). Here we show that APPC31 is toxic to neurons in primary culture, and that like APP, the APP family members APLP1 and possibly APLP2 are cleaved by caspases at their C-termini. The carboxy-terminal peptide derived from caspase cleavage of APLP1 shows a degree of neurotoxicity comparable to APPC31. Our results suggest that even though APLP1 and APLP2 cannot generate Abeta, they may potentially contribute to the pathology of AD by generating peptide fragments whose toxicity is comparable to that of APPC31.

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78.

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 stress-induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death.