M. Elizabeth Ross

doublecortin, a Brain-Specific Gene Mutated in Human X-Linked Lissencephaly and Double Cortex Syndrome, Encodes a Putative Signaling Protein

X-linked lissencephaly and double cortex are allelic human disorders mapping to Xq22.3-Xq23 associated with arrest of migrating cerebral cortical neurons. We identified a novel 10 kb brain-specific cDNA interrupted by a balanced translocation in an XLIS patient that encodes a novel 40 kDa predicted protein named Doublecortin. Four double cortex/X-linked lissencephaly families and three sporadic double cortex patients show independent doublecortin mutations, at least one of them a de novo mutation. Doublecortin contains a consensus Abl phosphorylation site and other sites of potential phosphorylation. Although Doublecortin does not contain a kinase domain, it is homologous to the amino terminus of a predicted kinase protein, indicating a likely role in signal transduction. Doublecortin, along with the newly characterized mDab1, may define an Abl-dependent pathway regulating neuronal migration.

Delayed Reduction of Ischemic Brain Injury and Neurological Deficits in Mice Lacking the Inducible Nitric Oxide Synthase Gene

Inducible nitric oxide synthase (iNOS), an enzyme that produces toxic amounts of nitric oxide, is expressed in a number of brain pathologies, including cerebral ischemia. We used mice with a null mutation of the iNOS gene to study the role of iNOS in ischemic brain damage. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCA). In wild-type mice, iNOS mRNA expression in the post-ischemic brain begun between 24 and 48 hr peaked at 96 hr and subsided 7 d after MCA occlusion. iNOS mRNA induction was associated with expression of iNOS protein and enzymatic activity. In contrast, mice lacking the iNOS gene did not express iNOS message or protein after MCA occlusion. The infarct and the motor deficits produced by MCA occlusion were smaller in iNOS knockouts than in wild-type mice (p < 0.05). Such reduction in ischemic damage and neurological deficits was observed 96 hr after ischemia but not at 24 hr, when iNOS is not yet expressed in wild-type mice. The decreased susceptibility to cerebral ischemia in iNOS knockouts could not be attributed to differences in the degree of ischemia or vascular reactivity between wild-type and knockout mice. These findings indicate that iNOS expression is one of the factors contributing to the expansion of the brain damage that occurs in the post-ischemic period. iNOS inhibition may provide a novel therapeutic strategy targeted specifically at the secondary progression of ischemic brain injury.

Inducible Nitric Oxide Synthase Gene Expression in Vascular Cells After Transient Focal Cerebral Ischemia

BACKGROUND AND PURPOSEnWe investigated whether inducible nitric oxide synthase (iNOS) is expressed after transient cerebral ischemia and, if so, we sought to define the temporal profile and cellular localization of the expression and the role of iNOS in the mechanism of ischemic brain injury.nnnMETHODSnThe middle cerebral artery in rats was occluded for 2 hours by an intraluminal filament. The occurrence of transient ischemia and reperfusion was confirmed by laser-Doppler flowmetry (n = 5). iNOS message in the ischemic neocortex was determined by reverse-transcription polymerase chain reaction. iNOS enzymatic activity was assessed by citrulline assay. The cellular localization of iNOS expression was determined by immunohistochemistry.nnnRESULTSniNOS mRNA was maximally expressed in postischemic brain at 12 hours and was not present at 4 days (n = 3 per time point). iNOS mRNA was not observed in the contralateral cerebral cortex. iNOS enzymatic activity developed in the postischemic brain between 12 and 24 hours (P < .05) and subsided at 4 days (n = 4 to 8 per time point). iNOS immunoreactivity in the ischemic region was restricted to the wall of capillaries and of larger blood vessels at 12 to 24 hours. In regions of early necrosis, inflammatory cells were iNOS positive. Treatment with the iNOS inhibitor aminoguanidine (n = 5; 100 mg/kg IP, BID for 4 days), starting 6 hours after ischemia, reduced infarct size in neocortex by 36 +/- 7% in comparison with vehicle-treated controls (n = 5) (P < .05).nnnCONCLUSIONSnTransient focal ischemia leads to iNOS expression in postischemic brain. However, the spatial and temporal patterns of expression differ from those occurring in permanent ischemia: iNOS is induced earlier and predominantly in vascular cells rather than in neutrophils. Thus, the temporal profile and localization of postischemic iNOS expression depend on the nature of the ischemic insult. The finding that aminoguanidine reduces infarct size adds further support to the hypothesis that postischemic iNOS expression contributes to ischemic brain damage.

Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia

We studied the effect of focal cerebral ischemia on inducible (iNOS) and constitutive (cNOS) nitric oxide synthase enzymatic activities in the affected brain. The middle cerebral artery (MCA) was occluded in spontaneously hypertensive rats. Animals were killed 1, 2, 4, and 7 days later. cNOS and iNOS enzymatic activities were determined in the infarcted cortex using the assay of Bredt and Snyder. cNOS was assayed in the presence of calcium, whereas iNOS was assayed in the absence of calcium and in the presence of tetrahydrobiopterin. The validity of the iNOS assay was verified in rats treated with bacterial lipopolysaccharide. In these animals, the magnitude of the induction of iNOS enzymatic activity in lung, spleen, and brain paralleled the expression of iNOS mRNA, assessed by reverse-transcription polymerase chain reaction. After MCA occlusion, calcium-dependent (cNOS) activity was markedly reduced only in lesioned cerebral cortex at days 1–7 (p < 0.001; analysis of variance and Tukeys test). In contrast to cNOS, calcium-independent (iNOS) activity was induced substantially in the infarct (p < 0.005) but not in the contralateral intact cortex (p > 0.05). iNOS activity peaked at day 2 and was not different from baseline at day 7 (p > 0.05). No NADPH diaphorase-positive neurons were observed in the area of the lesion at days 1–7. Macrophages appeared at day 2 and invaded the infarcted tissue by day 7. At this time, numerous glial fibrillary acidic protein-positive astrocytes were observed within the lesion. The results suggest that the decline in calcium-dependent (cNOS) activity reflects loss of NOS neurons within the lesion. The induction of calcium-independent activity is likely to reflect induction of iNOS in nonneuronal cells. Sustained nitric oxide production by iNOS may contribute to the late phase of tissue damage in focal cerebral ischemia.

Aminoguanidine Ameliorates and l-Arginine Worsens Brain Damage From Intraluminal Middle Cerebral Artery Occlusion

BACKGROUND AND PURPOSEnWe studied whether the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine reduces focal cerebral ischemic damage in a relatively noninvasive stroke model in which the rat middle cerebral artery (MCA) is occluded using an intravascular filament.nnnMETHODSnIn rats anesthetized with halothane, a nylon filament was advanced into the internal carotid artery until its tip occluded the origin of the MCA. The filament was left in place for 2 hours and then withdrawn. Twenty-four hours later, rats received intraperitoneal injections of aminoguanidine (100 mg/kg BID; n = 7), aminoguanidine+L-arginine (300 mg/kg QID; n = 7), L-arginine alone (n = 6), D-arginine alone (n = 6), or vehicle (n = 10). Drugs were administered for 3 consecutive days. Infarct volume was determined by image analysis in thionin-stained brain sections 4 days after ischemia. iNOS mRNA was detected with the use of reverse transcription polymerase chain reaction.nnnRESULTSnCerebral ischemia led to iNOS mRNA expression in the affected brain 48 hours after induction of ischemia. Administration of aminoguanidine reduced neocortical infarct volume by 26% (P < .05 versus vehicle, ANOVA and Tukeys test), a reduction that was antagonized by coadministration of L-arginine (P > .05 versus vehicle). Administration of L-arginine alone, but not D-arginine, enlarged the infarct by 29% (P < .05). Aminoguanidine or L-arginine did not influence the increase in water content in the postischemic brain, indicating that the effect on infarct volume is not related to modulation of ischemic edema.nnnCONCLUSIONSnThese results demonstrate that cerebral ischemia is also associated with iNOS expression in a minimally invasive model of transient MCA occlusion and that iNOS inhibition reduces focal ischemic damage. The findings support the hypothesis that nitric oxide produced by iNOS contributes to ischemic brain damage and that inhibition of iNOS may be a valuable tool in the management of cerebral ischemia.

Cyclooxygenase-1 Participates in Selected Vasodilator Responses of the Cerebral Circulation

Abstract— Cyclooxygenase (COX) is a prostanoid-synthesizing enzyme present in 2 isoforms: COX-1 and COX-2. Although it has long been hypothesized that prostanoids participate in cerebrovascular regulation, the lack of adequate pharmacological tools has led to conflicting results and has not permitted investigators to define the relative contribution of COX-1 and COX-2. We used the COX-1 inhibitor SC-560 and COX-1-null (COX-1−/−) mice to investigate whether COX-1 plays a role in cerebrovascular regulation. Mice were anesthetized (urethane and chloralose) and equipped with a cranial window. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry or by the 14C-iodoantipyrine technique with quantitative autoradiography. In wild-type mice, SC-560 (25 &mgr;mol/L) reduced resting CBF by 21±4% and attenuated the CBF increase produced by topical application of bradykinin (−59%) or calcium ionophore A23187 (−49%) and by systemic hypercapnia (−58%) (P <0.05 to 0.01). However, SC-560 did not reduce responses to acetylcholine or the increase in somatosensory cortex blood flow produced by vibrissal stimulation. In COX-1−/− mice, resting CBF assessed by 14C-iodoantipyrine was reduced (−13% to −20%) in cerebral cortex and other telencephalic regions (P <0.05). The CBF increase produced by bradykinin, A23187, and hypercapnia, but not acetylcholine or vibrissal stimulation, were attenuated (P <0.05 to 0.01). The free radical scavenger superoxide dismutase attenuated responses to bradykinin and A23187 in wild-type mice but not in COX-1−/− mice, suggesting that COX-1 is the source of the reactive oxygen species known to mediate these responses. The data provide evidence for a critical role of COX-1 in maintaining resting vascular tone and in selected vasodilator responses of the cerebral microcirculation.

Inducible nitric oxide synthase expression in human cerebral infarcts

Abstract The inducible or “immunological” isoform of nitric oxide synthase (iNOS) is induced in many cell types by inflammatory stimuli and synthesizes toxic amounts of NO. In rodent models of focal cerebral ischemia, iNOS is expressed in neutrophils invading the injured brain and in local blood vessels. Studies with iNOS inhibitors and iNOS null mice indicate that NO produced by iNOS contributes to ischemic brain injury. In the present study, we sought to determine whether iNOS is also expressed in the human brain after ischemic stroke. Studies were conducted using immunohistochemistry on autopsy brains with neuropathological evidence of acute cerebral infarction. iNOS immunoreactivity was observed in neutrophils infiltrating the ischemic brain and in blood vessels within the ischemic territory. iNOS-positive cells also were immunoreactive for nitrotyrosine, reflecting protein nitration by NO-derived peroxynitrite and nitrites. iNOS or nitrotyrosine immunoreactivity was not detected outside the region of the infarct. These observations provide evidence that iNOS is expressed in the human brain after ischemic infarction and support the hypothesis that iNOS inhibitors may be useful in the treatment of ischemic stroke in humans.

Cell division and the nervous system: regulating the cycle from neural differentiation to death

It has long been recognized that the balance between cellular proliferation and cell death during embryogenesis is a key factor in formation of the CNS. The recent definition of molecular mechanisms that drive the cell-division cycle and programmed cell death provides an opportunity to investigate the molecular interactions that co-ordinate cell-cycle regulation with CNS-pattern formation, neural differentiation and histogenesis. It is proposed that not only is the cell-division cycle regulated by developmentally controlled molecular signals to halt or proceed, but gene products that drive the cycle can also influence the course of neural differentiation and apoptosis.

Mutation analysis of the DCX gene and genotype/phenotype correlation in subcortical band heterotopia

Subcortical band heterotopia (SBH) comprises part of a spectrum of phenotypes associated with classical lissencephaly (LIS). LIS and SBH are caused by alterations in at least two genes: LIS1 (PAFAH1B1) at 17p13.3 and DCX (doublecortin) at Xq22.3–q23. DCX mutations predominantly cause LIS in hemizygous males and SBH in heterozygous females, and we have evaluated several families with LIS male and SBH female siblings. In this study, we performed detailed DCX mutation analysis and genotype–phenotype correlation in a large cohort with typical SBH. We screened 26xa0sporadic SBH females and 11xa0LIS/SBH families for DCX mutations by direct sequencing. We found 29xa0mutations in 22xa0sporadic patients and 11xa0pedigrees, including five deletions, four nonsense mutations, 19xa0missense mutations and one splice donor site mutation. The DCX mutation prevalence was 84.6% (22 of 26) in sporadic SBH patients and 100% (11 of 11) in SBH pedigrees. Maternal germline mosaicism was found in one family. Significant differences in genotype were found in relation to band thickness and familial vs sporadic status.

The Cyclooxygenase-2 Inhibitor NS-398 Ameliorates Ischemic Brain Injury in Wild-Type Mice but not in Mice with Deletion of the Inducible Nitric Oxide Synthase Gene

The authors investigated the role of the prostaglandin-synthesizing enzyme cyclooxygenase-2 (COX-2) in the mechanisms of focal cerebral ischemia and its interaction with inducible nitric oxide synthase (iNOS). Focal cerebral ischemia was produced by permanent occlusion of the middle cerebral artery (MCA) in mice. Infarct volume was measured 96 hours later by computer-assisted planimetry in thionin-stained brain sections. The highly selective COX-2 inhibitor NS398 (20 mg/kg; intraperitoneally), administered twice a day starting 6 hours after MCA occlusion, reduced total infarct volume in C57BL/6 (–23%) and 129/SVeV mice (–21%), and ameliorated the motor deficits produced by MCA occlusion (P < .05). However, NS398 did not influence infarct volume in mice with deletion of the iNOS gene (P > .05). In contrast, the neuronal NOS inhibitor 7-NI (50 mg/kg; intraperitoneally), administered once 5 minutes after MCA occlusion, reduced neocortical infarct volume by 20% in iNOS −/− mice (P < .05). NS398 did not affect arterial pressure, resting CBF or the CBF reactivity to hypercapnia in anesthetized iNOS null mice (P > .05). The data suggest that COX-2 reaction products, in mouse as in rat, contribute to ischemic brain injury. However, the failure of NS398 to reduce infarct volume in iNOS null mice suggests that iNOS-derived NO is required for the deleterious effects of COX-2 to occur. Thus, COX-2 reaction products may be another mechanism by which iNOS-derived NO contributes to ischemic brain injury.