Prenatal ontogeny of the epidermal growth factor receptor and its ligand,transforming growth factor alpha,in the rat brain

Transforming growth factor alpha (TGFα) interacts with the epidermal growth factor receptor (EGF-R) to produce its biological effects. TGFα induces the proliferation and differentiation of central nervous system (CNS) astrocytes and pluripotent stem cells, as well as the survival and differentiation of postmitotic CNS neurons. Both TGFα and EGF-R have been localized to the postnatal CNS. As the majority of CNS neuronal proliferation and migration occurs antenatally, we have examined the ontogeny of TGFα and EGF-R in the embryonic rat brain by in situ hybridization. EGF-R mRNA was expressed in the brain as early as embryonic day 11 (E11; the earliest age examined). It was initially detected in the midbrain, with subsequent expression first in multiple germinal zones, followed by expression in numerous cells throughout the brain. In many brain areas, EGF-R mRNA appeared in germinal centers during the later stages of neurogenesis and the early stages of gliogenesis. In the midbrain, the distribution of EGF-R mRNA overlapped extensively with that of tyrosine hydroxylase mRNA, suggesting that fetal dopaminergic neurons express EGF-R. Immunocytochemistry was used to demonstrate the presence of EGF-R-immunoreactive protein in brain areas that expressed EGF-R mRNA on E15 and E20. The expression of TGFα in many brain structures preceded that of EGF-R mRNA. TGFα mRNA was distributed throughout many non-germinal centers of the brain on E12 and later. Some brain areas, such as the external granule cell layer of the cerebellum, expressed EGF-R, but not TGFα mRNA. Northern blot analysis demonstrated that mRNA species for both TGFα and EGF-R were similar in embryos and adults. These data indicate that TGFα and EGF-R are positioned to have a role in the genesis, differentiation, migration, or survival of numerous cell populations in the embryonic brain. J. Comp. Neurol. 380:243–261, 1997. © 1997 Wiley-Liss, Inc.     

小鼠局灶性脑损伤组织中PDGF-A、B链及其β受体表达

目的 了解血小板衍化生长因子(PDGF)在中枢神经损伤及修复中的作用.方法 制备小白鼠颅脑损伤模型,分别在伤后1、4、7和14 d处死,采用原位杂交和免疫组化染色法检测脑组织中的PDGF-A、B链mRNA及其β受体的表达.结果 伤后1 d,PDGF-A链mRNA的表达较对侧脑组织增加,持续到第4 d,第7 d达高峰,14 d后略有下降;PDGF-B链mRNA的表达于伤后第1 d较对侧脑组织增加,第4 d较第1 d略有下降,7 d后达到高峰,至14 d后仍持续在高峰状态.PDGF-β受体的表达在伤后第1 d较对侧脑无明显变化,第4 d有所增加,第7 d达高峰,14 d略有下降.结论 血小板衍化生长因子参与了中枢神经损伤的修复过程.     

Abnormal astrocyte development and neuronal death in mice lacking the epidermal growth factor receptor

Stimulation of the epidermal growth factor receptor (EGF-R) produces numerous effects on central nervous system (CNS) cells in vitro including neuronal survival and differentiation, astrocyte proliferation and the proliferation of multipotent progenitors. However, the in vivo role of EGF-R is less well understood. In the present study, we demonstrate that EGF-R null mice generated on a 129Sv/J Swiss Black background undergo focal but massive degeneration the olfactory bulb, piriform cortex, neocortex, and thalamus between postnatal days 5 and 8 which is due, at least in part, to apoptosis. Some of the neuronal populations that degenerate do not normally express EGF-R, indicating an indirect mechanism of neuronal death. There were also delays in GFAP expression within the glia limitans and within structures outside the germinal zones in early postnatal ages. At or just prior to the onset of the degeneration, however, there was an increase in GFAP expression in these areas. The brains of EGF-R −/− animals were smaller but cytoarchitecturally normal at birth and neuronal populations appeared to be intact, including striatal GABAergic and midbrain dopaminergic neurons which have previously been shown to express EGF-R. Multipotent progenitors and astrocytes derived from EGF-R −/− mice were capable of proliferating in response to FGF-2. These data demonstrate that EGF-R expression is critical for the maintenance of large portions of the postnatal mouse forebrain as well as the normal development of astrocytes. J. Neurosci. Res. 53:697–717, 1998. © 1998 Wiley-Liss, Inc.     

Differential regulation of the low-affinity nerve growth factor receptor during postnatal development of the rat brain.

We studied the temporal and spatial localization of the low-affinity nerve growth factor receptor (LNGF-R) during the early postnatal period in rat brain in order to understand better the relationship between nerve growth factor (NGF)-like responsiveness and the development of specific central neuronal populations. Four different developmental patterns of LNGF-R mRNA hybridization were found in this study. First, some neurons contain high levels of LNGF-R mRNA from postnatal time points into adulthood, as exemplified by neurons of the cholinergic basal forebrain and mesencephalic trigeminal nucleus. Second, several cell groups exhibit robust hybridization during the early postnatal period but contain much reduced levels of LNGF-R mRNA in the adult brain. These include striatal neurons, Purkinje cells of the cerebellum, and several medullary nuclei. A third group of cells produces the LNGF-R transiently during development, including cranial nerve nuclei of the brainstem, the periolivary nuclei complex, the reticular formation, and the deep cerebellar nuclei. Finally, cell populations which may exist only transiently during central nervous system (CNS) development, such as subplate neurons of the cerebral cortex, appear to express the LNGF-R during only a brief period. These results show that the LNGF-R gene is differentially regulated in a cell type-specific manner during development, and suggests that diverse neuronal populations require only transient growth factor sensitivity, while others exhibit NGF-like responsitivity into maturity.     

Effects of pentobarbital on the expression of GABAA receptor β1 mRNA in the hippocampus: Differential responses of CA1 and CA3

Effects of barbiturates have been linked to the inhibitory GABA_A receptor in the brain. The present study examines changes in the expression of GABA_A receptor in the hippocampus of pentobarbital treated rat. Intraperitoneal pentobarbital injections were administered once daily for 9 days at an increasing dose schedule, 30 mg/kg at day 1–3, 60 mg/kg day 4–6, and 90 mg/kg day 7–9. Within each of the three dosage periods, the duration of sleep and extent of reduction in body temperature of the rats decreased with time. Two hours after the 9th injection, ³H-muscimol binding of the hippocampal homogenates of the animals showed that the maximal number of binding sites (B_max), 10.2 ± 1.6 pmol/mg protein, was not significantly greater than 9.5 ± 1.2 of saline control, but strikingly about 7-fold control level of β₁ mRNA was seen in the pyramidal cells of CA1 and CA2, as revealed by in situ hybridization analysis with digoxigenin-cRNA probes. However, when the rats were withdrawn from pentobarbital injection for 24 hours and 7 days, the B_max of the hippocampi was lowered to 7.3 ± 1.0 and 5.1 ± 0.7, respectively, and the expression of β₁ mRNA in CA1–2 returned toward control. The pentobarbital treatment did not significantly alter the affinity of the radioligand to the receptor in the hippocampus and the expression of β₁ mRNA in CA3 and CA4. The results suggest the plasticity of the β₁ mRNA in CA1–2 as well as differential involvement of CA1–2 and CA3–4 in response to the pentobarbital perturbation. Synapse 29:371–378, 1998. © 1998 Wiley-Liss, Inc.     

GABAA/benzodiazepine receptor α6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: Expression in developing and mutant mice

The gamma aminobutyric acid_A/benzodiazepine (GABA_A/BZ) receptor is a multisubunit (α, β, γ, δ, and ρ) ligand-gated chloride channel; there are several variants of the α, β, and γ subunits, each of which has been localized throughout the central nervous system. A large number of GABA_A/BZ subunit variants are expressed within the cerebellar cortex. In previous studies from other laboratories, α₆ subunit mRNA has been reported to be present exclusively in cerebellar granule cells. The developmental expression of α₆ mRNA in cerebellar and cochlear granule cells is of interest because it has been suggested that each of these cell types is derived from a common precursor pool. The polymerase chain reaction was used to generate a cDNA fragment encoding a portion of the M3–M4 intracellular loop of the α₆ subunit of the GABA_A/BZ receptor. A [³⁵S] riboprobe, transcribed from this cDNA fragment, was used to examine the expression of the α₆ subunit mRNA by in situ hybridization in developing normal mice and in adult mutant mice with known deficits in synaptic circuitry. A strong hybridization signal was observed over the granule cell layers of both the cerebellum and cochlear nuclei in adult mice. The signal over the cochlear nuclei appeared after birth toward the end of postnatal week 1, coinciding with the appearance of labeling in the cerebellar cortex. The intensity of the hybridization signal in both regions increased rapidly until postnatal day 14, after which it increased more gradually, reaching adult levels during postnatal week 3. In the weaver mutant, α₆ labeling was detected in surviving granule cells, but not in cerebellar regions devoid of granule cells. Significant levels of the α₆ hybridization signal were also present in cerebellar granule cells of Purkinje cell degeneration, lurcher, and staggerer mutants, suggesting that aberrations in synaptic circuitry do not prevent α₆ subunit gene expression. Our results demonstrate that α₆ subunit mRNA is not limited to the cerebellum, but is expressed in other neurons which share a common cellular precursor pool. These data also suggest that these granule cell precursors may be intrinsically programmed to acquire a specific form of the GABA_A/BZ receptor, irrespective of their final location and lack of connections with target neurons. © 1994 Wiley-Liss, Inc.     

Different expressions of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptor subunit mRNAs between visceromotor and somatomotor neurons of the rat lumbosacral spinal cord

The glutamatergic transmission system plays a key role in afferent and efferent pathways involved in micturition. By in situ hybridization combined with retrograde Fast Blue labeling, expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR-A to -D) and N-methyl-D -aspartate (NMDA) receptor (NR1 and NR2A-D) subunit mRNAs were examined in visceromotor and somatomotor neurons of the rat lumbosacral spinal cord. Parasympathetic preganglionic neurons (PGNs) in the intermediolateral nucleus highly expressed GluR-A and GluR-B subunit mRNAs, with very low levels for GluR-C and GluR-D subunits. As for the NMDA receptor, PGNs were associated with abundant signals for NR1 subunit mRNA, but without any NR2 subunit mRNAs. On the other hand, somatomotor neurons in the ventral horn (dorsolateral nucleus) express all four AMPA receptor subunit mRNAs, showing relatively abundant expressions of GluR-C and GluR-D subunit mRNA compared with PGNs. In addition to high levels of NR1 subunit mRNA, dorsolateral nucleus neurons moderately expressed NR2A and NR2B subunit mRNAs. These results suggest that molecular organization of both AMPA and NMDA receptor channels are distinct between PGNs and dorsolateral nucleus neurons. Considering that native NMDA receptors are heteromeric channels composed of NR1 and NR2 subunits, it seems likely that dorsolateral nucleus neurons, not PGNs, are provided with functional NMDA receptors, which could induce activity-dependent changes in synaptic transmission in the efferent pathway for the lower urinary tract. J. Comp. Neurol. 404:172–182, 1999. © 1999 Wiley-Liss, Inc.     

Interleukin-1β, but not IL-1α, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1β (IL-1β). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1α and IL-1β) and the antagonist (IL-1ra)—members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [¹²⁵I] IL-1β to cultured astrocytes was saturable and of high affinity. Mean values for the K_D and B_max were calculated to be 60.7±7.4 pM and 2.5±0.1 fmol mg^-1 protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1α (K_i of 341.1 pM) and IL-1β (K_i 59.9 pM), as well as the antagonist IL-1ra (K_i 257.6 pM), displaced specific [¹²⁵I] IL-1β binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [¹²⁵I] IL-1β binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1α and IL-1β revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1β (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1α by itself had no stimulatory action on NGF release. The simultaneous application of IL-1α and IL-1β elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1β. We concluded that IL-1β-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1α and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.     

Developmental study of the gene expression for α and γ subunits of enolase in the rat brain by in situ hybridization histochemistry

The gene expression for α and γ subunits of enolase, a dimeric enzyme in the glycolytic pathway, was examined in the developing brain of rats by in situ hybridization. The expression for the γ subunit of enolase was first detected in post-mitotic neurons settled in the mantle zone at E13, and it increased progressively until the adult stage. Expression signals for the α subunit were discerned in two discrete regions showing different developmental changes: the signals in the proliferative ventricular zone were intense at E13 and decreased and eventually disappeared around birth, whereas the signals in the mantle zone persisted until the adult stage. In the adult brain, mRNAs for the α and γ subunits were expressed widely in neurons, resulting in almost similar temporal patterns in the brain except for the cerebellum. Expression levels of the α subunit in adult glial cells were below the detection threshold of the in situ hybridization analysis. These findings suggest that both α and γ enolase subunits participate in energy production in neurons of the mature brain and that marked changes in the subunit composition of enolase occur according to both neuron type and maturation. (c) 1993 Wiley-Liss, Inc.     

Selective upregulation of Tα1 α-tubulin and neuropeptide Y mRNAs after intermittent excitatory stimulation in adult rat hippocampus in vivo

Adult central neurons exhibit significant structural and moledular chnges in epilepsy. We have examined changesin two markers of morphological and physiological plasticuity, Tα1 α-tubkulin (Tα1) and neuropeptide Y (NPY) mRNAs, inresponse tointermittent (20 Hz, 10 seconds), 1 stimulation of therat perforant path in vivo. Stimulus trins elicited brief (0.5–3 seconds) afterdischarges intheipsilateral dentate gyrus (DG). Four hours of stimulation caused no significant loss ofinhibitiobn intthe DG 40–48 hours after stimulationceased. Hiowever, it did lead to an increase in NPY mRNA in neurons of the ipsilateral and to a lesser extent, contralateral DGs and Ammon's Horn. Many of these were presumably interneurons tht normally express NPY. However, dentate granule cells (DGCs), which do not normally express this peptide, also expressed robust levels of NPY mRNA bilaterally. NPY mRNA levels peaked at 4–24 hours and returned to baseline by 48 hours poststimulation. Although 24 hourz of stimulation induced a similar increse in interneurons, DGCs showed no detecstable NPY mRNA. Afterdischarges were necessaryto elevate NPY mRNA exprssion. Four hours of stimulation evelvated Tα1 mRNA expsression in both ipsilateral and, to a lesser extent, contralteral DGCs; this elevation peaked at 24 hours poststimulation and declined to baseline by 72 hours. Stimulation for 24 hors caused broader changes in Tα1 mRNA expression, with incrases in DGCs and in CA3 pyramidal cells bilterally. Acute denervattion of the DG did not affect Tα1 mRNA level in the hippocampal formation. Elevated sysnaptic input resulting in afterdischarges, but not necessarily in excitbility changes in the DG, led to altrations in the exspression of molecuar markers of plasticity. These changes may rflect adaptive rsponses to physiological activation. © 1996 Wiley-Liss, Inc.     

Differential expression of Na,K-ATPase α-isoform mRNAs in aging rat cerebellum

Age-dependent changes in the expression of Na,K-ATPase α1- and α3-mRNAs were analyzed in the rat cerebellum by in situ hybridization. In young rats, α1-mRNA showed prominent labeling in the granular layer (GL) with moderate fine distribution in the molecular layer (ML), Purkinje cell layer (PCL), and white matter (WM) but no clusters over Purkinje cells (PCs). In old rats, α1-mRNA remained unchanged in ML and PCL, but declined by 43% (P < 0.0001) in GL and increased by 624% (P < 0.0001) in WM. α3-mRNA in young rats showed large clusters of label on stellate, basket, Golgi, and PCs and fine grains diffusely in ML, GL, and WM. In old rats, α3-mRNA declined by 87% in ML, 83% in PCL, 84% per PC, and 89% in GL and increased by 111% in WM (all values P < 0.0001) relative to young rats. PC numbers were reduced by 30%, but the average area of PC profiles did not change significantly. In old rats, the specific cluster-like label related to α3-mRNA on PCs, stellate, basket, and Golgi cells was lost. Immunocytochemistry of cerebellum and hippocampus showed no age-related change in the distribution and density of total catalytic polypeptide. Thus, the discordance between changes in the levels of mRNAs in neuronal layers and WM in the face of constant polypeptide levels indicates age-related changes in polypeptide turnover. Cell- and isoform-specificity of α-isoform mRNAs in aging rat cerebellum may reflect differential regulation underlying age-related impairments in signal transduction and motor learning. J. Neurosci. Res. 47:287–299, 1997. © 1997 Wiley-Liss, Inc.     

Tumor necrosis factor α and transforming growth factor β upregulate astrocyte expression of monocyte chemoattractant protein-1

Astrocytes participate in the pathophysiology of central nervous system (CNS) inflammatory disease. Astrocyte expression of adhesion molecules, cytokines, and major histocompatibility complex antigens may contribute to these inflammatory processes. In addition, recent data suggested that astrocytes may be a source of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a member of the chemokine family of small cytokines and functions both as a chemoattractant as well as a stimulator of monocytes. To further characterize the role of astrocytes in CNS inflammation, we examined the effect of inflammatory cytokines on MCP-1 expression by astrocytes. Results of these studies demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFa) upregulates MCP-1 message and protein expression. The pleiotropic cytokine transforming growth factor beta (TGFβ) also stimulated MCP-1 expression. When astrocytes were exposed to both cytokines simultaneously, an additive effect on MCP-1 message, but not MCP-1 protein expression, was observed. These data suggest that TNFa and TGFβ, each present during CNS inflammatory disease, may upregulate the expression of MCP-1 which, in turn, may function to both recruit monocytes to the site of inflammation as well as to activate those monocytes already present in an inflammatory lesion.     

Amplification of the epidermal growth factor receptor in astrocytic tumours by chromogenic in situ hybridization: association with clinicopathological features and patient survival

Chromogenic in situ hybridization (CISH) was used to detect amplification of the epidermal growth factor receptor (EGFR) gene in tissue microarrays of tumours derived from 287 patients with grade II-IV diffuse astrocytomas. Amplification was found in 32% of the tumours with a highly significant association with histological grade (4% in grade II, 21% in grade III and 39% in grade IV; P < 0.001). Amplification of the EGFR gene was more common in primary than in secondary glioblastomas (41%vs. 16%, P = 0.033). Overexpression of EGFR mRNA and protein (wild-type and vIII variant) was found to correlate with EGFR gene amplification (P = 0.028, P = 0.035 and P = 0.014 respectively), but wild-type EGFR protein was also frequently overexpressed in tumours without EGFR gene amplification. Patients with older age (P < 0.001) and tumours with lack of p53 overexpression (P = 0.03) and higher apoptosis rate (P < 0.001) had significantly more EGFR gene amplifications than their counterparts. No such correlation with apoptosis was found in glioblastomas. The survival of patients with EGFR gene-amplified grade III tumours was significantly shorter than in those with grade III non-amplified tumours (P = 0.03). No such difference was noted in glioblastomas (grade IV tumours). Our data verify the central role of EGFR in the pathobiology of astrocytic tumours, and highlight the advantages of CISH as a simple and practical assay to screen for EGFR gene amplification in astrocytic tumours.     

Distribution and levels of insulin-like growth factor I mRNA across the life span in the Brown Norway×Fischer 344 rat brain

Previous studies have reported changes in insulin-like growth factor I (IGF-I) mRNA expression during early postnatal development of the rat brain. Although changes in IGF-I gene expression have been documented in a wide range of central nervous system structures during early development and investigated in the hippocampus during aging, no study has compared changes in IGF-I gene expression in different brain regions across the life span. The present study assessed the distribution of IGF-I gene expression using in situ hybridization in rats aged 2–30 months. Dot blots were used as a quantitative assessment of cortical IGF-I mRNA. Results indicate that both the distribution and levels of brain IGF-I mRNA do not change significantly between 2 and 30 months of age in the rat. However, in spite of relatively constant levels of mRNA, other studies from our laboratory have demonstrated that cortical IGF-I protein levels decrease 36.6% between 11 and 32 months of age, suggesting that IGF-I function is decreased with increasing age.     

Regional distribution of the cells expressing glycine receptor β subunit mRNA in the rat brain

The expression of mRNA of the β subunit of the glycine receptor was investigated in the rat by in situ hybridization histochemistry using an oligonucleotide probe specific to the sequence of the β subunit. Neurons expressing β subunit mRNA were widely and abundantly distributed in the rat brain from the olfactory bulb to the spinal cord. The pattern of distribution of cells containing β subunit mRNA in the lower brainstem was very similar to that of cells containinga₁ subunit mRNA. In addition, β subunit mRNA was strongly expressed by the neurons of the cerebral cortex, hippocampal formation and diencephalon as well as by the Purkinje cells wherea₁ subunit mRNA expression is rare. These findings indicated that the glycine receptor is heterogeneous. The sites where strong labeling was noted were as follows. In the forebrain and diencephalon, strongly labeled neurons were abundant in the olfactory region, hippocampal formation, cerebral cortex, and thalamus. In the hippocampal formation, neurons in the subiculum, pyramidal cells in Ammon's horn, and neurons in the polymorphic layer of the dentate gyrus were strongly labeled. In the thalamus, the anterodorsal, reticular, parafascicular, and the subthalamic nuclei were strongly labeled. In the brainstem, the red nucleus, almost all of the motor neurons in the cranial motor nuclei innervating striated muscles, the trigeminal mesencephalic nucleus, the ventral tegmental nucleus of Gudden, and the pontine nucleus were strongly labeled. In the cerebellum, Purkinje cells in the Purkinje cell layer and all of the cerebellar nuclei were strongly labeled.     

Cellular signaling roles of TGFβ, TNFα and βAPP in brain injury responses and Alzheimer's disease

β-Amyloid precursor protein (βAPP), transforming growth factor β (TGFβ), and tumor necrosis factor-α (TNFα) are remarkably pleiotropic neural cytokines/neurotrophic factors that orchestrate intricate injury-related cellular and molecular interactions. The links between these three factors include: their responses to injury; their interactive effects on astrocytes, microglia and neurons; their ability to induce cytoprotective responses in neurons; and their association with cytopathological alterations in Alzheimer's disease. Astrocytes and microglia each produce and respond to TGFβ and TNFα in characteristic ways when the brain is injured. TGFβ, TNFα and secreted forms of βAPP (sAPP) can protect neurons against excitotoxic, metabolic and oxidative insults and may thereby serve neuroprotective roles. On the other hand, under certain conditions TNFα and the fibrillogenic amyloid β-peptide (Aβ) derivative of βAPP can promote damage of neuronal and glial cells, and may play roles in neurodegenerative disorders. Studies of genetically manipulated mice in which TGFβ, TNFα or βAPP ligand or receptor levels are altered suggest important roles for each factor in cellular responses to brain injury and indicate that mediators of neural injury responses also have the potential to enhance amyloidogenesis and/or to interfere with neuroregeneration if expressed at abnormal levels or modified by strategic point mutations. Recent studies have elucidated signal transduction pathways of TGFβ (serine/threonine kinase cascades), TNFα (p55 receptor linked to a sphingomyelin-ceramide-NFκB pathway), and secreted forms of βAPP (sAPP; receptor guanylate cyclase-cGMP-cGMP-dependent kinase-K⁺ channel activation). Knowledge of these signaling pathways is revealing novel molecular targets on which to focus neuroprotective therapeutic strategies in disorders ranging from stroke to Alzheimer's disease.