Wei Hua Lee

ALTERATIONS IN GLUT1 AND GLUT3 GLUCOSE TRANSPORTER GENE EXPRESSION FOLLOWING UNILATERAL HYPOXIA-ISCHEMIA IN THE IMMATURE RAT BRAIN

The brain damage produced by unilateral cerebral hypoxia-ischemia in the immature rat results from major alterations in cerebral energy metabolism and glucose utilization which begin during the course of the insult and proceed into the recovery period. Consistent with a lack of pathology, the alterations in the hemisphere contralateral to the carotid artery ligation are transient and return to normal within 24 h of recovery, whereas the hemisphere ipsilateral to the ligation exhibits both early and late responses, and infarction. The facilitative glucose transporter proteins mediate glucose transport across the blood-brain barrier (55 kDa GLUT1), and into neurons and glia (GLUT3 and 45 kDa GLUT1), and demonstrate both early and late responses to perinatal hypoxia-ischemia. This study employed in situ hybridization histochemistry to investigate the temporal and regional patterns of GLUT1 and GLUT3 gene expression following a severe (2.5 h) hypoxic-ischemic insult in the 7-day old rat brain. Enhanced GLUT1 mRNA expression was apparent in cerebral microvessels of both hemispheres and remained elevated in the ipsilateral hemisphere through 24 h of recovery, consistent with our previous observation of increased microvascular 55 kDa GLUT1 protein. The expression of the neuronal isoform, GLUT3, was enhanced in penumbral regions, such as piriform cortex and amygdala, but was rapidly reduced in the affected areas of cortex, hippocampus and thalamus, reflecting necrosis. The late response, observed at 72 h of recovery, was characterized by extensive necrosis in the ipsilateral hemisphere, loss of GLUT3 expression, and a gliotic reaction including increased GLUT1 in GFAP-positive astrocytes. This study demonstrates that cerebral hypoxia-ischemia in the immature rat produces both immediate-early and long-term effects on the glucose transporter proteins at the level of gene expression.

High K+ and IGF-1 protect cerebellar granule neurons via distinct signaling pathways

In culture, cerebellar granule neurons die of apoptosis in serum‐free media containing a physiologic level of K+ but survive in a depolarizing concentration of K+ or when insulin‐like growth factor 1 (IGF‐1) is added. Both Akt/PKB activation and caspase‐3 inhibition were implicated as the underlying neuroprotective mechanisms. The duration of high K+, however, induced survival effects that outlasted its transient activation of Akt, and granule neurons derived from caspase‐3 knockout mice died to the same extent as did those from wild‐type mice, suggesting that additional mechanisms are involved. To delineate these survival mechanisms, we compared the activities of two major survival pathways after high K+‐induced depolarization or IGF‐1 stimulation. Although IGF‐1 promoted neuronal survival by activating its tyrosine kinase receptor, high K+ depolarization provided the same effect by increasing the Ca2+ influx through the L Ca2+ channel. Moreover, high K+‐induced depolarization resulted in sustained activation of MAP kinase, whereas IGF‐1 activated Akt in 4 hr. Inhibition of MEK (MAP kinase kinase) by either PD98059 or UO126 abolished the protective effect of high K+‐induced depolarization, but not that of IGF‐1, suggesting that activation of the MAP kinase pathway is necessary for high K+ neuroprotective effects. We demonstrated also that high K+‐induced depolarization, but not IGF‐1, increased phosphorylation of cAMP‐response element‐binding protein (CREB) and protein synthesis, both of which can be blocked by UO126. Overall, our findings suggested that high K+‐induced depolarization, unlike IGF‐1, promoted neuronal survival via activating MAP kinase, possibly by increasing CREB‐dependent transcriptional activation of specific proteins that promote neuronal survival.

Detection of apoptosis in weaver cerebellum by electron microscopic in situ end-labeling of fragmented DNA

Massive degeneration of granule cell precursors occurs perinatally in the cerebellum of weaver mutant mice. We have studied the electron microscopic (EM) features of granule cell death in weaver and control mice, using an in situ end-labeling (ISEL) technique for detecting DNA fragmentation, a hallmark of apoptosis. In all animals, EM-ISEL revealed the pattern of apoptosis, with an enhanced expression in weaver mice. The weaver gene appears to accelerate the death program, most likely through potassium channel-mediated signals.

Insulin-like growth factor-I protects granule neurons from apoptosis and improves ataxia in weaver mice

Most cerebellar granule neurons in weaver mice undergo premature apoptosis during the first 3 postnatal weeks, subsequently leading to severe ataxia. The death of these granule neurons appears to result from a point mutation in the GIRK2 gene, which encodes a G protein‐activated, inwardly rectifying K+ channel protein. Although the genetic defect was identified, the molecular mechanism by which the mutant K+ channel selectively attacks granule neurons in weaver mice is unclear. Before their demise, weaver granule neurons express abnormally high levels of insulin‐like growth factor (IGF) binding protein 5 (IGFBP5). IGF‐I is essential for the survival of cerebellar neurons during their differentiation. Because IGFBP5 has the capacity to block IGF‐I activity, we hypothesized that reduced IGF‐I availability resulting from excess IGFBP5 accelerates the apoptosis of weaver granule neurons. We found that, consistently with this hypothesis, exogenous IGF‐I partially protected cultured weaver granule neurons from apoptosis by activating Akt and decreasing caspase‐3 activity. To determine whether IGF‐I protects granule neurons in vivo, we cross‐bred weaver mice with transgenic mice that overexpress IGF‐I in the cerebellum. The cerebellar volume was increased in weaver mice carrying the IGF‐I transgene, predominantly because of an increased number of surviving granule neurons. The presence of the IGF‐I transgene resulted in improved muscle strength and a reduction in ataxia, indicating that the surviving granule neurons are functionally integrated into the cerebellar neuronal circuitry. These results confirm our previous suggestion that a lack of IGF‐I activity contributes to apoptosis of weaver granule neurons in vivo and supports IGF‐Is potential therapeutic use in neurodegenerative disease.

Potential Role of IGF-I in Hypoxia Tolerance Using A Rat Hypoxic-Ischemic Model: Activation of Hypoxia-Inducible Factor 1α

Hypoxia preconditioning and subsequent tolerance to hypoxia-ischemia damage is a well-known phenomenon and has significant implications in clinical medicine. In this investigation, we tested the hypothesis that the transcriptional activation of IGF-I is one of the underlying mechanisms for hypoxia-induced neuroprotection. In a rodent model of hypoxia-ischemia, hypoxia preconditioning improved neuronal survival as demonstrated by decreased hypoxia-ischemia-induced neuronal apoptosis. To study the role of IGF-I in hypoxia tolerance, we used in situ hybridization to examine IGF-I mRNA distribution on adjacent tissue sections. In cerebral cortex and hippocampus, hypoxia preconditioning resulted in an increase in neuronal IGF-I mRNA levels with or without hypoxia-ischemia. To test its direct effects, we added IGF-I to primary neuronal culture under varying oxygen concentrations. As oxygen concentration decreased, neuronal survival also decreased, which could be reversed by IGF-I, especially at the lowest oxygen concentration. Interestingly, IGF-I treatment resulted in an activation of hypoxia-inducible factor 1α (HIF-1α), a master transcription factor for hypoxia-induced metabolic adaptation. To evaluate whether IGF-I transcriptional activation correlates with HIF-1α activity, we studied the time course of HIF-1α DNA binding activity in the same rat model of hypoxia-ischemia. After hypoxia-ischemia, there was an increase in HIF-1α DNA binding activity in cortical tissues, with the highest increase around 24 h. Like IGF-I mRNA levels, hypoxia preconditioning increased HIF-1α DNA binding activity alone or with subsequent hypoxia ischemia. Overall, our results suggest that IGF-I transcriptional activation is one of the metabolic adaptive responses to hypoxia, which is likely mediated by a direct activation of HIF-1α.

Decreased IGF-I gene expression during the apoptosis of Purkinje cells in pcd mice

Insulin-like growth factor I (IGF-I) plays a potential functional role in cerebellar development in the rat, as indicated by its spatio-temporally coordinated expression with the IGF-I receptor (IGFR-I), IGF binding protein (IGFBP) 2 and 5 during the postnatal critical growth period. Although IGF-I promotes the survival of cultured cerebellar neurons, its role during cerebellar development in vivo is unclear. Growth factor deprivation has been shown to trigger apoptosis, the developmental cell death which, if abnormal, may lead to various pathological states. To understand the involvement of IGF-I in Purkinje cell survival, we examined mRNA expression of IGF-I, IGFR-I, IGFBP 2 and 5 in the Purkinje cell degeneration (pcd) mice. During pcd cerebellar development, Purkinje cells rapidly degenerate leading to their almost complete depletion by adult life. IGF system mRNA expression was studied during Purkinje cell death in the pcd mutants (pcd/pcd) at postnatal day (D) 11, 17, 24 and adult. At D11 and D17, no significant difference of the IGF-I system mRNA expression was observed between the normal and pcd/pcd cerebellum. At D24, a significant decrease of IGF-I mRNA was found in the apoptotic Purkinje cells in the pcd/pcd cerebellum, which was accompanied by a severe astrogliosis and activation of astrocytic IGF-I expression. In the adult pcd/pcd cerebellum, with few Purkinje cells remaining, many granule cells underwent apoptosis. In conclusion, decreased IGF-I mRNA expression was correlated with Purkinje cell apoptosis in pcd cerebellum. Whether the decrease of IGF-I mRNA expression is the cause or result of the Purkinje cell degeneration needs to be further elucidated.

Inhibition of insulin‐like growth factor I activity contributes to the premature apoptosis of cerebellar granule neuron in weaver mutant mice: In vitro analysis

Evidence from transgenic mice and cultured cerebellar neurons supports an important role for insulin‐like growth factor I (IGF‐I) in the formation of cerebellar cytoarchitecture. To understand IGF‐Is function during cerebellar development, we examined the involvement of IGF‐I in the premature apoptosis of granule neurons derived from the cerebella of weaver (wv) mutant mice. Before their demise, wv granule neurons increased the expression and secretion of IGFBP5 in a gene dose‐dependent manner. Because IGFBP5 may interfere with the interaction of IGF‐I and its receptor, the abnormally high IGFBP5 levels in wv granule neurons suggest that a lack of IGF‐I activation may contribute to their premature apoptosis. This hypothesis is supported by a gene dose‐dependent decrease in IGF‐I receptor (IGF‐IR) phosphorylation. More importantly, there is a parallel gene dose‐dependent decrease in Akt activity, which was inversely correlated with the activity levels of caspase 3. On the other hand, adding IGFBP5 antibody into culture media increased the survival of wv granule neurons, whereas adding IGFBP5 decreased the survival of wild‐type granule neurons. To delineate the interaction between IGF‐I and IGFBP5 on wv granule neurons, we examined neuronal survival after treating with IGF‐I, des(1–3) IGF‐I, or IGFBP5 antibody. At the same concentration, des(1–3) IGF‐I was more effective than IGF‐I in promoting survival, in increasing Akt activity, and in decreasing caspase 3 activity. These results indicate that IGF‐Is actions on wv granule neurons are normally inhibited by excess IGFBP5, and sufficient IGF‐I receptor activation rescues wv granule neurons via stimulating the Akt signaling pathway.

Altered IGFBP5 gene expression in the cerebellar external germinal layer of weaver mutant mice

The IGF system components play important roles in cerebellar development as demonstrated by their specific spatial-temporal expression. IGF-I, type I IGF receptor (IGFR-I), IGFBP2 and IGFBP5 mRNA are localized in distinct cell populations, and all are expressed at the highest levels at the peak of Purkinje cell growth, active synaptogenesis and dendritic formation. To understand IGF-Is action at the cellular level, in situ hybridization was employed to investigate the distribution of IGF system gene transcripts in the cerebellum of weaver mutant mice (wv/wv). Although located ectopically, the surviving Purkinje cells express IGF-I mRNA at the same level in wv/wv as in +/+. No alteration in the cellular distribution or mRNA levels was observed with IGFBP2, or IGFR-I mRNAs. However, the pattern of IGFBP5 expression is altered in the external germinal layer of wv/wv mice. Not only is IGFBP5 expressed by more granule cell precursors of wv/wv cerebellum, but its mRNA level is 2.3 fold that of +/+. The altered IGFBP5 gene expression in granule cell precursors may modulate the interaction of IGF-I with IGFR-I in ways that contribute to their massive death occurring in the development of wv/wv cerebellum.

Lack of spontaneous ocular neovascularization and attenuated laser-induced choroidal neovascularization in IGF-I overexpression transgenic mice

Robust IGF-I overexpression induces ocular angiogenesis in mice. To investigate the effect of subtle IGF-I overexpression, we examined the ocular phenotype of IGF-II promoter-driven IGF-I transgenic mice. Despite 2.5-fold elevation of IGF-I mRNA in the retina and 29 and 52% increase of IGF-I protein in the retina and aqueous humor, respectively, no ocular abnormality was observed in these transgenics. This was correlated with unaltered VEGF mRNA levels in the transgenic retina. The transgene was also associated with an attenuated laser-induced choroidal neovascularization. Differential expression levels and pattern of IGF-I gene may underlie the different retinal phenotypes in different transgenic lines.