Shih-Hung Yang

Functional axonal regeneration through astrocytic scar genetically modified to digest chondroitin sulfate proteoglycans.

Axotomized neurons within the damaged CNS are thought to be prevented from functional regeneration by inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors. Here, we provide a transgenic test of the role of CSPGs in limiting regeneration, using the gfap promotor to express a CSPG-degrading enzyme chondroitinase ABC (ChABC) in astrocytes. Corticospinal axons extend within the lesion site, but not caudal to it, after dorsal hemisection in the transgenic mice. The presence of the gfap–ChABC transgene yields no significant improvement in motor function recovery in this model. In contrast, functionally significant sensory axon regeneration is observed after dorsal rhizotomy in transgenic mice. These transgenic studies confirm a local efficacy for reduced CSPG to enhance CNS axon growth after traumatic injury. CSPGs appear to function in a spatially distinct role from myelin inhibitors, implying that combination-based therapy will be especially advantageous for CNS injuries.

Clinical Significance of Posterior Circulation Changes after Revascularization in Patients with Moyamoya Disease

Objective: It has been noted that the posterior circulation serves as an important source of collateral blood supply in moyamoya disease. Since most of the literature has focused on non-operative cases and many symptomatic patients receive surgical revascularization, we evaluated the posterior circulation changes after revascularization and found that progressive posterior cerebral artery (PCA) steno-occlusive changes after revascularization caused cerebral hemodynamic compromise and clinical deterioration in a significant portion of patients. Methods: Twenty-three moyamoya disease patients with ischemic presentation who received revascularization with complete angiography and xenon CT during a minimum of 3 years’ clinical follow-up were enrolled. Revascularization was performed in 38 hemispheres. Pre- and postoperative angiography were reviewed to determine the internal carotid artery (ICA) stage, PCA stage, leptomeningeal collateral (LMC) grade, and Matsushima synangiosis grade. The postoperative regional cerebral blood flow (CBF) and cerebral vascular reserve (CVR) were recorded and correlated with angiographic findings and clinical outcome. Results: Progression of ICA staging was noted in 23 sides (55.2%), and progression of PCA staging was noted in 18 sides (47.4%). Among the 18 cases of PCA stage progression, an associated decrease in LMC grade was noted in 12 sides (66.7%). These changes were associated with decreased regional CBF and CVR, which also explained the recurrent ischemic symptoms in 27.8% of these patients. In contrast, LMC grade increased in 15 (65.2%) sides of patients with ICA progression. Conclusions: Progressive steno-occlusive change in the PCA after revascularization is associated with a reduction in LMC blood flow and cerebral ischemia in moyamoya patients. This phenomenon might cause recurrent ischemic symptoms in 27.8% of patients.

Epidemiology of Moyamoya Disease in Taiwan A Nationwide Population-Based Study

Background and Purpose— Previous studies have shown regional and temporal variations in epidemiological features of moyamoya disease, but population-based studies in regions other than Japan are limited. We investigated the incidence and patients characteristics of moyamoya disease during 12 years in Taiwan using claims databases of a universal health insurance system. Methods— From the inpatient databases of the Taiwan National Health Insurance program, we identified subjects who had an initial hospitalization with moyamoya disease and had been underwent cerebral angiography as incidence cases during 2000 to 2011. The incidence and the patient characteristics were described by age and time periods of the hospitalization. Results— During the 12-year period, 422 patients were identified, representing an annual incidence of 0.15 per 100 000 person-years. Adults exhibited an upward trend in incidence with an incidence rate ratio of 1.74 (95% confidence interval [1.17–2.58]) in years 2010 to 2011 comparing with years 2000 to 2001. However, children had a decreased incidence except a slightly increase in the last 2 years. Compared with patients hospitalized during 2000 to 2005, patients identified during 2006 to 2011 had greater women-to-men ratio (1.7 versus 1.1, P=0.048). Children were more likely to have comorbid epilepsy than were adult patients (25.0% versus 3.4%, P=0.002). Hemorrhagic stroke was rare among pediatric patients but presented more frequently in adults. However, ischemic stroke was more prevalent in both groups. Conclusions— The incidence of moyamoya disease has increased in adults but not in children from 2000 to 2011 in Taiwan. Sex ratio and comorbid conditions differed by age and study period.

Brain stem cavernous malformations

We retrospectively reviewed the clinical experience of 30 patients with brain stem cavernous malformations (BSCM) treated operatively and non-operatively at our hospital between 1983 and 2005 to elucidate the natural history of BSCM and the factors that affect surgical outcome. Inpatient charts, imaging studies, operative records, and follow-up results were evaluated. The average follow up was 48.5 months. Twenty-two patients (73.3%) received surgical extirpation and of these 86.4% improved or stabilized and 13.6% deteriorated with permanent or severe morbidity. There was no mortality. Size, preoperative status, and surgical timing were factors related to surgical outcome. In the non-operative group, 50% of the patients were the same or better, 25% deteriorated, and 25% died. With appropriate patient selection, resection of BSCM can be achieved with acceptable morbidity compared with the ominous natural history of these lesions.

The interaction of estrogen receptor α and caveolin-3 regulates connexin43 phosphorylation in metabolic inhibition-treated rat cardiomyocytes

Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor alpha in cardiomyocytes, we investigate whether the membrane estrogen receptor alpha associates with caveolin-3 and whether this association is linked to the 17beta-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor alpha co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor alpha was not seen following treatment with the cholesterol-depleting agent methyl-beta-cyclodextrin (5mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor alpha. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor alpha, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17beta-estradiol (200 nM). The effect of 17beta-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor alpha docking at caveolae, which provide a unique compartment for conveying 17beta-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.

Role of Thy‐1 in in vivo and in vitro neural development and regeneration of dorsal root ganglionic neurons

We have examined the expression of Thy‐1, an abundant glycosylphosphatidylinositol (GPI)‐anchored glycoprotein, in dorsal root ganglia (DRG) and associated nerve fascicles, during postnatal development and following a nerve crush. The expression levels of Thy‐1 in DRG neurons, dorsal roots, and central processes in spinal cord were rather low at postnatal day 2, and gradually increased as DRG neurons matured. During early development, the expression of Thy‐1 within DRG neurons was low and equally distributed between plasma membrane and cytosol. With maturation, the staining intensities of Thy‐1 in both the plasma membrane and the cytosol of DRG neurons became increased. We also studied Thy‐1 expression in the regeneration of mature DRG neurons following the crush injury of sciatic nerve. Two days after the crush injury, Thy‐1 expression dramatically decreased in the DRG neurons on the lesion side. Between 4 and 7 days after the injury, the expression of Thy‐1 gradually increased and returned to a normal level 1 week after the sciatic nerve crush. The time course of the up‐regulation of Thy‐1 expression during regeneration matched that of the recovery of sensory functions, such as pain withdraw reflex, placing reflex, and the score of Basso–Beattie–Bresnahan Locomotor Rating Scale. Taken together, our results suggest that Thy‐1 expression is developmentally regulated and is closely associated with the functional maturation of DRG neurons during both postnatal development and nerve regeneration. Furthermore, perturbation of Thy‐1 function with anti‐Thy‐1 antibodies promoted neurite outgrowth from primary cultured DRG neurons, again confirming the inhibitory role of Thy‐1 on neurite outgrowth.

Cordycepin induces apoptosis of C6 glioma cells through the adenosine 2A receptor-p53-caspase-7-PARP pathway.

Cordycepin, 3-deoxyadenosine from Cordyceps sinensis, has been shown to exert anti-tumor effects in several cancer cell lines. This study investigated the effect of cordycepin on a rat glioma cell line. Cordycepin caused apoptosis in C6 glioma cells in a time- and concentration-dependent manner, but did not affect the survival of primary cultured rat astrocytes. Cordycepin increased the total protein levels of p53 and phosphorylated p53 in the C6 cells. Levels of cleaved caspase-7 and poly (ADP-ribose) polymerase (PARP), but not cleaved caspase-3, were also increased after cordycepin treatment. Specific inhibitors for p53 and caspases abrogated cordycepin-induced caspase-7 and PARP cleavage, and prevented cordycepin-induced apoptosis. Moreover, siRNA knockdown of p53 blocked cordycepin-induced cleavage of caspase-7 and PARP. Both adenosine 2A receptor (A2AR) antagonist and small interference RNA (siRNA) knockdown of A2AR blocked cordycepin-induced apoptosis, p53 activation, and caspase-7 and PARP cleavage. These may provide a new strategy of cordycepin for glioma therapy in the future.

Role of Pka in the Anti-Thy-1 Antibody-Induced Neurite Outgrowth of Dorsal Root Ganglionic Neurons

Thy‐1 is highly expressed in the mammalian nervous system. Our previous study showed that addition of anti‐Thy‐1 antibody to cultured dorsal root ganglionic (DRG) neurons promotes neurite outgrowth. In this study, we identified a novel signaling pathway mediating this event. Treatment with function‐blocking anti‐Thy‐1 antibodies enhanced neurite outgrowth of DRG neurons in terms of total neurite length, longest neurite length, and total neurite branching points. To elucidate the possible signal transduction pathway involved, activation of kinases was evaluated by Western blotting. Transient phosphorylation of protein kinase A (PKA) and mitogen‐activated kinase kinase (MEK) was induced after 15 min of anti‐Thy‐1 antibody treatment. Pretreatment with a PKA inhibitor (PKI) or an MEK inhibitor, PD98059, significantly decreased the neurite outgrowth response triggered by anti‐Thy‐1 antibody, indicating the involvement of both kinases. In addition, anti‐Thy‐1 antibody treatment also induced transient phosphorylation of cyclic AMP‐response element‐binding protein (CREB) and this effect was also blocked by a PKI or PD98059. Furthermore, the fact that PKI abolished anti‐Thy‐1 antibody‐induced MEK phosphorylation showed that PKA acts upstream of the MEK‐CREB cascade. In summary, the PKA‐MEK‐CREB pathway is a new pathway involved in the neurite outgrowth‐promoting effect of anti‐Thy‐1 antibody. J. Cell. Biochem. 101: 566–575, 2007.

Effects of 18-glycyrrhetinic acid on serine 368 phosphorylation of connexin43 in rat neonatal cardiomyocytes.

18β‐Glycyrrhetinic acid (18β‐GA) regulates serine/threonine dephosphorylation of connexin43 (Cx43). Phospho‐specific antibodies were used here to determine the effect of 18β‐GA on serine 368‐phosphorylated Cx43 (pSer368Cx43) in cultured rat neonatal cardiomyocytes by immunofluorescence microscopy and immunoblot analyses. 18β‐GA caused a time‐dependent increase in pSer368Cx43 levels and induced gap junction disassembly, shown by a change in pSer368Cx43 immunostaining from large aggregates to dispersed punctates at cell—cell contact areas. 18β‐GA also induced a time‐dependent increase in the levels of serine 729‐phosphorylated PKCɛ, the active form of PKCɛ. The 18β‐GA‐induced increase in pSer368Cx43 levels and changes in pSer368Cx43 staining pattern were abolished by the PKC inhibitor, chelerythrine. Furthermore, 18β‐GA increased the co‐immunoprecipitation of Cx43 with PKCɛ. However, the 18β‐GA‐induced increase in pSer368Cx43 levels and increased association of Cx43 with PKCɛ were inhibited by co‐treatment with the protein phosphatase type 1 and type 2A inhibitor, calyculin A. We conclude that 18β‐GA induces Ser368 phosphorylation of Cx43 via PKCɛ.

Daidzein induces neuritogenesis in DRG neuronal cultures

AbsractBackgroundDaidzein, a phytoestrogen found in isoflavone, is known to exert neurotrophic and neuroprotective effects on the nervous system. Using primary rat dorsal root ganglion (DRG) neuronal cultures, we have examined the potential neurite outgrowth effect of daidzein.MethodsDissociated dorsal root ganglia (DRG) cultures were used to study the signaling mechanism of daidzein-induced neuritogenesis by immunocytochemistry and Western blotting.ResultsIn response to daidzein treatment, DRG neurons showed a significant increase in total neurite length and in tip number per neuron. The neuritogenic effect of daidzein was significantly hampered by specific blockers for Src, protein kinase C delta (PKCδ) and mitogen-activated protein kinase/extracellular signal-regulated kinase kinases (MEK/ERK), but not by those for estrogen receptor (ER). Moreover, daidzein induced phosphorylation of Src, PKCδ and ERK. The activation of PKCδ by daidzein was attenuated in the presence of a Src kinase inhibitor, and that of ERK by daidzein was diminished in the presence of either a Src or PKCδ inhibitor.ConclusionDaidzein may stimulate neurite outgrowth of DRG neurons depending on Src kinase, PKCδ and ERK signaling pathway.