Chung-Jiuan Jeng

Dominant-negative effects of episodic ataxia type 2 mutations involve disruption of membrane trafficking of human P/Q-type Ca2+ channels.

Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder associated with mutations in the gene encoding pore‐forming α1A subunits of human P/Q‐type calcium (CaV2.1) channels. The exact mechanism of how mutant channels cause such clinical EA2 features as cerebellar dysfunctions, however, remains unclear. Our previous functional studies in Xenopus oocytes support the idea that EA2 mutants may exert prominent dominant‐negative effects on wild‐type CaV2.1 channels. To further pursue the mechanism underlying this dominant‐negative effect, we examined the effects of EA2 mutants on the subcellular localization pattern of GFP‐tagged wild‐type CaV2.1 channels in HEK293T cells. In the presence of EA2 mutants, wild‐type channels displayed a significant deficiency in membrane targeting and a concurrent increase in cytoplasm retention. Moreover, the cytoplasmic fraction of wild‐type channels co‐localized with an endoplasmic reticulum (ER) marker, suggesting that a significant amount of wild‐type CaV2.1 channels was trapped in the ER. This EA2 mutant‐induced ER retention pattern was reversed by lowering the cell incubation temperature from 37 to 27°C. We also inspected the effects of untagged EA2 mutants on the functional expression of GFP‐tagged wild‐type CaV2.1 channels in HEK293T cells. Whole‐cell current density of wild‐type channels was diminished in the presence of EA2 mutants, which was also reversed by 27°C incubation. Finally, biochemical analyses indicated that EA2 mutants did not significantly affect the protein expression level of wild‐type channels. Taken together, our data suggest that EA2 mutants induce significant ER retention of their wild‐type counterparts, thereby suppressing the functional expression of CaV2.1 channels. J. Cell. Physiol. 214: 422–433, 2008.

Differential localization of rat Eag1 and Eag2 K+ channels in hippocampal neurons.

Two isoforms of rat ether-à-go-go (Eag) K+ channels, rEag1 and rEag2, are widely expressed in many regions of the brain. The neurophysiological roles of these channels, however, are unclear. We addressed this issue by studying their subcellular localizations in hippocampal neurons. Immunofluorescence studies using markers for different compartments of neurons demonstrated a differential expression pattern of rEag1 and rEag2 K+ channels in the somatodendritic region. Furthermore, rEag1 K+ channels were in close proximity to synaptophysin and densin-180, but not GAD65. Our data suggest that both rEag1 and rEag2 K+ channels may play a pivotal role in the regulation of the excitability of dendrites and somas, and that rEag1 K+ channels may modulate the postsynaptic signaling of glutamatergic synapses.

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.

Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway.

The Roles of CD4

Activation of CD8+ cytotoxic T cells has long been regarded as a major antitumor mechanism of the immune system. Emerging evidence suggests that CD4+ T cells are required for the generation and maintenance of effective CD8+ cytotoxic and memory T cells, a phenomenon known as CD4+ T-cell help. CD4+ T-cell help facilitates the optimal expansion, trafficking, and effector function of CD8+ T cells, thereby enhancing tumor destruction. In addition, a specialized subset of CD4+ T cells, CD4+CD25+ regulatory T cells (TRegs), effectively hampers anti-tumor immune responses, which has been proposed to be one of the major tumor immune evasion mechanisms. Here, we review recent advances in deciphering how anti-tumor immune responses are orchestrated by CD4+ T cells. We will also discuss the immunotherapeutic potential of CD4+ T-cell manipulation in anti-tumor immune response.

Dehydroeburicoic acid induces calcium- and calpain-dependent necrosis in human U87MG glioblastomas.

Dehydroeburicoic acid (DeEA) is a triterpene purified from medicinal fungi such as Antrodia camphorate, the crude extract of which is known to exert cytotoxic effects against several types of cancer cells. We aim to test the hypothesis that DeEA possesses significant cytotoxic effects against glioblastomas, one of the most frequent and malignant brain tumors in adults. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays indicated that DeEA inhibited the proliferation of the human glioblastoma cell U87MG. In addition, Annexin V and propidium iodide staining showed that DeEA treatment led to a rapid increase of glioblastomas in the necrotic/late apoptotic fraction, whereas cell cycle analysis revealed that DeEA failed to significantly enhance the population of U87MG cells in the hypodiploid (sub-G1) fraction. Using electron microscopy, we found that DeEA induced significant cell enlargements, massive cytoplasmic vacuolization, and loss of mitochondrial membrane integrity. DeEA treatment triggered an intracellular Ca(2+) increase, and DeEA-induced cell death was significantly attenuated by BAPTA-AM but not ethylenediaminetetraacetic acid or ethylene glycol tetraacetic acid. DeEA instigated a reduction of both mitochondrial transmembrane potential and intracellular ATP level. Moreover, DeEA induced proteolysis of alpha-spectrin by calpain, and DeEA cytotoxicity in U87MG cells was caspase-independent but was effectively blocked by calpain inhibitor. Interestingly, DeEA also caused autophagic response that was prevented by calpain inhibitor. Taken together, these results suggest that in human glioblastomas, DeEA induces necrotic cell death that involves Ca(2+) overload, mitochondrial dysfunction, and calpain activation.

Location of the C-terminus of titin at the Z-line region in the sarcomere

Limited proteolysis of titin with trypsin yielded a number of polypeptides which were electrophoresed and transferred to a nitrocellulose membrane. Proteolytic removal of the C-terminal residues on the nitrocellulose-bound polypeptides was achieved by using carboxypeptidase Y. The species of the polypeptides left after the digestion was quantified by immunoblotting with two distinct monoclonal anti-titin antibodies A2 and A12 of which the epitopes were located at 0.74 micron and 0.69 micron away from the center of an A-band, respectively. Two polypeptides (266 kd and 84 kd) reactive to both antibodies were identified in the control group. Fifteen minutes after the digestion, the immunoreactivities of A2 on 266 kd and 84 kd polypeptides were disappeared, while those of A12 on these polypeptides were not affected. The results indicate that the C-terminal end of titin is located near the Z-line region and the N-terminal end at the M-line region in the sarcomere.

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.

Differential localization of rat Eag1 and Eag2 potassium channels in the retina.

Despite of their wide expression in the brain, the precise neurophysiological role of rat Eag1 (rEag1) and Eag2 (rEag2) K(+) channels remains elusive. Our previous studies in hippocampal pyramidal neurons demonstrate a somatodendritic localization of rEag1 and rEag2 channels, suggesting that the two channel isoforms may contribute to setting the membrane excitability of somas and dendrites. Here, we aim to further characterize the cellular and subcellular localization patterns of rEag1 and rEag2 proteins by studying their laminar distribution in the retina. Confocal microscopic analyses of immunofluorescence data revealed that rEag1 and rEag2 K(+) channels exhibit distinct cellular expression pattern in the retina. rEag1 immunoreactivity was most prominent in the outer half of the inner plexiform layer, whereas strong rEag2 immunostain was found in the outer and inner segments of photoreceptor cells, the outer plexiform layer, and the inner nuclear layer. These results suggest that rEag1 and rEag2 K(+) channels may play a significant role in the transmission of electrical signals along the retinal neuronal circuits. We also performed double-labeling experiments to demonstrate that rEag1 and rEag2 are predominantly expressed in the somatodendritic compartment of retinal neurons. In addition, we presented evidence suggesting that rEag1 channels may be expressed in the GABAergic amacrine cell. Finally, based on their different immunostaining patterns over the inner region of the retina, we propose that compared to rEag2, rEag1 expression encompasses a significantly broader range of the somatodendritic compartment of the retinal ganglion cell.

The Subfamily-specific Assembly of Eag and Erg K+ Channels Is Determined by Both the Amino and the Carboxyl Recognition Domains

Background: K+ channel subunits from different ether-à-go-go subfamilies cannot form heterotetramers. Results: Reversal of subfamily specificity was only possible when we exchanged both amino and carboxyl termini between two ether-à-go-go subunits. Conclusion: Both recognition domains are required for subfamily-specific assembly. Significance: This is the first evidence suggesting that the amino terminus of ether-à-go-go K+ channels governs subunit interaction specificity. A functional voltage-gated K+ (Kv) channel comprises four pore-forming α-subunits, and only members of the same Kv channel subfamily may co-assemble to form heterotetramers. The ether-à-go-go family of Kv channels (KCNH) encompasses three distinct subfamilies: Eag (Kv10), Erg (Kv11), and Elk (Kv12). Members of different ether-à-go-go subfamilies, such as Eag and Erg, fail to form heterotetramers. Although a short stretch of amino acid sequences in the distal C-terminal section has been implicated in subfamily-specific subunit assembly, it remains unclear whether this region serves as the sole and/or principal subfamily recognition domain for Eag and Erg. Here we aim to ascertain the structural basis underlying the subfamily specificity of ether-à-go-go channels by generating various chimeric constructs between rat Eag1 and human Erg subunits. Biochemical and electrophysiological characterizations of the subunit interaction properties of a series of different chimeric and truncation constructs over the C terminus suggested that the putative C-terminal recognition domain is dispensable for subfamily-specific assembly. Further chimeric analyses over the N terminus revealed that the N-terminal region may also harbor a subfamily recognition domain. Importantly, exchanging either the N-terminal or the C-terminal domain alone led to a virtual loss of the intersubfamily assembly boundary. By contrast, simultaneously swapping both recognition domains resulted in a reversal of subfamily specificity. Our observations are consistent with the notion that both the N-terminal and the C-terminal recognition domains are required to sustain the subfamily-specific assembly of rat Eag1 and human Erg.