In-depth correlation analysis between tear glucose and blood glucose using a wireless smart contact lens.

Tears have emerged as a promising alternative to blood for diagnosing diabetes. Despite increasing attempts to measure tear glucose using smart contact lenses, the controversy surrounding the correlation between tear glucose and blood glucose still limits the clinical usage of tears. Herein, we present an in-depth investigation of the correlation between tear glucose and blood glucose using a wireless and soft smart contact lens for continuous monitoring of tear glucose. This smart contact lens is capable of quantitatively monitoring the tear glucose levels in basal tears excluding the effect of reflex tears which might weaken the relationship with blood glucose. Furthermore, this smart contact lens can provide an unprecedented level of continuous tear glucose data acquisition at sub-minute intervals. These advantages allow the precise estimation of lag time, enabling the establishment of the concept called 'personalized lag time'. This demonstration considers individual differences and is successfully applied to both non-diabetic and diabetic humans, as well as in animal models, resulting in a high correlation.

Power-integrated, wireless neural recording systems on the cranium using a direct printing method for deep-brain analysis.

Conventional power-integrated wireless neural recording devices suffer from bulky, rigid batteries in head-mounted configurations, hindering the precise interpretation of the subject's natural behaviors. These power sources also pose risks of material leakage and overheating. We present the direct printing of a power-integrated wireless neural recording system that seamlessly conforms to the cranium. A quasi-solid-state Zn-ion microbattery was 3D-printed as a built-in power source geometrically synchronized to the shape of a mouse skull. Soft deep-brain neural probes, interconnections, and auxiliary electronics were also printed using liquid metals on the cranium with high resolutions. In vivo studies using mice demonstrated the reliability and biocompatibility of this wireless neural recording system, enabling the monitoring of neural activities across extensive brain regions without notable heat generation. This all-printed neural interface system revolutionizes brain research, providing bio-conformable, customizable configurations for improved data quality and naturalistic experimentation.

In-vivo integration of soft neural probes through high-resolution printing of liquid electronics on the cranium.

Current soft neural probes are still operated by bulky, rigid electronics mounted to a body, which deteriorate the integrity of the device to biological systems and restrict the free behavior of a subject. We report a soft, conformable neural interface system that can monitor the single-unit activities of neurons with long-term stability. The system implements soft neural probes in the brain, and their subsidiary electronics which are directly printed on the cranial surface. The high-resolution printing of liquid metals forms soft neural probes with a cellular-scale diameter and adaptable lengths. Also, the printing of liquid metal-based circuits and interconnections along the curvature of the cranium enables the conformal integration of electronics to the body, and the cranial circuit delivers neural signals to a smartphone wirelessly. In the in-vivo studies using mice, the system demonstrates long-term recording (33 weeks) of neural activities in arbitrary brain regions. In T-maze behavioral tests, the system shows the behavior-induced activation of neurons in multiple brain regions.

Real-time in vivo monitoring of intraocular pressure distribution in the anterior chamber and vitreous chamber for diagnosis of glaucoma.

Glaucoma causes irreversible vision loss due to optic nerve damage and retinal cell degeneration. Since high intraocular pressure (IOP) is a major risk factor for glaucoma development, accurate IOP measurement is crucial, especially intravitreal IOP affecting the optical nerve and cells. However, conventional methods have limits in selectively and directly detecting local retina pressure. Here, we present continuous measurements of local IOP values in the anterior chamber and vitreous chamber of living animals using minimally invasive probes with pressure-sensitive transistors. After inducing glaucoma in animal models, we compared the local IOP distribution between normal and glaucomatous eyes. We also compared IOP values detected in the cornea using tonometry measurements. Our findings revealed that glaucoma induced higher IOP in the vitreous chamber than in the anterior chamber, indicating that measuring IOP in the vitreous chamber is key to the glaucoma model. This progress offers future directions for diagnosis and treatment of glaucoma.

Smart Contact Lenses as Wearable Ophthalmic Devices for Disease Monitoring and Health Management.

The eye contains a complex network of physiological information and biomarkers for monitoring disease and managing health, and ocular devices can be used to effectively perform point-of-care diagnosis and disease management. This comprehensive review describes the target biomarkers and various diseases, including ophthalmic diseases, metabolic diseases, and neurological diseases, based on the physiological and anatomical background of the eye. This review also includes the recent technologies utilized in eye-wearable medical devices and the latest trends in wearable ophthalmic devices, specifically smart contact lenses for the purpose of disease management. After introducing other ocular devices such as the retinal prosthesis, we further discuss the current challenges and potential possibilities of smart contact lenses.

Human MicroRNAs Attenuate the Expression of Immediate Early Proteins and HCMV Replication during Lytic and Latent Infection in Connection with Enhancement of Phosphorylated RelA/p65 (Serine 536) That Binds to MIEP.

Attenuating the expression of immediate early (IE) proteins is essential for controlling the lytic replication of human cytomegalovirus (HCMV). The human microRNAs (hsa-miRs), miR-200b-3p and miR-200c-3p, have been identified to bind the 3'-untranslated region (3'-UTR) of the mRNA encoding IE proteins. However, whether hsa-miRs can reduce IE72 expression and HCMV viral load or exhibit a crosstalk with the host cellular signaling machinery, most importantly the NF-κB cascade, has not been evaluated. In this study, argonaute-crosslinking and immunoprecipitation-seq revealed that miR-200b-3p and miR-200c-3p bind the 3'-UTR of UL123, which is a gene that encodes IE72. The binding of these miRNAs to the 3'-UTR of UL123 was verified in transfected cells stably expressing GFP. We used miR-200b-3p/miR-200c-3p mimics to counteract the downregulation of these miRNA after acute HCMV infection. This resulted in reduced IE72/IE86 expression and HCMV VL during lytic infection. We determined that IE72/IE86 alone can inhibit the phosphorylation of RelA/p65 at the Ser536 residue and that p-Ser536 RelA/p65 binds to the major IE promoter/enhancer (MIEP). The upregulation of miR-200b-3p and miR-200c-3p resulted in the phosphorylation of RelA/p65 at Ser536 through the downregulation of IE, and the binding of the resultant p-Ser536 RelA/p65 to MIEP resulted in a decreased production of pro-inflammatory cytokines. Overall, miR-200b-3p and miR-200c-3p-together with p-Ser536 RelA/p65-can prevent lytic HCMV replication during acute and latent infection.

De Novo Genotypic Heterogeneity in the UL56 Region in Cytomegalovirus-Infected Tissues: Implications for Primary Letermovir Resistance.

BACKGROUND: Letermovir, an inhibitor of unique long (UL)56-encoded cytomegalovirus (CMV)-terminase, shows prophylactic effects with low-grade adverse events in hematopoietic stem cell transplant recipients. Despite few case reports on acquired letermovir resistance, the frequency of de novo amino acid (A.A.) changes encoded by UL56 in CMV-infected tissues is unclear. METHODS: We analyzed CMV UL56 sequences between the conserved region IV and variable region I in 175 formalin-fixed, paraffin-embedded tissues obtained from 147 patients showing positive CMV immunochemical staining between November 2012 and October 2016. Nucleotides 552-1330 of the open reading frame of UL56 were amplified with 5 primers and sequenced by a dideoxy fluorescence-based cycle. RESULTS: Six (3.4%) tissues from 4 (2.7%) patients harbored A.A. substitutions. There were no known potent resistant mutations. However, we found C325Y in 2 tissues from 1 patient, along with other mutations. Four novel A.A. changes, which have not been observed in previous in vitro experiments, were identified (T244I, S301T, G312V, and M434I). Most (9 of 11, 81.8%) of the A.A. changes occurred between the codons 301 and 325 present between the conserved regions V and VI. CONCLUSIONS: The treatment difficulties associated with letermovir resistance in a clinical setting need to be verified before its widespread use.

Expression of human miR-200b-3p and -200c-3p in cytomegalovirus-infected tissues.

Human cytomegalovirus (HCMV) infection can cause inflammatory tissue-invasive end-organ diseases upon lytic replication. In humans, mature miR-200b-3p and -200c-3p suppress the synthesis of HCMV immediate early 2 (IE2) protein by binding to the 3'-UTR of the mRNA encoded by the unique long (UL) 122-123 region in human foreskin fibroblasts and pre-transplant peripheral blood mononuclear cells stimulated with HCMV. The present study aimed to quantitate the expression of Homo sapiens (hsa)-miR-200b-3p and 200c-3p in HCMV-infected tissues. We collected 240 HCMV-infected and 154 HCMV-non-infected, formalin-fixed, paraffin-embedded tissue samples of the gastrointestinal (GI) tract and bronchi/lungs. MiRNAs, HCMV, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantitated by quantitative reverse transcription-PCR (qRT-PCR) and quantitative PCR (qPCR) on the basis of standard curves generated using miRNA mimics, the HCMV strain from National Institute for Biological Standards and Control (NIBSC) 09/162, and GAPDH control. To avoid the effect of cell counts on the qRT-PCR and qPCR results, the data were normalized to GAPDH levels. HCMV-infected tissues had significantly lower levels of 200b-3p/GAPDH (3.03 ± 1.50 compared with 3.98 ± 1.08 log10 copies/µl, P<0.001) and 200c-3p/GAPDH (4.67 ± 1.84 compared with 6.35 ± 1.47 log10 copies/µl, P<0.001) than normal tissues. The values for 200b-3p/GAPDH (r = -0.51, P<0.001) and 200c-3p/GAPDH (r = -0.54, P<0.001) were significantly inversely correlated with HCMV load. Low tissue levels of 200b-3p and 200c-3p in humans are associated with cytopathic inflammation due to HCMV infection.

Moutan Cortex Radicis inhibits the nigrostriatal damage in a 6-OHDA-induced Parkinson's disease model.

The traditionally used oriental herbal medicine Moutan Cortex Radicis [MCR; Paeonia Suffruticosa Andrews (Paeoniaceae)] exerts anti-inflammatory, anti-spasmodic, and analgesic effects. In the present study, we investigated the therapeutic effects of differently fractioned MCR extracts in a 6-hydroxydopamine (OHDA)-induced Parkinson's disease model and neuro-blastoma B65 cells. Ethanol-extracted MCR was fractionated by n-hexane, butanol, and distilled water. Adult Sprague-Dawley rats were treated first with 20 µg of 6-OHDA, followed by three MCR extract fractions (100 or 200 mg·kg-1) for 14 consecutive days. In the behavioral rotation experiment, the MCR extract-treated groups showed significantly decreased number of net turns compared with the 6-OHDA control group. The three fractions also significantly inhibited the reduction in tyrosine hydroxylase-positive cells in the substantia nigra pars compacta following 6-OHDA neurotoxicity. Western blotting analysis revealed significantly reduced tyrosine hydroxylase expression in the substantia nigra pars compacta in the 6-OHDA-treated group, which was significantly inhibited by the n-hexane or distilled water fractions of MCR. B65 cells were exposed to the extract fractions for 24 h prior to addition of 6-OHDA for 30 min; treatment with n-hexane or distilled water fractions of MCR reduced apoptotic cell death induced by 6-OHDA neurotoxicity and inhibited nitric oxide production and neuronal nitric oxide synthase expression. These results showed that n-hexane- and distilled water-fractioned MCR extracts inhibited 6-OHDA-induced neurotoxicity by suppressing nitric oxide production and neuronal nitric oxide synthase activity, suggesting that MCR extracts could serve as a novel candidate treatment for the patients with Parkinson's disease.

Decreased expression of serum- and glucocorticoid-inducible kinase 1 (SGK1) promotes alpha-synuclein increase related with down-regulation of dopaminergic cell in the Substantia Nigra of chronic MPTP-induced Parkinsonism mice and in SH-SY5Y cells.

Parkinson's disease (PD) is a chronically progressive neurodegenerative disease, with its main pathological hallmarks being a dramatic loss of dopaminergic neurons predominantly in the Substantia Nigra (SN), and the formations of intracytoplasmic Lewy bodies and dystrophic neurites. Alpha-synuclein (α-syn), widely recognized as the most prominent element of the Lewy body, is one of the representative hallmarks in PD. However, the mechanisms behind the increased α-syn expression and aggregation have not yet been clarified. To examine what causes α-syn expression to increase, we analyzed the pattern of gene expression in the SN of mice intoxicated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), where down-regulation of dopaminergic cells occurred. We identified serum- and glucocorticoid-dependent kinase 1 (SGK1) as one of the genes that is evidently downregulated in chronic MPTP-intoxication. The results of Western blot analyses showed that, together with the down-regulation of dopaminergic cells, the decrease in SGK1 expression increased α-syn expression in the SN in a chronic MPTP-induced Parkinsonism mouse. For an examination of the expression correlation between SGK1 and α-syn, SH-5YSY cells were knocked down with SGK1 siRNA then, the downregulation of dopaminergic cells and the increase in the expression of α-syn were observed. These results suggest that decreased expression of SGK1 may play a critical role in increasing the expression of α-syn, which is related with dopaminergic cell death in the SN of chronic MPTP-induced Parkinsonism mice and in SH-SY5Y cells.

The Short Isoform of DNAJB6 Protects against 1-Methyl-4-phenylpridinium Ion-Induced Apoptosis in LN18 Cells via Inhibiting Both ROS Formation and Mitochondrial Membrane Potential Loss.

In a previous study, we found that the short isoform of DNAJB6 (DNAJB6(S)) had been decreased in the striatum of a mouse model of Parkinson's disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). DNAJB6, one of the heat shock proteins, has been implicated in the pathogenesis of PD. In this study, we explored the cytoprotective effect of DNAJB6(S) against 1-methyl-4-phenylpyridinium ion- (MPP+-) induced apoptosis and the underlying molecular mechanisms in cultured LN18 cells from astrocytic tumors. We observed that MPP+ significantly reduced the cell viability and induced apoptosis in LN18 glioblastoma cells. DNAJB6(S) protected LN18 cells against MPP+-induced apoptosis not only by suppressing Bax cleavage but also by inhibiting a series of apoptotic events including loss of mitochondrial membrane potential, increase in intracellular reactive oxygen species, and activation of caspase-9. These observations suggest that the cytoprotective effects of DNAJB6(S) may be mediated, at least in part, by the mitochondrial pathway of apoptosis.

Neuroprotective changes in degeneration-related gene expression in the substantia nigra following acupuncture in an MPTP mouse model of Parkinsonism: Microarray analysis.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of dopamine-generating cells in the substantia nigra (SN). Acupuncture stimulation results in an enhanced survival of dopaminergic neurons in the SN in Parkinsonism animal models. The present study investigated changes in gene expression profiles measured using whole transcript array in the SN region related to the inhibitory effects of acupuncture in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonism model. In this model, acupuncture stimulation at GB34 and LR3 attenuated the decrease in tyrosine hydroxylase in the SN region; stimulation at non-acupoints did not suppress this decrease. Gene array analysis revealed that 22 (10 annotated genes: Cdh1, Itih2, Mpzl2, Rdh9, Serping1, Slc6a13, Slc6a20a, Slc6a4, Tph2, and Ucma) probes that were up-regulated in MPTP animals relative to controls were exclusively down-regulated by acupuncture stimulation. In addition, 17 (two annotated genes: 4921530L21Rik and Gm13931) probes that were down-regulated in MPTP animals compared to controls were exclusively up-regulated by acupuncture stimulation. These findings indicate that the 39 probes (12 annotated genes) affected by MPTP and acupuncture may be responsible for the inhibitory effects of acupuncture on degeneration-related gene expression in the SN following damage induced by MPTP intoxication.

Neuroprotective changes of thalamic degeneration-related gene expression by acupuncture in an MPTP mouse model of parkinsonism: microarray analysis.

Acupuncture stimulations at GB34 and LR3 inhibit the reduction of tyrosine hydroxylase in the nigrostriatal dopaminergic neurons in the parkinsonism animal models. Especially, behavioral tests showed that acupuncture stimulations improved the motor dysfunction in a previous study by almost 87.7%. The thalamus is a crucial area for the motor circuit and has been identified as one of the most markedly damaged areas in Parkinson's disease (PD), so acupuncture stimulations might also have an effect on the thalamic damage. In this study, gene expression changes following acupuncture at the acupoints were investigated in the thalamus of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism model using a whole transcript array. It was confirmed that acupuncture at these acupoints could inhibit the decrease of tyrosine hydroxylase in the thalamic regions of the MPTP model, while acupuncture at the non-acupoints could not suppress this decrease by its level shown in the acupoints. GeneChip gene array analysis showed that 18 (5 annotated genes: Dnase1l2, Dusp4, Mafg, Ndph and Pgm5) of the probes down-regulated in MPTP, as compared to the control, were exclusively up-regulated by acupuncture at the acupoints, but not at the non-acupoints. In addition, 14 (3 annotated genes; Serinc2, Sp2 and Ucp2) of the probes up-regulated in MPTP, as compared to the control, were exclusively down-regulated by acupuncture at the acupoints, but not at the non-acupoints. The expression levels of the representative genes in the microarray were validated by real-time RT-PCR. These results suggest that the 32 probes (8 annotated genes) which are affected by MPTP and acupuncture may be responsible for exerting the inhibitory effect of acupuncture in the thalamus which can be damaged by MPTP intoxication.

Changes of gene expression profiles in the cervical spinal cord by acupuncture in an MPTP-intoxicated mouse model: microarray analysis.

It has been shown that acupuncture at acupoints GB34 and LR3 inhibits the degeneration of nigrostriatal neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. The degeneration of spinal cord was reported to be induced in the MPTP-treated pre-symptomatic mouse. In this study, the gene expression profile changes following acupuncture at the acupoints were investigated in the cervical spinal cord of an MPTP-induced parkinsonism model using a whole transcript array (Affymetrix GeneChip mouse gene 1.0 ST array). It was shown that 8 of the probes up-regulated in MPTP, as compared to the control, were down-regulated after acupuncture at the acupoints. Of these 8 probes, 6 probes (4 annotated genes in 6 probes: Ctla2a, EG383229, Ppbp and Ube2l6) were exclusively down-regulated by acupuncture at the specific acupoints except for 2 probes as these 2 probes were commonly down-regulated by acupuncture at both the acupoints and the non-acupoints. In addition, 11 of the probes down-regulated in MPTP, as compared to the control, were up-regulated by acupuncture at the acupoints. Of these 11 probes, 10 probes (5 annotated genes in 10 probes: EG665033, ENSMUSG00000055323, Obox6, Pbp2 and Tmem150) were exclusively up-regulated by acupuncture at the specific acupoints except for the Fut11 because the Fut11 was commonly up-regulated by acupuncture at both the acupoints and the non-acupoints. The expression levels of the representative genes in the microarray were validated by real-time RT-PCR. These data suggest that the expression of these exclusively regulated 16 probes (9 genes) may be, at least in part, affected by acupuncture at the acupoints in the cervical spinal cord which can be damaged by MPTP intoxication.

Neuroprotective changes of striatal degeneration-related gene expression by acupuncture in an MPTP mouse model of Parkinsonism: microarray analysis.

Acupuncture at acupoints GB34 and LR3 has been reported to inhibit nigrostriatal degeneration in Parkinsonism models, yet the genes related to this preventive effect of acupuncture on the nigrostriatal dopaminergic system remain elusive. This study investigated gene expression profile changes in the striatal region of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism models after acupuncture at the acupoints GB34 and LR3 using a whole transcript genechip microarray (Affymetrix genechip mouse gene 1.0 ST array). It was confirmed that acupuncture at these acupoints could inhibit the decrease of tyrosine hydroxylase and dopamine transporter in the nigrostriatal region of the MPTP model while acupuncture at the non-acupoints could not counteract this decrease. Genechip gene array analysis (fold change cutoff 1.3 and P < 0.05) showed that 12 of the 69 probes up-regulated in MPTP when compared to the control were down-regulated by acupuncture at the acupoints. Of these 12 probes, 11 probes (nine annotated genes) were exclusively down-regulated by acupuncture only at the acupoints; the Gfral gene was excluded because it was commonly down-regulated by acupuncture at both the acupoints and the non-acupoints. In addition, 28 of the 189 probes down-regulated in MPTP when compared to the control were up-regulated by acupuncture at the acupoints. Of these 28 probes, 19 probes (seven annotated genes) were exclusively up-regulated by acupuncture only at the acupoints while nine probes were commonly up-regulated by acupuncture at both the acupoints and the non-acupoints. The regulation patterns of representative genes in real-time RT-PCR correlated with those of the genes in the microarray. These results suggest that the 30 probes (16 annotated genes), which are affected by MPTP and acupuncture only at the acupoints, are responsible for exerting in the striatal regions the inhibitory effect of acupuncture at the acupoints on MPTP-induced striatal degeneration.

Microarray analysis of gene expression profile by treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cells.

It has been reported that components of Cinnamomi Ramulus (CR) demonstrate an anti-inflammatory effect by inhibiting the expression of inducible nitric oxide synthesis (iNOS) and cyclooxygenase-2 (COX-2) and by suppressing nitric oxide (NO) production in the central nervous system (CNS) as well as in the periphery. In this study, microarray analysis was performed to investigate the effect of CR on the gene expression and associated pathways of lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Microglia plays an important role in the processes of several inflammation-mediated neurodegenerative diseases. Activated microglia can produce various pro-inflammatory cytokines, chemokines, and toxic mediators, which may initiate or amplify the inflammatory responses in the CNS. In the present study, the negative control group was cultured in normal medium, the positive control group was activated with 1 microg/ml of LPS, and the CR group was previously treated with 10 microg/ml of CR before LPS stimulation. With the cutoff value of 1.5-fold change in the expression, 341 genes including pro-inflammatory cytokines, chemokines, and transcription factors were found to be up-regulated in LPS-stimulated BV-2 cells (Supplemental Table 2). CR reduced the LPS-induced up-regulation of such inflammatory genes as Ccl5, Cd80, Cxcl10, Grin2a, Ifi203, Ifit1, Il1alpha, Il6, Lilrb3, Nos2 (iNOS), Rab2b, Rsad2 and Vpreb1. This resulted in a full list of 38 and 37 annotated genes whose expression is up- and down-regulated by CR respectively (Supplemental Table 3). RT-PCR analysis showed that the expression of LPS-induced TNF-alpha, IL-1beta, IL-6, and iNOS mRNAs were attenuated in the presence of the CR extract. The results imply that CR has the anti-inflammatory effect of down-regulating the expression of various genes related to inflammatory responses in LPS-stimulated BV-2 cells, and that CR could be a candidate for the prevention or therapeutic treatment of inflammation-mediated neurodegenerative diseases.

Inhibition of nNOS and COX-2 expression by lutein in acute retinal ischemia.

OBJECTIVE: Lutein is a major nutrient in the retina. Lutein has an antioxidative effect and protects against macular degeneration and retinal degenerative disease. However, the mechanism of lutein is not clear in retinal degeneration, and a role for lutein is not known in ischemic injury. In this study, an ischemia-induced rat retina was examined to determine the effect of the lutein on ischemic retinal cell death. METHODS: We used a transient ischemia model of high intraocular pressure in the rat. Lutein (Kemin Foods, LC) was injected into the intraperitoneal or intravitreous before ischemia. Retinal degeneration was observed by light microscopy 24 h after ischemia. Expressions of neuronal nitric oxide synthase (nNOS) and cyclo-oxygenase-2 (COX-2) were detected by western blot analysis at various times after retinal ischemia. RESULTS: The nNOS and COX-2 expression levels were increased early in ischemic control retinas, but these increases were inhibited by lutein. In addition, the inhibitory effect of lutein was observed to be dose dependent. Further, ischemia-induced cell death was inhibited by lutein. Lutein-injected ischemic retina appeared similar to normal retina. CONCLUSION: This study investigated the protective effect of lutein on retinal ischemia and the inhibitory effect of nNOS and COX-2 expressions. These results suggest that a supplement with lutein may have the potential to inhibit ischemic cell death by this mechanism in the neural retina.

Calcium binding of ARC mediates regulation of caspase 8 and cell death.

Apoptosis repressor with CARD (ARC) possesses the ability not only to block activation of caspase 8 but to modulate caspase-independent mitochondrial events associated with cell death. However, it is not known how ARC modulates both caspase-dependent and caspase-independent cell death. Here, we report that ARC is a Ca(2+)-dependent regulator of caspase 8 and cell death. We found that in Ca(2+) overlay and Stains-all assays, ARC protein bound to Ca(2+) through the C-terminal proline/glutamate-rich (P/E-rich) domain. ARC expression reduced not only cytosolic Ca(2+) transients but also cytotoxic effects of thapsigargin, A23187, and ionomycin, for which the Ca(2+)-binding domain of ARC was indispensable. Conversely, direct interference of endogenous ARC synthesis by targeting ARC enhanced such Ca(2+)-mediated cell death. In addition, binding and immunoprecipitation analyses revealed that the protein-protein interaction between ARC and caspase 8 was decreased by the increase of Ca(2+) concentration in vitro and by the treatment of HEK293 cells with thapsigargin in vivo. Caspase 8 activation was also required for the thapsigargin-induced cell death and suppressed by the ectopic expression of ARC. These results suggest that calcium binding mediates regulation of caspase 8 and cell death by ARC.

Induced inhibition of ischemic/hypoxic injury by APIP, a novel Apaf-1-interacting protein.

We describe the isolation and characterization of a new apaf-1-interacting protein (APIP) as a negative regulator of ischemic injury. APIP is highly expressed in skeletal muscle and heart and binds to the CARD of Apaf-1 in competition with caspase-9. Exogenous APIP inhibits cytochrome c-induced activation of caspase-3 and caspase-9, and suppresses cell death triggered by mitochondrial apoptotic stimuli through inhibiting the downstream activity of cytochrome c released from mitochondria. Conversely, reduction of APIP expression potentiates mitochondrial apoptosis. APIP expression is highly induced in mouse muscle affected by ischemia produced by interruption of the artery in the hindlimb and in C2C12 myotubes created by hypoxia in vitro, and the blockade of APIP up-regulation results in TUNEL-positive ischemic damage. Furthermore, forced expression of APIP suppresses ischemia/hypoxia-induced death of skeletal muscle cells. Taken together, these results suggest that APIP functions to inhibit muscle ischemic damage by binding to Apaf-1 in the Apaf-1/caspase-9 apoptosis pathway.

Induction of pro-apoptotic calsenilin/DREAM/KChIP3 in Alzheimer's disease and cultured neurons after amyloid-beta exposure.

Calsenilin/DREAM/KChIP3 was identified as a calcium-binding protein that interacts with presenilins, serves as a transcription repressor, and binds to the A-type potassium channel. In this study, we hypothesized that calsenilin might be involved in the neurodegeneration of Alzheimer's disease and examined calsenilin expression in Alzheimer's disease. Calsenilin levels were elevated in the cortex region of Alzheimer's patient brains and in the neocortex and the hippocampus of Swedish mutant beta-amyloid precursor protein transgenic mice brains. Induction of calsenilin was also observed in the activated astroglia as well as in the neurons surrounding beta-amyloid (Abeta)- and Congo red-positive plaques. Exposing cultured cortical and hippocampal neurons to Abeta42, an amyloid-beta peptide whose deposition in the brain is a characteristic of Alzheimer's disease, induced both calsenilin protein and mRNA expression, and cell death. Moreover, blocking the calsenilin expression protected the neuronal cells from Abeta toxicity. These findings suggest that chronic up-regulation of calsenilin may be a risk factor for developing Alzheimer's disease, perhaps by facilitating calsenilin-mediated neurodegeneration.