ATF4 regulates arsenic trioxide-mediated NADPH oxidase, ER-mitochondrial crosstalk and apoptosis

Arsenic is a mitochondrial toxin, and its derivatives, such as arsenic trioxide (ATO), can trigger endoplasmic reticulum (ER) and the associated unfolded protein response (UPR). Here, we show that arsenic induction of the UPR triggers ATF4, which is involved in regulating this ER-mitochondrial crosstalk that is important for the molecular pathogenesis of arsenic toxicity. Employing ATF4+/+ and ATF4−/− MEFs, we show that ATO induces UPR and impairs mitochondrial integrity in ATF4+/+ MEF cells which is largely ablated upon loss of ATF4. Following ATO treatment, ATF4 activates NADPH oxidase by promoting assembly of the enzyme components Rac-1/P47phox/P67phox, which generates ROS/superoxides. Furthermore, ATF4 is required for triggering Ca++/calpain/caspase-12-mediated apoptosis following ATO treatment. The IP3R inhibitor attenuates Ca++/calpain-dependent apoptosis, as well as reduces m-ROS and MMP disruption, suggesting that ER-mitochondria crosstalk involves IP3R-regulated Ca++ signaling. Blockade of m-Ca++ entry by inhibiting m-VDAC reduces ATO-mediated UPR in ATF4+/+ cells. Additionally, ATO treatment leads to p53-regulated mitochondrial apoptosis, where p53 phosphorylation plays a key role. Together, these findings indicate that ATO-mediated apoptosis is regulated by both ER and mitochondria events that are facilitated by ATF4 and the UPR. Thus, we describe novel mechanisms by which ATO orchestrates cytotoxic responses involving interplay of ER and mitochondria.

GADD34 Ablation Exacerbates Retinal Degeneration in P23H RHO Mice

The UPR is sustainably activated in degenerating retinas, leading to translational inhibition via p-eIF2α. Recent findings have demonstrated that ablation of growth arrest and DNA damage-inducible protein 34 (GADD34), a protein phosphatase 1 regulatory subunit permitting translational machinery operation through p-eIF2α elevation, does not impact the rate of translation in fast-degenerating rd16 mice. The current study aimed to validate whether P23H RHO mice degenerating at a slower pace manifest translational attenuation and whether GADD34 ablation impacts the rate of retinal degeneration via further suppression of retinal protein synthesis and apoptotic cell death. For this study, mice were examined with ERG and histological analyses. The molecular assessment was conducted in the naïve and LPS-challenged mice using Western blot and qRT-PCR analyses. Thus, this study demonstrates that the P23H RHO retinas manifest translational attenuation. However, GADD34 ablation resulted in a more prominent p-eIF2a increase without impacting the translation rate. GADD34 deficiency also led to a reduction in scotopic ERG amplitudes and an increased number of TUNEL-positive cells. Molecular analysis revealed that GADD34 deficiency reduces the expression of p-STAT3 and Il-6 while increasing the expression of Tnfa. Overall, the data indicate that GADD34 plays a multifunctional role. Under chronic UPR activation, GADD34 acts as a feedback player, dephosphorylating p-eIF2a, although this role does not seem to be critical. Additionally, GADD34 controls cytokine expression and STAT3 activation. Perhaps these molecular events are particularly important in controlling the pace of retinal degeneration

The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons.

Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the endoplasmic reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work, we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose-regulated protein 78 (GRP78) could be related to the development of Parkinson's disease. We first determined that old (24 months) rats exhibit significantly lower levels of GRP78 protein in the nigrostriatal system as compared with young (2 months) animals. Then using recombinant adeno-associate virus-mediated gene transfer, we found that GRP78 downregulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α-syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of 9 months protected aging nigral DA neurons in the α-syn-induced rat model of Parkinson's-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration

The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons.

Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the endoplasmic reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work, we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose-regulated protein 78 (GRP78) could be related to the development of Parkinson's disease. We first determined that old (24 months) rats exhibit significantly lower levels of GRP78 protein in the nigrostriatal system as compared with young (2 months) animals. Then using recombinant adeno-associate virus-mediated gene transfer, we found that GRP78 downregulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α-syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of 9 months protected aging nigral DA neurons in the α-syn-induced rat model of Parkinson's-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration

ER stress in retinal degeneration in S334ter Rho rats.

The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12-P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells

Ablation of C/EBP Homologous Protein Does Not Protect T17M <i>RHO</i> Mice from Retinal Degeneration

<div><p></p><p>Despite the proposed link between ablation of the CHOP protein and delay of the onset of ER stress-mediated disorders including diabetes, Alzheimer Disease, and cardiac hypertrophy, the role of CHOP protein in photoreceptor cell death associated with Autosomal Dominant Retinitis Pigmentosa (ADRP) has not been investigated. T17M <i>RHO</i> transgenic mice carry a mutated human rhodopsin transgene, the expression of which in retina leads to protein misfolding, activation of UPR and progressive retinal degeneration. The purpose of this study is to investigate the role of CHOP protein in T17M <i>RHO</i> retina. Wild-type, CHOP−/−, T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/−mice were used in the study. Evaluation of the impact of CHOP ablation was performed using electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), quantitative Real-Time PCR (qRT-PCR) and western blot analysis. Dark-adapted ERG analysis demonstrated that by 1 month, the T17M <i>RHO</i> CHOP−/− mice had a 70% reduction of the a-wave amplitude compared to the T17M <i>RHO</i> mice. The loss of function in T17M <i>RHO</i> CHOP−/− photoreceptors was associated with a 22–24% decline in the thickness of the outer nuclear layer. These mice had significant reduction in the expression of transcription factors, <i>Crx</i> and <i>Nrl</i>, and also in mouse <i>Rho</i>, and human <i>RHO.</i> The reduction was associated with an 8-fold elevation of the UPR marker, p-eIf2α protein and 30% down-regulation of sXbp1 protein. In addition, the histone deacetylase 1 (Hdac1) protein was 2-fold elevated in the T17M <i>RHO</i> CHOP−/− retina. The ablation of CHOP led to a reduction in the expression of photoreceptor-specific transcriptional factors, and both endogenous and exogenous <i>RHO</i> mRNA. Thus, despite its role in promoting apoptosis, CHOP protects rod photoreceptors carrying an ADRP mutation.</p> </div

ER Stress in Retinal Degeneration in S334ter Rho Rats

The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12– P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur i

The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons

Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the Endoplasmic Reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose regulated protein GRP78 (GRP78) could be related to the development of Parkinson’s disease (PD). We first determined that old (24 month) rats exhibit significantly lower levels of GRP78 protein in the nigrastriatal system as compared to young (2 month) animals. Then using recombinant adeno-associate virus (rAAV) mediated gene transfer, we found that GRP78 down-regulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α–syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of nine months protected aging nigral DA neurons in the α–syn-induced rat model of Parkinson’s-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration

CHOP ablation in P30 T17M <b><i>RHO</i></b><b> retinas modulates the expression of Rhodopsin (m-Rho and h-Rho) and photoreceptor-specific transcription factor (Crx and Nrl) genes.</b>

<p>We analyzed 4 groups of animals (N = 6) and found differences in the expression levels of <i>Crx</i>, <i>Nrl</i> and <i>RHO</i> (mRho and T17M <i>RHO</i>). A: mRho and T17M <i>RHO</i> expression was modulated in T17M <i>RHO</i> CHOP−/− retinas. The relative expression of endogenous mouse <i>Rho</i> was 0.9±0.03 in wild-type; 0.9±0.06 in CHOP−/−; 0.4±0.03 in T17M <i>RHO</i>; and 0.02±0.01 in T17M <i>RHO</i> CHOP−/− mice. The differences between wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were statistically significant (<i>P</i><0.001). The 96% reduction of endogenous <i>RHO</i> gene expression, observed in T17M <i>RHO</i> CHOP−/− mice compared to T17M <i>RHO</i> mice was statistically significant (<i>P</i><0.001). No difference in <i>Crx</i> gene expression was observed when the expression of mRho mRNA was compared in wild-type and CHOP retinas. The expression of the human T17M <i>RHO</i> transgene was also modulated in T17M <i>RHO</i> CHOP−/− mice, with a value of 1.1±0.06 in T17M <i>RHO</i> mice vs. 0.04±0.01 in T17M <i>RHO</i> CHOP−/− mice. The observed 96% reduction of transgene expression was statistically significant (<i>P</i><0.0001). B: Modified Expressions of <i>Crx</i> and <i>Nrl</i> in the T17M <i>RHO</i> CHOP−/− retina. The relative <i>Crx</i> gene expression was 1.0±0.059 in wild-type; 1.1±0.1 in CHOP−/−; 0.7±0.03 in T17M <i>RHO;</i> and 0.3±0.02 in T17M <i>RHO</i>. The differences between the wild-type or CHOP−/− and T17M <i>RHO</i> groups were statistically significant (<i>P</i><0.01), as were the differences between wild-type or CHOP−/− and T17M <i>RHO</i> CHOP−/− (<i>P</i><0.001). The 60% reduction of <i>Crx</i> gene expression detected in T17M <i>RHO</i> CHOP−/− retinas compared to T17M <i>RHO</i> was statistically significant (P<0.001). No difference was observed when the wild-type and CHOP−/− retinas were compared. The level of <i>Nrl</i> gene expression was 1.2±0.2 in wild-type; 1.04±0.2 in CHOP−/−; 0.4±0.15 in T17M <i>RHO;</i> and 0.02±0.03 in T17M RHO CHOP−/−. The differences between wild-type and T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> CHOP−/− were significant at the <i>P</i><0.001 level, while those between wild-type and T17M <i>RHO,</i> T17M <i>RHO</i> and CHOP−/−, and T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− were significant at the <i>P</i><0.01 level. The 95% reduction of <i>Nrl</i> gene expression observed in the T17M <i>RHO</i> CHOP−/− compared to the T17M RHO retina was statistically significant (<i>P</i><0.05). No difference in <i>Nrl</i> gene expression was observed when wild-type and CHOP−/− retinas were compared.</p

Ablation of CHOP protein in P30 T17M <b><i>RHO</i></b><b> retinas led to modulation of the PERK and IRE1 pathways of the UPR.</b>

<p>A: We analyzed T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− retinas (N = 4) and found that the expression of phosphorylated eIF2α protein was increased by over 8 fold in T17M <i>RHO</i> CHOP−/− mice (0.5±0.1 a.u.vs. 0.04±0.1 a.u., <i>P</i> = 0.01). The level of spliced Xbp1 in the T17M <i>RHO</i> retina was decreased by 30%, presenting a value of 0.64±0.027 a.u. vs. 0.4±0.03 a.u.in T17M <i>RHO</i> CHOP−/− mice (<i>P</i> = 0.004). B: Representative images of western blots treated with antibodies against Xbp1, peIF2α and β-actin proteins.</p