Ppp1r3d deficiency preferentially inhibits neuronal and cardiac Lafora body formation in a mouse model of the fatal epilepsy Lafora disease.

Mammalian glycogen chain lengths are subject to complex regulation, including by seven proteins (protein phosphatase-1 regulatory subunit 3, PPP1R3A through PPP1R3G) that target protein phosphatase-1 (PP1) to glycogen to activate the glycogen chain-elongating enzyme glycogen synthase and inactivate the chain-shortening glycogen phosphorylase. Lafora disease is a fatal neurodegenerative epilepsy caused by aggregates of long-chained, and as a result insoluble, glycogen, termed Lafora bodies (LBs). We previously eliminated PPP1R3C from a Lafora disease mouse model and studied the effect on LB formation. In the present work, we eliminate and study the effect of absent PPP1R3D. In the interim, brain cell type levels of all PPP1R3 genes have been published, and brain cell type localization of LBs clarified. Integrating these data we find that PPP1R3C is the major isoform in most tissues including brain. In the brain, PPP1R3C is expressed at 15-fold higher levels than PPP1R3D in astrocytes, the cell type where most LBs form. PPP1R3C deficiency eliminates ~90% of brain LBs. PPP1R3D is quantitatively a minor isoform, but possesses unique MAPK, CaMK2 and 14-3-3 binding domains and appears to have an important functional niche in murine neurons and cardiomyocytes. In neurons, it is expressed equally to PPP1R3C, and its deficiency eliminates ~50% of neuronal LBs. In heart, it is expressed at 25% of PPP1R3C where its deficiency eliminates ~90% of LBs. This work studies the role of a second (PPP1R3D) of seven PP1 subunits that regulate the structure of glycogen, toward better understanding of brain glycogen metabolism generally, and in Lafora disease.

Knockdown of GADD34 in neonatal mutant SOD1 mice ameliorates ALS.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause ~20% of familial ALS (FALS), which comprises 10% of total ALS cases. In mutant SOD1- (mtSOD1-) induced ALS, misfolded aggregates of SOD1 lead to activation of the unfolded protein response/integrated stress response (UPR/ISR). Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a kinase that phosphorylates eukaryotic translation initiator factor 2α (p-eIF2α), coordinates the response by causing a global suppression of protein synthesis. Growth arrest and DNA damage 34 (GADD34) dephosphorylates p-eIF2α, allowing protein synthesis to return to normal. If the UPR/ISR is overwhelmed by the amount of misfolded protein, CCAAT/enhancer-binding homologous protein (CHOP) is activated leading to apoptosis. In the current study we investigated the effect of knocking down CHOP and GADD34 on disease of G93A and G85R mtSOD1 mice. Although a CHOP antisense oligonucleotide had no effect on survival, an intravenous injection of GADD34 shRNA encoded in adeno-associated virus 9 (AAV9) into neonatal G93A as well as neonatal G85R mtSOD1 mice led to a significantly increased survival. G85R mtSOD1 mice had a reduction in SOD1 aggregates/load, astrocytosis, and microgliosis. In contrast, there was no change in disease phenotype when GADD34 shRNA was delivered to older G93A mtSOD1 mice. Our current study shows that GADD34 shRNA is effective in ameliorating disease when administered to neonatal mtSOD1 mice. Targeting the UPR/ISR may be beneficial in mtSOD1-induced ALS as well as other neurodegenerative diseases in which misfolded proteins and ER stress have been implicated.

Rearrangement of the Protein Phosphatase 1 Interactome During Heart Failure Progression.

BACKGROUND: Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis, but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study, we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors. METHODS: Mice were subjected to transverse aortic constriction and grouped on the basis of ejection fraction into sham, hypertrophy, moderate HF (ejection fraction, 30%-40%), and severe HF (ejection fraction <30%). Cardiac lysates were subjected to affinity purification with anti-PP1c antibodies followed by high-resolution mass spectrometry. PP1 regulatory subunit 7 (Ppp1r7) was knocked down in mouse cardiomyocytes and HeLa cells with adeno-associated virus serotype 9 and siRNA, respectively. Calcium imaging was performed on isolated ventricular myocytes. RESULTS: Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome data set ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression, including 2 known (Ppp1r7 and Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum. CONCLUSIONS: PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The 9 key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular "sponge" of PP1c.

Chronic oxidative stress promotes GADD34-mediated phosphorylation of the TAR DNA-binding protein TDP-43, a modification linked to neurodegeneration.

Oxidative and endoplasmic reticulum (ER) stresses are hallmarks of the pathophysiology of ALS and other neurodegenerative diseases. In these stresses, different kinases phosphorylate eukaryotic initiation factor eIF2α, enabling the translation of stress response genes; among these is GADD34, the protein product of which recruits the α-isoform of protein phosphatase 1 catalytic subunit (PP1α) and eIF2α to assemble a phosphatase complex catalyzing eIF2α dephosphorylation and resumption of protein synthesis. Aberrations in this pathway underlie the aforementioned disorders. Previous observations indicating that GADD34 is induced by arsenite, a thiol-directed oxidative stressor, in the absence of eIF2α phosphorylation suggest other roles for GADD34. Here, we report that arsenite-induced oxidative stress differs from thapsigargin- or tunicamycin-induced ER stress in promoting GADD34 transcription and the preferential translation of its mRNA in the absence of eIF2α phosphorylation. Arsenite also stabilized GADD34 protein, slowing its degradation. In response to oxidative stress, but not ER stress, GADD34 recruited TDP-43, and enhanced cytoplasmic distribution and cysteine modifications of TDP-43 promoted its binding to GADD34. Arsenite also recruited a TDP-43 kinase, casein kinase-1ϵ (CK1ϵ), to GADD34. Concomitant with TDP-43 aggregation and proteolysis after prolonged arsenite exposure, GADD34-bound CK1ϵ catalyzed TDP-43 phosphorylations at serines 409/410, which were diminished or absent in GADD34-/- cells. Our findings highlight that the phosphatase regulator, GADD34, also functions as a kinase scaffold in response to chronic oxidative stress and recruits CK1ϵ and oxidized TDP-43 to facilitate its phosphorylation, as seen in TDP-43 proteinopathies.

Oxidative stress promotes SIRT1 recruitment to the GADD34/PP1α complex to activate its deacetylase function.

Phosphorylation of the eukaryotic translation initiation factor, eIF2α, by stress-activated protein kinases and dephosphorylation by the growth arrest and DNA damage-inducible protein (GADD34)-containing phosphatase is a central node in the integrated stress response. Mass spectrometry demonstrated GADD34 acetylation at multiple lysines. Substituting K315 and K322 with alanines or glutamines did not impair GADD34's ability to recruit protein phosphatase 1α (PP1α) or eIF2α, suggesting that GADD34 acetylation did not modulate eIF2α phosphatase activity. Arsenite (Ars)-induced oxidative stress increased cellular GADD34 levels and enhanced Sirtuin 1 (SIRT1) recruitment to assemble a cytoplasmic complex containing GADD34, PP1α, eIF2α and SIRT1. Induction of GADD34 in WT MEFs paralleled the dephosphorylation of eIF2α (phosphoserine-51) and SIRT1 (phosphoserine-47). By comparison, eIF2α and SIRT1 were persistently phosphorylated in Ars-treated GADD34-/- MEFs. Expressing WT GADD34, but not a mutant unable to bind PP1α in GADD34-/- MEFs restored both eIF2α and SIRT1 dephosphorylation. SIRT1 dephosphorylation increased its deacetylase activity, measured in vitro and in cells. Loss of function of GADD34 or SIRT1 enhanced cellular p-eIF2α levels and attenuated cell death following Ars exposure. These results highlighted a novel role for the GADD34/PP1α complex in coordinating the dephosphorylation and reactivation of eIF2α and SIRT1 to determine cell fate following oxidative stress.

Ablation of PPP1R3G reduces glycogen deposition and mitigates high-fat diet induced obesity.

Glycogen and triglyceride are two major forms of energy storage in the body and provide the fuel during different phases of food deprivation. However, how glycogen metabolism is linked to fat deposition in adipose tissue has not been clearly characterized. We generated a mouse model with whole-body deletion of PPP1R3G, a glycogen-targeting subunit of protein phosphatase-1 required for glycogen synthesis. Upon feeding with high-fat diet, the body weight and fat composition are significantly reduced in the PPP1R3G-/- mice compared to the wild type controls. The metabolic rate of the mice as measured by O2 consumption and CO2 production is accelerated by PPP1R3G deletion. The high-fat diet-induced liver steatosis is also slightly relieved by PPP1R3G deletion. The glycogen level in adipose tissue is reduced by PPP1R3G deletion. In 3T3L1 cells, overexpression of PPP1R3G leads to increases of both glycogen and triglyceride levels. In conclusion, our study indicates that glycogen is actively involved in fat accumulation in adipose tissue and obesity development upon high-fat diet. Our study also suggests that PPP1R3G is an important player that links glycogen metabolism to lipid metabolism in vivo.

Dual role of the integrated stress response in medulloblastoma tumorigenesis.

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). Phosphorylated eIF2α is quickly dephosphorylated by the protein phosphatase 1 and growth arrest and DNA damage 34 (GADD34) complex. Data indicate that the ISR can either promote or suppress tumor development. Our previous studies showed that the ISR is activated in medulloblastoma in both human patients and animal models, and that the decreased ISR via PERK heterozygous deficiency attenuates medulloblastoma formation in Patched1 heterozygous deficient (Ptch1+/-) mice by enhancing apoptosis of pre-malignant granule cell precursors (GCPs) during cell transformation. We showed here that GADD34 heterozygous mutation moderately enhanced the ISR and noticeably increased the incidence of medulloblastoma in adult Ptch1+/- mice. Surprisingly, GADD34 homozygous mutation strongly enhanced the ISR, but significantly decreased the incidence of medulloblastoma in adult Ptch1+/- mice. Intriguingly, GADD34 homozygous mutation significantly enhanced pre-malignant GCP apoptosis in cerebellar hyperplastic lesions and reduced the lesion numbers in young Ptch1+/- mice. Nevertheless, neither GADD34 heterozygous mutation nor GADD34 homozygous mutation had a significant effect on medulloblastoma cells in adult Ptch1+/- mice. Collectively, these data imply the dual role of the ISR, promoting and inhibiting, in medulloblastoma tumorigenesis by regulating apoptosis of pre-malignant GCPs during the course of malignant transformation.

Infantile Cirrhosis, Growth Impairment, and Neurodevelopmental Anomalies Associated with Deficiency of PPP1R15B.

OBJECTIVE: To assess the utility of whole-exome sequencing (WES) in a sibling pair with undetermined liver disease and describe the phenotype associated with mutations discovered therein. STUDY DESIGN: Next-generation WES was performed on 2 siblings (S1 and S2) who were born to nonconsanguineous parents of European extraction. Both siblings developed cirrhosis of indeterminate etiology and required liver transplantation; S1 at 7 months and S2 at 22 months. RESULTS: Sequencing of germline DNA identified compound heterozygous mutations in PPP1R15B resulting in increased levels of phosphorylated eukaryotic translation initiation factor 2α. CONCLUSIONS: The first demonstration of PPP1R15B associated with liver disease expands the phenotypic spectrum of PPP1R15B related diseases. Our findings validate the application of WES in the diagnosis of children with undetermined liver disease. Understanding the genetic basis of liver disease may allow the development of targeted therapies for treatment and adequate counseling of families.

GADD34-deficient mice develop obesity, nonalcoholic fatty liver disease, hepatic carcinoma and insulin resistance.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the prevalence of obesity. DNA damage-inducible protein 34 (GADD34/Ppp1r15a), originally isolated from UV-inducible transcripts in Chinese hamster ovary (CHO) cells, dephosphorylates several kinases that function in important signaling cascades, including dephosphorylation of eIF2α. We examined the effects of GADD34 on natural life span by using GADD34-deficient mice. Here we observed for the first time that with age GADD34-deficient mice become obese, developing fatty liver followed by liver cirrhosis, hepatocellular carcinoma, and insulin resistance. We found that myofibroblasts and immune cells infiltrated the portal veins of aged GADD34-deficient mouse livers. A high-fat diet (HFD) induced a higher level of steatosis in young GADD34-deficient mice compared with WT mice. Differentiation into fat is dependent on insulin signaling. Insulin signaling in young GADD34-deficient mice was higher than that in WT mice, which explained the higher fat differentiation of mouse embryonic fibroblasts (MEFs) observed in GADD34-deficient mice. Through aging or a HFD, insulin signaling in GADD34-deficient liver converted to be down regulated compared with WT mice. We found that a HFD or palmitate treatment converted insulin signaling by up-regulating TNF-α and JNK.

Restoring endoplasmic reticulum homeostasis improves functional recovery after spinal cord injury.

The endoplasmic reticulum (ER) stress response (ERSR) is activated to maintain protein homeostasis or induce apoptosis in the ER in response to distinct cellular insults including hypoxia, inflammation, and oxidative damage. Recently, we showed ERSR activation in a mouse model of a contusive spinal cord injury (SCI) and an improved hindlimb locomotor function following SCI when the pro-apoptotic arm of ERSR was genetically inhibited. The objective of the current study was to explore if the pharmacological enhancement of the homeostatic arm of the ERSR pathway can improve the functional outcome after SCI. Salubrinal enhances the homeostatic arm of the ERSR by increasing phosphorylation of eIF2α. Salubrinal significantly enhanced the levels of phosphorylated eIF2α protein and modulated the downstream ERSR effectors assessed at the lesion epicenter 6h post-SCI. Hindlimb locomotion showed significant improvement in animals treated with salubrinal. Treadmill-based-gait assessment showed a significant increase in maximum speed of coordinated walking and a decrease in rear stance time and stride length in salubrinal-treated animals. This improved functional recovery corresponded with increased white matter sparing and decreased oligodendrocyte apoptosis. In addition, salubrinal protected cultured mouse oligodendrocyte progenitor cells against the ER stress-inducing toxin tunicamycin. These data suggest that boosting the homeostatic arm of the ERSR reduces oligodendrocyte loss after traumatic SCI and support the contention that pharmacological targeting of the ERSR after CNS trauma is a therapeutically viable approach.

Significant expression levels of transgenic PPP1CC2 in testis and sperm are required to overcome the male infertility phenotype of Ppp1cc null mice.

PPP1CC2, one of four isoforms of the ser/thr protein phosphatase PP1, is a mammalian-specific splice variant of the Ppp1cc gene, and the only isoform whose expression is confined almost completely to spermatogenic cells. Additionally, PPP1CC2 is the sole isoform found in mammalian spermatozoa. Although PPP1CC1, the other Ppp1cc product, is expressed in many tissues including testis, the only phenotype resulting from deletion of Ppp1cc gene is male infertility. To determine which of the products of Ppp1cc is essential for male fertility, we created two PPP1CC2 transgenes, eTg-G2 and pTg-G2, where Ppp1cc2 expression was driven by the putative endogenous promoter of Ppp1cc or by the testis specific human Pgk2 promoter, respectively. Our results demonstrate that the 2.6-kb genomic region directly upstream of the Ppp1cc structural gene can drive expression of Ppp1cc2, and recapitulate the wild-type tissue specificity of PPP1CC2 in transgenic mice. More importantly, we show that expression of PPP1CC2 alone, via either promoter, is able not only to restore normal spermatogenesis, but the fertility of Ppp1cc null mice as well, provided that transgenic PPP1CC2 expression in testis reaches at least a lower threshold level equivalent to approximately 50% of its expression by a Ppp1cc +/- male. We conclude that the endogenous Ppp1cc promoter normally functions in the testis to maintain a sufficient level of PPP1CC2 expression for normal spermatogenesis to occur, and that production of spermatozoa capable of fertilization in vivo can take place in the complete absence of PPP1CC1 expression.

The catalytic subunit of protein phosphatase 1 gamma regulates thrombin-induced murine platelet alpha(IIb)beta(3) function.

BACKGROUND: Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin alpha(IIb)beta(3). Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with alpha(IIb)beta(3), the role of PP1c in platelet reactivity is unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using gamma isoform of PP1c deficient mice (PP1cgamma(-/-)), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cgamma(-/-) platelets showed decreased alpha(IIb)beta(3) activation despite comparable levels of alpha(IIb)beta(3), PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin alpha(IIb)beta(3) signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cgamma(-/-) platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cgamma(-/-) mice. Phosphorylation of glycogen synthase kinase (GSK3)beta-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cgamma(-/-) platelets by an AKT independent mechanism. Inhibition of GSK3beta partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cgamma(-/-) platelets. CONCLUSIONS/SIGNIFICANCE: These studies illustrate a role for PP1cgamma in maintaining GSK3beta-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.