Expression of glycosylated α-dystroglycan in newborn skeletal and cardiac muscles of fukutin related protein (FKRP) mutant mice.

INTRODUCTION: Mutations in the Fukutin related protein (FKRP) gene are characterized by a lack of functionally glycosylated α-dystroglycan (F-α-DG) in muscles. A small number of fibers retain the capacity to produce strong IIH6 reactive glycosylated-α-DG (g-α-DG) in muscles of both FKRP mutant animals and patients. METHODS: We examined the expression of g-α-DG in limb, diaphragm, and cardiac muscles of newborn FKRP mutants and LARGEmyd mice with IIH6 antibody. RESULTS: Near-normal levels of g-α-DG were detected in all 3 muscles in the FKRP448LNeo- mutant. Expression was limited within the first 8 postnatal days with decreasing levels. No expression was identified in LARGEmyd mice. CONCLUSIONS: Temporary expression of glycosylated-α-DG in newborn FKRP mutant muscles is LARGE- and mutant FKRP-dependent. The capability of mutant FKRP with a severe clinic phenotype to produce glycosylated-α-DG provides a new perspective for possible approaches to mitigate FKRP deficiency. Muscle Nerve 55: 582-590, 2017.

Restoration of Functional Glycosylation of α-Dystroglycan in FKRP Mutant Mice Is Associated with Muscle Regeneration.

Mutations in fukutin-related protein (FKRP) gene are characterized with lack of functionally glycosylated α-dystroglycan (F-α-DG). Surprisingly, a few muscle fibers express strong F-α-DG. Herein, we investigated the restoration of F-α-DG in the FKRP mutant muscles and showed that the restoration of glycosylation is associated with muscle regeneration and dependent on the expression of both like-glycosyltransferase (LARGE) and partially functional FKRP. F-α-DG in the regenerating fibers reaches up to normal levels and lasts for >4 weeks, but no up-regulation of the LARGE and FKRP is detected during the regeneration process. The FKRP protein with P448L mutation is sufficient for functional glycosylation of α-DG in regenerating fibers, but not in mature fibers. Thus, factors other than FKRP enable regenerating fibers to produce functional α-DG, compensating for the defect in FKRP function. Identification of factors other than LARGE and FKRP could generate new approaches for restoration of F-α-DG in mature muscle fibers with defects in FKRP functions.

Adeno-associated virus-mediated overexpression of LARGE rescues α-dystroglycan function in dystrophic mice with mutations in the fukutin-related protein.

Multiple genes (e.g., POMT1, POMT2, POMGnT1, ISPD, GTDC2, B3GALNT2, FKTN, FKRP, and LARGE) are known to be involved in the glycosylation pathway of α-dystroglycan (α-DG). Mutations of these genes result in muscular dystrophies with wide phenotypic variability. Abnormal glycosylation of α-DG with decreased extracellular ligand binding activity is a common biochemical feature of these genetic diseases. While it is known that LARGE overexpression can compensate for defects in a few aforementioned genes, it is unclear whether it can also rescue defects in FKRP function. We examined adeno-associated virus (AAV)-mediated LARGE or FKRP overexpression in two dystrophic mouse models with loss-of-function mutations: (1) Large(myd) (LARGE gene) and (2) FKRP(P448L) (FKRP gene). The results agree with previous findings that overexpression of LARGE can ameliorate the dystrophic phenotypes of Large(myd) mice. In addition, LARGE overexpression in the FKRP(P448L) mice effectively generated functional glycosylation (hyperglycosylation) of α-DG and improved dystrophic pathologies in treated muscles. Conversely, FKRP transgene overexpression failed to rescue the defect in glycosylation and improve the phenotypes of the Large(myd) mice. Our findings suggest that AAV-mediated LARGE gene therapy may still be a viable therapeutic strategy for dystroglycanopathies with FKRP deficiency.

Adeno-associated virus 9 mediated FKRP gene therapy restores functional glycosylation of α-dystroglycan and improves muscle functions.

Mutations in the FKRP gene are associated with a wide range of muscular dystrophies from mild limb-girdle muscular dystrophy (LGMD) 2I to severe Walker-Warburg syndrome and muscle-eye-brain disease. The characteristic biochemical feature of these diseases is the hypoglycosylation of α-dystroglycan (α-DG). Currently there is no effective treatment available. In this study, we examined the adeno-associated virus serotype 9 vector (AAV9)-mediated gene therapy in the FKRP mutant mouse model with a proline to leucine missense mutation (P448L). Our results showed that intraperitoneal administration of AAV9-FKRP resulted in systemic FKRP expression in all striated muscles examined with the highest levels in cardiac muscle. Consistent with our previous observations, FKRP protein is localized in the Golgi apparatus in myofibers. Expression of FKRP consequently restored functional glycosylation of α-DG in the skeletal and cardiac muscles. Significant improvement in dystrophic pathology, serum creatine kinase levels and muscle function was observed. Only limited FKRP transgene expression was detected in kidney and liver with no detectable toxicity. Our results provided evidence for the utility of AAV-mediated gene replacement therapy for FKRP-related muscular dystrophies.

Mouse models of fukutin-related protein mutations show a wide range of disease phenotypes.

Dystroglycanopathies are characterized by a reduction in the glycosylation of alpha-dystroglycan (α-DG). A common cause for this subset of muscular dystrophies is mutations in the gene of fukutin-related protein (FKRP). FKRP mutations have been associated with a wide spectrum of clinical severity from severe Walker-Warburg syndrome and muscle-eye-brain disease with brain and eye defects to mild limb-girdle muscular dystrophy 2I with myopathy only. To examine the affects of FKRP mutations on the severity of the disease, we have generated homozygous and compound heterozygous mouse models with human mutations in the murine FKRP gene. P448Lneo+ and E310delneo+ mutations result in severe dystrophic and embryonic lethal phenotypes, respectively. P448Lneo+/E310delneo+ compound heterozygotes exhibit brain defects and severe muscular dystrophies with near absence of α-DG glycosylation. Removal of the Neo(r) cassette from the P448Lneo+ homozygous mice eliminates overt brain and eye defects, and reduces severity of dystrophic phenotypes. Furthermore, introduction of the common L276I mutation to generate transgenic L276Ineo+ homozygous and L276Ineo+/P448Lneo+ and L276Ineo+/E310delneo+ compound heterozygotes results in mice displaying milder dystrophies with reduced α-DG glycosylation and no apparent brain defects. Limited sampling and variation in functionally glycosylated α-DG levels between and within muscles may explain the difficulties in correlating FKRP expression levels with phenotype in clinics. The nature of individual mutations, expression levels and status of muscle differentiation all contribute to the phenotypic manifestation. These mutant FKRP mice are useful models for the study of disease mechanism(s) and experimental therapies.

Required roles of Bax and JNKs in central and peripheral nervous system death of retinoblastoma-deficient mice.

Retinoblastoma-deficient mice show massive neuronal damage and deficits in both CNS and PNS tissue. Previous work in the field has shown that death is regulated through distinct processes where CNS tissue undergoes death regulated by the tumor suppressor p53 and the apoptosome component, APAF1. Death in the PNS, however, is independent of p53 and reliant on the death protease, caspase 3. In the present study, we more carefully delineated the common and distinct mechanisms of death regulation by examining the stress-activated kinases, JNK2 and 3, the conserved Bcl-2 member Bax, and the relationship among these elements including p53. By use of genetic modeling, we show that death in various regions of the CNS and DRGs of the PNS is reliant on Bax. In the CNS, Bax acts downstream of p53. The relevance of the JNKs is more complex, however. Surprisingly, JNK3 deficiency by itself does not inhibit c-Jun phosphorylation and instead, aggravates death in both CNS and PNS tissue. However, JNK2/3 double deficiency blocks death due to Rb loss in both the PNS and CNS. Importantly, the relationships between JNKs, p53, and Bax exhibit regional differences. In the medulla region of the hindbrain in the CNS, JNK2/3 deficiency blocks p53 activation. Moreover, Bax deficiency does not affect c-Jun phosphorylation. This indicates that a JNK-p53-Bax pathway is central in the hindbrain. However, in the diencephalon regions of the forebrain (thalamus), Bax deficiency blocks c-Jun activation, indicating that a Bax-JNK pathway of death is more relevant. In the DRGs of the PNS, a third pathway is present. In this case, a JNK-Bax pathway, independent of p53, regulates damage. Accordingly, our results show that a death regulator Bax is common to death in both PNS and CNS tissue. However, it is regulated by or itself regulates different effectors including the JNKs and p53 depending upon the specific region of the nervous system.

c-Jun N-terminal kinase 3 deficiency protects neurons from axotomy-induced death in vivo through mechanisms independent of c-Jun phosphorylation.

Both the transcription factor c-Jun and the c-Jun N-terminal kinases (JNKs) have been associated with neuronal loss in several death paradigms. JNK are key regulators of c-Jun and a common accepted model has been that JNKs mediate neuronal death through modulation of c-Jun activation. In the present study, we examined whether JNK2 and -3 (JNK members most associated with neuronal loss) deficiency can rescue neuronal loss caused by facial and sciatic nerve axotomy in the neonate in vivo. JNK2, JNK3, and JNK2/3 double-deficient neurons displayed significantly less death in the facial nerves of the CNS when compared with controls. JNK2 and JNK2/3 double-deficient animals also showed reduced c-Jun phosphorylation and induction following axotomy, consistent with the model that JNK acts to regulate death by activating c-Jun. Of significance, however, protection of facial nerves in JNK3-deficient animals was not accompanied by reduction in c-Jun activation. These results suggest that JNKs can mediate death independently of c-Jun. Importantly, the lack of correlation between JNK3 deficiency and c-Jun induction was not universal. In a sciatic axotomy model of neuronal injury in the neonate, death of DRG neurons was also reduced by JNK3 deficiency. However, in this case, c-Jun activation was also eliminated.

Ataxia telangiectasia-mutated protein can regulate p53 and neuronal death independent of Chk2 in response to DNA damage.

DNA damage is a key initiator of neuronal death. We have previously shown that the tumor suppressor p53, in conjunction with cyclin-dependent kinases (CDKs), regulates the mitochondrial pathway of death in neurons exposed to genotoxic agents. However, the mechanisms by which p53 is regulated is unclear. Presently, we show that p53 is phosphorylated on Ser-15 following DNA damage and this occurs independently of the CDK pathway. Instead, we show that p53 phosphorylation, stability, as well as neuronal death is regulated, in part, by the ataxia telangiectasia-mutated (ATM) protein. Previous reports have suggested that ATM regulation of p53 occurs through Chk2. However, in our present paradigms, we show that ATM functions separately from Chk2 to regulate p53 stability and neuronal death. Chk2 deficiency does not affect p53 stability or neuronal death induced by Topoisomerase I or II inhibition. Taken together, our results provide a model by which DNA damage can activate an ATM-dependent, Chk2-independent pathway of p53-mediated neuronal death.

Calpains mediate p53 activation and neuronal death evoked by DNA damage.

DNA damage is an initiator of neuronal death implicated in neuropathological conditions such as stroke. Previous evidence has shown that apoptotic death of embryonic cortical neurons treated with the DNA damaging agent camptothecin is dependent upon the tumor suppressor p53, an upstream death mediator, and more distal death effectors such as caspases. We show here that the calcium-regulated cysteine proteases, calpains, are activated during DNA damage induced by camptothecin treatment. Moreover, calpain deficiency, calpastatin expression, or pharmacological calpain inhibitors prevent the death of embryonic cortical neurons, indicating the important role of calpain in DNA damage-induced death. Calpain inhibition also significantly reduced and delayed the induction of p53. Consistent with the actions of calpains upstream of p53 and the proximal nature of p53 death signaling, calpain inhibition inhibited cytochrome c release and DEVD-AFC cleavage activity. Taken together, our results indicate that calpains are a key mediator of p53 induction and consequent caspase-dependent neuronal death due to DNA damage.

Interaction of the c-Jun/JNK pathway and cyclin-dependent kinases in death of embryonic cortical neurons evoked by DNA damage.

DNA damage, an important initiator of neuronal death, has been implicated in numerous neurodegenerative conditions. We previously delineated several pathways that control embryonic cortical neuronal death evoked by the DNA-damaging agent, camptothecin. In this model, the tumor suppressor p53 and cyclin-dependent kinases (CDKs) are activated independently and cooperate to mediate the conserved death pathway. To further our understanding, we presently examined whether the c-Jun/JNK pathway modulates death and whether this pathway is regulated by CDKs, p53, and Bax. We show that c-Jun/JNK is activated following DNA damage. Moreover, the c-Jun pathway is one mediator of death, because expression of dominant negative c-Jun and cdc42, and JNK pathway inhibitors are neuroprotective. Although previous evidences indicate that JNK3 is required for neuronal death under certain conditions, we show that JNK3 deficiency only partially mediates c-Jun phosphorylation and its deficiency does not protect neurons from death. Interestingly, we provide evidence that CDK activity regulates c-Jun but does not affect upstream pathways that lead to JNK phosphorylation. Finally, c-Jun activation is independent of p53 and Bax. Accordingly, we propose that c-Jun is regulated by the JNK and CDK pathways and that both must be activated for efficient c-Jun activation to occur.