Eva Andres-Mateos

DJ-1 gene deletion reveals that DJ-1 is an atypical peroxiredoxin-like peroxidase

Parkinsons disease (PD) is a common neurodegenerative movement disorder. Whereas the majority of PD cases are sporadic, rare genetic defects have been linked to this prevalent movement disorder. Mutations in DJ-1 are associated with autosomal recessive early-onset PD. The exact biochemical function of DJ-1 has remained elusive. Here we report the generation of DJ-1 knockout (KO) mice by targeted deletion of exon 2 and exon 3. There is no observable degeneration of the central dopaminergic pathways, and the mice are anatomically and behaviorally similar to WT mice. Fluorescent Amplex red measurements of H2O2 indicate that isolated mitochondria from young and old DJ-1 KO mice have a 2-fold increase in H2O2. DJ-1 KO mice of 2–3 months of age have a 60% reduction in mitochondrial aconitase activity without compromising other mitochondrial processes. At an early age there are no differences in antioxidant enzymes, but in older mice there is an up-regulation of mitochondrial manganese superoxide dismutase and glutathione peroxidase and a 2-fold increase in mitochondrial glutathione peroxidase activity. Mutational analysis and mass spectrometry reveal that DJ-1 is an atypical peroxiredoxin-like peroxidase that scavenges H2O2 through oxidation of Cys-106. In vivo there is an increase of DJ-1 oxidized at Cys-106 after 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine intoxication of WT mice. Taken together these data indicate that the DJ-1 KO mice have a deficit in scavenging mitochondrial H2O2 due to the physiological function of DJ-1 as an atypical peroxiredoxin-like peroxidase.

Losartan restores skeletal muscle remodeling and protects against disuse atrophy in sarcopenia.

Losartan improves muscle remodeling and protects against immobilization atrophy by mediating pathways critical for muscle homeostasis. Losartan Comes of Age The Little Old Lady from Pasadena describes a diminutive woman of advanced years who aggressively drives her Dodge around a southern California city. In popular culture, people link long life spans with being “little”; yet, shortened stature is only one physical change associated with aging. Another, less jocular, transformation is loss of muscle mass and strength—called sarcopenia—which can cause disability and predicts impending death in older adults. Burks et al. now identify losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, as a new drug candidate for treating sarcopenia. Although the causes of sarcopenia are poorly understood, transforming growth factor–β (TGF-β) may contribute to faulty repair in aged muscle. Burks et al. used losartan to antagonize TGF-β signaling in an aged mouse model of sarcopenia. Losartan treatment improved muscle remodeling after injury and protected sarcopenic muscle from further loss of muscle mass caused by immobilization; these effects were mediated via two signaling circuits critical for skeletal muscle homeostasis: the TGF-β and insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathways. These observations suggest that treatment with losartan, a Food and Drug Administration (FDA)–approved drug, may benefit sarcopenia patients and allow little old ladies everywhere to continue their street racing for many years to come. Go granny go. Sarcopenia, a critical loss of muscle mass and function because of the physiological process of aging, contributes to disability and mortality in older adults. It increases the incidence of pathologic fractures, causing prolonged periods of hospitalization and rehabilitation. The molecular mechanisms underlying sarcopenia are poorly understood, but recent evidence suggests that increased transforming growth factor–β (TGF-β) signaling contributes to impaired satellite cell function and muscle repair in aged skeletal muscle. We therefore evaluated whether antagonism of TGF-β signaling via losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, had a beneficial impact on the muscle remodeling process of sarcopenic mice. We demonstrated that mice treated with losartan developed significantly less fibrosis and exhibited improved in vivo muscle function after cardiotoxin-induced injury. We found that losartan not only blunted the canonical TGF-β signaling cascade but also modulated the noncanonical TGF-β mitogen-activated protein kinase pathway. We next assessed whether losartan was able to combat disuse atrophy in aged mice that were subjected to hindlimb immobilization. We showed that immobilized mice treated with losartan were protected against loss of muscle mass. Unexpectedly, this protective mechanism was not mediated by TGF-β signaling but was due to an increased activation of the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. Thus, blockade of the AT1 (angiotensin II type I) receptor improved muscle remodeling and protected against disuse atrophy by differentially regulating the TGF-β and IGF-1/Akt/mTOR signaling cascades, two pathways critical for skeletal muscle homeostasis. Thus, losartan, a Food and Drug Administration–approved drug, may prove to have clinical benefits to combat injury-related muscle remodeling and provide protection against disuse atrophy in humans with sarcopenia.

Unexpected Lack of Hypersensitivity in LRRK2 Knock-Out Mice to MPTP (1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine)

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known cause of Parkinsons disease (PD). Whether loss of LRRK2 function accounts for neurodegeneration of dopamine neurons in PD is not known, nor is it known whether LRRK2 kinase activity modulates the susceptibility of dopamine (DA) neurons to the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To better understand the role of LRRK2 in DA neuronal survival and its role in the susceptibility of DA neurons to MPTP, we generated LRRK2 knock-out (KO) mice lacking the kinase domain of LRRK2. Here, we show that LRRK2 KO mice are viable and have no major abnormalities and live to adulthood. The dopaminergic system is normal in LRRK2 KO mice as assessed via HPLC for DA and its metabolites and via stereologic assessment of DA neuron number in young and aged mice. Importantly, there is no significant difference in the susceptibility of LRRK2 KO and wild-type mice to MPTP. These results suggest that LRRK2 plays little if any role in the development and survival of DA neurons under physiologic conditions. Thus, PD due to LRRK2 mutations are likely not due to a loss of function. Moreover, LRRK2 is not required for the susceptibility of DA neurons to MPTP.

In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

Activation of serum/glucocorticoid-induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy

Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non‐hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non‐hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy.

Hibernating squirrel muscle activates the endurance exercise pathway despite prolonged immobilization

Skeletal muscle atrophy is a very common clinical challenge in many disuse conditions. Maintenance of muscle mass is crucial to combat debilitating functional consequences evoked from these clinical conditions. In contrast, hibernation represents a physiological state in which there is natural protection against disuse atrophy despite prolonged periods of immobilization and lack of nutrient intake. Even though peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1-α (PGC-1α) is a central mediator in muscle remodeling pathways, its role in the preservation of skeletal muscle mass during hibernation remains unclear. Since PGC-1α regulates muscle fiber type formation and mitochondrial biogenesis, we analyzed muscles of 13-lined ground squirrels. We find that animals in torpor exhibit a shift to slow-twitch Type I muscle fibers. This switch is accompanied by activation of the PGC-1α-mediated endurance exercise pathway. In addition, we observe increased antioxidant capacity without evidence of oxidative stress, a marked decline in apoptotic susceptibility, and enhanced mitochondrial abundance and metabolism. These results show that activation of the endurance exercise pathway can be achieved in vivo despite prolonged periods of immobilization, and therefore might be an important mechanism for skeletal muscle preservation during hibernation. This PGC-1α regulated pathway may be a potential therapeutic target promoting skeletal muscle homeostasis and oxidative balance to prevent muscle loss in a variety of inherited and acquired neuromuscular disease conditions.

Loss of sarcolemmal nNOS is common in acquired and inherited neuromuscular disorders

Objective: Neuronal nitric oxide synthase (nNOS), normally expressed at the sarcolemmal membrane, is known to be mislocalized to the sarcoplasm in several forms of muscular dystrophy. Our objectives were to characterize further the range of patients manifesting aberrant nNOS sarcolemmal immunolocalization and to study nNOS localization in animal models of nondystrophic myopathy. Methods: We carried out a retrospective cross-sectional study. We performed immunofluorescent staining for nNOS on biopsy specimens from 161 patients with acquired and nondystrophin inherited neuromuscular conditions. The localization of sarcolemmal nNOS correlated with mobility and functional status. Muscle specimens from mouse models of steroid-induced and starvation-related atrophy were studied for qualitative and quantitative nNOS expression. Results: Sarcolemmal nNOS staining was abnormal in 42% of patients with inherited myopathic conditions, 25% with acquired myopathic conditions, 57% with neurogenic conditions, and 93% with hypotonia. Interestingly, we found significant associations between mobility status or muscle function and sarcolemmal nNOS expression. Furthermore, mouse models of catabolic stress also demonstrated mislocalization of sarcolemmal nNOS. Conclusion: Our analyses indicate that nNOS mislocalization is observed in a broad range of nondystrophic neuromuscular conditions associated with impaired mobility status and catabolic stress. Our findings suggest that the assessment of sarcolemmal localization of nNOS represents an important tool for the evaluation of muscle biopsies of patients with a variety of inherited and acquired forms of neuromuscular disorders.

Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.

Impaired Skeletal Muscle Regeneration in the Absence of Fibrosis during Hibernation in 13-Lined Ground Squirrels

Skeletal muscle atrophy can occur as a consequence of immobilization and/or starvation in the majority of vertebrates studied. In contrast, hibernating mammals are protected against the loss of muscle mass despite long periods of inactivity and lack of food intake. Resident muscle-specific stem cells (satellite cells) are known to be activated by muscle injury and their activation contributes to the regeneration of muscle, but whether satellite cells play a role in hibernation is unknown. In the hibernating 13-lined ground squirrel we show that muscles ablated of satellite cells were still protected against atrophy, demonstrating that satellite cells are not involved in the maintenance of skeletal muscle during hibernation. Additionally, hibernating skeletal muscle showed extremely slow regeneration in response to injury, due to repression of satellite cell activation and myoblast differentiation caused by a fine-tuned interplay of p21, myostatin, MAPK, and Wnt signaling pathways. Interestingly, despite long periods of inflammation and lack of efficient regeneration, injured skeletal muscle from hibernating animals did not develop fibrosis and was capable of complete recovery when animals emerged naturally from hibernation. We propose that hibernating squirrels represent a new model system that permits evaluation of impaired skeletal muscle remodeling in the absence of formation of tissue fibrosis.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.