Parkinson's 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 H 2O2 indicate that isolated mitochondria from young and old DJ-1 KO mice have a 2-fold increase in H 2O2. 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 H 2O2 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 H 2O2 due to the physiological function of DJ-1 as an atypical peroxiredoxin-like peroxidase.glutathione peroxidase ͉ mitochondria ͉ manganese superoxide dismutase ͉ PARK7 ͉ Parkinson's disease P arkinson's disease (PD) is a common progressive neurodegenerative movement disorder (1) caused by the selective loss of dopaminergic neurons in the substantia nigra, pars compacta (2, 3). Although in most cases the etiology of PD is not known, its pathogenesis may involve deficits in mitochondrial function, oxidative stress, excitotoxicity, inflammation, accumulation of aberrant or misfolded proteins (Lewy bodies), and ubiquitin-proteosome system dysfunction (2, 3). PD is essentially a sporadic disorder of the aging brain, but Ϸ10% of all cases are linked to a variety of genetic defects (4, 5). The identification of some of these genes has opened new areas of research (4, 5). In 2003, Bonifati et al. (6) found that loss-of-function mutations in the DJ-1 locus were associated with rare forms of autosomal recessive early-onset parkinsonism with psychiatric and behavioral disturbances, slow progression, and a good response to treatment with levodopa. DJ-1 mutations account for 1-2% of all early-onset PD (7-9), with a number of different pathogenic mutations, including exonic deletions, truncations, and homozygous and heterozygous point mutations.DJ-1 is a highly conserved protein that belongs to the DJ-1/ Thi/PfpI protein superfamily. In vertebrates it is expressed in a variety of tissues including brain (10), and at a subcellular level it is fou...

SUMMARY Adeno-associated viral vectors (AAV) 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 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 AAV, 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 9 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.

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.

related to MMA filed by the NIH on their behalf. Lina Li is an employee of LLN Consultant Inc. and founder of CELiD Biotechnologies Inc. GME is a consultant for Moderna Therapeutics, LogicBio Therapeutics, Horizon Pharma, and Natera; a data-monitoring committee member for BioMarin, Audentes Therapeutics, Amicus, RegenxBio, and NeuroVia; and an investigator on clinical trials for Aeglea, BioElectron, and Stealth Therapeutics. LHV is an inventor of AncAAV and other AAV technologies, which are licensed to various biotechnology and pharmaceutical entities. LHV is a consultant to a number of companies with gene therapy interest, including Selecta Biosciences and Lonza, licensees of AncAAV technology. CPV has received funding to support AAV gene therapy research for MMA from Selecta Biosciences and LogicBio Therapeutics.

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.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known cause of Parkinson's 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. IntroductionProgressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) is one of the main neuropathological findings in Parkinson's disease (PD). Although PD is mostly a sporadic disorder and the etiology is not known, its pathogenesis may involve genetic susceptibility and environmental factors, that will cause oxidative stress, impairments in mitochondria and the proteosome, or aggregation of misfolded proteins (Dawson and Dawson, 2003;Vila and Przedborski, 2004;Moore et al., 2005).Only a 10% of the PD cases are linked to Mendelian genetic defects, and mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most frequent of the familial forms of PD (Gasser, 2007;Lees et al., 2009). Several missense mutations distributed along the protein have been reported, but the role of LRRK2 in the pathophysiology of PD is still not well known. The kinase activity may be a link between LRRK2 and PD West et al., , 2007 as attenuation of LRRK2 kinase activity prevents LRRK2 toxicity in cellular models of PD. LRRK2 also has independent GTPase activity that can regulate its kinase activity (Greggio et al., 2008). It is assumed that LRRK2 mutations linked to PD are gain of function mutations in which the kinase domain is critical for the toxic actions of mutant LRRK2 (Biskup and West, 2009).Previous studies showed that LRRK2 is associated with the mitochondria (Biskup et al., 2006), and that the toxicity mediated by LRRK2 mutants could be due to mitochondria-dependent apoptosis (Iaccarino et al., 2007). Wild-type (WT) LRRK2, but not the mutants, attenuate hydrogen peroxide (H 2 O 2 )-induced oxidative stress suggesting a protective role for LRRK2 (Liou et al., 2008). Moreover, data generated in lines of Caenorhabditis elegans express...

The neuronal ␣7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dup␣7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dup␣7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dup␣7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dup␣7 mRNA into oocytes failed to generate functional receptors, but when co-injected with ␣7 mRNA at ␣7/dup␣7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited ␣7 current generated in control oocytes (␣7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional ␣7 receptors reaching the oocyte membrane, as deduced from ␣-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dup␣7 on ␣7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with ␣7 mRNA, basal dup␣7 mRNA levels are substantial in human cerebral cortex and higher in macrophages.(ii) dup␣7 mRNA levels in macrophages are down-regulated by IL-1␤, LPS, and nicotine. Thus, dup␣7 could modulate ␣7 receptor-mediated synaptic transmission and cholinergic antiinflammatory response.Neuronal ␣7 nicotinic acetylcholine receptors (␣7 nAChRs) 4 are widely expressed in the central and peripheral nervous systems. In neurons, homomeric ␣7 nAChRs, composed of five ␣7 subunits, modulate neurotransmitter release in presynaptic nerve terminals and induce excitatory impulses in postsynaptic neurons (1-4). Signaling through ␣7 nAChRs in the central nervous system has been associated with neuronal plasticity and cell survival (5-7), although impaired activity of this receptor has been implicated in the pathogenesis of schizophrenia, Alzheimer disease, and depression (8 -12). The presence of ␣7 nAChRs has also been reported in non-neuronal cells such as vascular and brain en-

Sepsis-associated encephalopathy (SAE) is a frequent but poorly understood neurological complication in sepsis that negatively influences survival. Here we present clinical and experimental evidence that this brain dysfunction may be related to altered neurotransmission produced by inflammatory mediators. Compared with septic patients, SAE patients had higher interleukin-1␤ (IL-1␤) plasma levels; interestingly, these levels decreased once the encephalopathy was resolved. A putative IL-1␤ effect on type A ␥-aminobutyric acid receptors (GABA A Rs), which mediate fast synaptic transmission in most cerebral inhibitory synapses in mammals, was investigated in cultured hippocampal neurons and in Xenopus oocytes expressing native or foreign rat brain GABA A Rs, respectively. Confocal images in both cell types revealed that IL-1␤ increases recruitment of GABA A Rs to the cell surface. Moreover, brief applications of IL-1␤ to voltage-clamped oocytes yielded a delayed potentiation of the GABA-elicited chloride currents (I GABA ); this effect was suppressed by IL-1ra, the natural IL-1 receptor (IL-1RI) antagonist. Western blot analysis combined with I GABA recording and confocal images of GABA A Rs in oocytes showed that IL-1␤ stimulates the IL-1RI-dependent phosphatidylinositol 3-kinase activation and the consequent facilitation of phospho-Akt-mediated insertion of GABA A Rs into the cell surface. The interruption of this signaling pathway by specific phosphatidylinositol 3-kinase or Akt inhibitors suppresses the cytokine-mediated effects on GABA A R, whereas activation of the conditionally active form of Akt1 (myr-Akt1.ER*) with 4-hydroxytamoxifen reproduces the effects. These findings point to a previously unrecognized signaling pathway that connects IL-1␤ with increased "GABAergic tone." We propose that through this mechanism IL-1␤ might alter synaptic strength at central GABAergic synapses and so contribute to the cognitive dysfunction observed in SAE.