A focused and efficient genetic screening strategy in the mouse: identification of mutations that disrupt cortical development

Although the mechanisms that regulate development of the cerebral cortex have begun to emerge, in large part through the analysis of mutant mice (Boncinelli et al. 2000; Molnar and Hannan 2000; Walsh and Goffinet 2000), many questions remain unanswered. To provide resources for further dissecting cortical development, we have carried out a focused screen for recessive mutations that disrupt cortical development. One aim of the screen was to identify mutants that disrupt the tangential migration of interneurons into the cortex. At the same time, we also screened for mutations that altered the growth or morphology of the cerebral cortex. We report here the identification of thirteen mutants with defects in aspects of cortical development ranging from the establishment of epithelial polarity to the invasion of thalamocortical axons. Among the collection are three novel alleles of genes for which mutant alleles had already been used to explore forebrain development, and four mutants with defects in interneuron migration. The mutants that we describe here will aid in deciphering the molecules and mechanisms that regulate cortical development. Our results also highlight the utility of focused screens in the mouse, in addition to the large-scale and broadly targeted screens that are being carried out at mutagenesis centers.     

DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation

Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.     

Cetacean morbillivirus: A Land-to-Sea Journey and Back?

<正>Cetacean morbillivirus(CeMV), the most relevant pathogen impacting the health and conservation of several already threatened cetacean populations worldwide(Van Bressem et al. 2014), has shown in recent years an apparently increased tendency to cross ‘‘interspecies barriers' '(Jo et al. 2018 a), thereby giving rise to disease and mortality outbreaks in free-ranging dolphins and whales     

Evaluation of Antibody-Dependent Enhancement of SARS-CoV Infection in Rhesus Macaques Immunized with an Inactivated SARS-CoV Vaccine

正Dear Editor,In 2003,severe acute respiratory syndrome coronavirus(SARS-CoV)emerged in Guangdong Province,China,infected more than 8000 individuals,and resulted in a 10%mortality rate(Rota et al.2003).Later,in 2012,a novel CoV,Middle East respiratory syndrome coronavirus(MERS-CoV),was isolated from the sputum of a man in Saudi Arabia(Perl et al.2014).Notably,MERS-CoV     

On the Centenary of the Spanish Flu: Being Prepared for the Next Pandemic

正Influenza is one of the oldest infectious diseases affecting humans. Every influenza pandemic in history has ended with disastrous outcomes regarding public health and the social economy. This year marks the 100th anniversary of the Spanish flu (H1N1) outbreak of 1918, which is recognized as the most lethal natural event in recent history. In     

Genomic Characterization of the First Parechovirus in Bats

<正>Dear Editor,Parechoviruses (PeVs) are non-enveloped, spherical viruses of genus Parechovirus and family Picornaviridae.Within the capsid is a naked monopartite, linear, singlestranded positive-sense RNA genome of 7.3 kb, comprising a single long open reading frame (ORF) encoding a     

Topology and robustness in the Drosophila segment polarity network

A complex hierarchy of genetic interactions converts a single-celled Drosophila melanogaster egg into a multicellular embryo with 14 segments. Previously, von Dassow et al. reported that a mathematical model of the genetic interactions that defined the polarity of segments (the segment polarity network) was robust (von Dassow et al. 2000). As quantitative information about the system was unavailable, parameters were sampled randomly. A surprisingly large fraction of these parameter sets allowed the model to maintain and elaborate on the segment polarity pattern. This robustness is due to the positive feedback of gene products on their own expression, which induces individual cells in a model segment to adopt different stable expression states (bistability) corresponding to different cell types in the segment polarity pattern. A positive feedback loop will only yield multiple stable states when the parameters that describe it satisfy a particular inequality. By testing which random parameter sets satisfy these inequalities, I show that bistability is necessary to form the segment polarity pattern and serves as a strong predictor of which parameter sets will succeed in forming the pattern. Although the original model was robust to parameter variation, it could not reproduce the observed effects of cell division on the pattern of gene expression. I present a modified version that incorporates recent experimental evidence and does successfully mimic the consequences of cell division. The behavior of this modified model can also be understood in terms of bistability in positive feedback of gene expression. I discuss how this topological property of networks provides robust pattern formation and how large changes in parameters can change the specific pattern produced by a network.     

Neural activity when people solve verbal problems with insight

People sometimes solve problems with a unique process called insight, accompanied by an “Aha!” experience. It has long been unclear whether different cognitive and neural processes lead to insight versus noninsight solutions, or if solutions differ only in subsequent subjective feeling. Recent behavioral studies indicate distinct patterns of performance and suggest differential hemispheric involvement for insight and noninsight solutions. Subjects solved verbal problems, and after each correct solution indicated whether they solved with or without insight. We observed two objective neural correlates of insight. Functional magnetic resonance imaging (Experiment 1) revealed increased activity in the right hemisphere anterior superior temporal gyrus for insight relative to noninsight solutions. The same region was active during initial solving efforts. Scalp electroencephalogram recordings (Experiment 2) revealed a sudden burst of high-frequency (gamma-band) neural activity in the same area beginning 0.3 s prior to insight solutions. This right anterior temporal area is associated with making connections across distantly related information during comprehension. Although all problem solving relies on a largely shared cortical network, the sudden flash of insight occurs when solvers engage distinct neural and cognitive processes that allow them to see connections that previously eluded them.     

Oxidizable Residues Mediating Protein Stability and Cytoprotective Interaction of DJ-1 with Apoptosis Signal-regulating Kinase 1

Parkinson disease (PD)-associated genomic deletions and the destabilizing L166P point mutation lead to loss of the cytoprotective DJ-1 protein. The effects of other PD-associated point mutations are less clear. Here we demonstrate that the M26I mutation reduces DJ-1 expression, particularly in a null background (knockout mouse embryonic fibroblasts). Thus, homozygous M26I mutation causes loss of DJ-1 protein. To determine the cellular consequences, we measured suppression of apoptosis signal-regulating kinase 1 (ASK1) and cytotoxicity for [M26I]DJ-1, and systematically all other DJ-1 methionine and cysteine mutants. C106A mutation of the central redox site specifically abolished binding to ASK1 and the cytoprotective activity of DJ-1. DJ-1 was apparently recruited into the ASK1 signalosome via Cys-106-linked mixed disulfides. The designed higher order oxidation mimicking [C106DD]DJ-1 non-covalently bound to ASK1 even in the absence of hydrogen peroxide and conferred partial cytoprotection. Interestingly, mutations of peripheral redox sites (C46A and C53A) and M26I also led to constitutive ASK1 binding. Cytoprotective [wt]DJ-1 bound to the ASK1 N terminus (which is known to bind another negative regulator, thioredoxin 1), whereas [M26I]DJ-1 bound to aberrant C-terminal site(s). Consequently, the peripheral cysteine mutants retained cytoprotective activity, whereas the PD-associated mutant [M26I]DJ-1 failed to suppress ASK1 activity and nuclear export of the death domain-associated protein Daxx and did not promote cytoprotection. Thus, cytoprotective binding of DJ-1 to ASK1 depends on the central redox-sensitive Cys-106 and may be modulated by peripheral cysteine residues. We suggest that impairments in oxidative conformation changes of DJ-1 might contribute to PD neurodegeneration.Loss-of-function mutations in the DJ-1 gene (PARK7) cause autosomal-recessive hereditary Parkinson disease (PD)² (1). The most dramatic PD-associated mutation L166P impairs DJ-1 dimer formation and dramatically destabilizes the protein (2–7). Other mutations such as M26I (8) and E64D (9) have more subtle defects with unclear cellular consequences (4, 7, 10, 11). In addition to this genetic association, DJ-1 is neuropathologically linked to PD. DJ-1 is up-regulated in reactive astrocytes, and it is oxidatively modified in brains of sporadic PD patients (12–14).DJ-1 protects against oxidative stress and mitochondrial toxins in cell culture (15–17) as well as in diverse animal models (18–21). The cytoprotective effects of DJ-1 may be stimulated by oxidation and mediated by molecular chaperoning (22, 23), and/or facilitation of the pro-survival Akt and suppression of apoptosis signal-regulating kinase 1 (ASK1) pathways (6, 24, 25). The cytoprotective activity of DJ-1 against oxidative stress depends on its cysteine residues (15, 17, 26). Among the three cysteine residues of DJ-1, the most prominent one is the easiest oxidizable Cys-106 (27) that is in a constrained conformation (28), but the other cysteine residues Cys-46 and Cys-53 have been implicated with DJ-1 activity as well (22). However, the molecular basis of oxidation-mediated cytoprotective activity of DJ-1 is not clear. Moreover, the roles of PD-mutated and in vivo oxidized methionines are not known.Here we have mutagenized all oxidizable residues within DJ-1 and studied the effects on protein stability and function. The PD-associated mutation M26I within the DJ-1 dimer interface selectively reduced protein expression as well as ASK1 suppression and cytoprotective activity in oxidatively stressed cells. These cell culture results support a pathogenic effect of the clinical M26I mutation (8). Furthermore, oxidation-defective C106A mutation abolished binding to ASK1 and cytoprotective activity of DJ-1, whereas the designed higher order oxidation mimicking mutant [C106DD]DJ-1 bound to ASK1 even in the absence of H₂O₂ and conferred partial cytoprotection. The peripheral cysteine mutants [C46A]DJ-1 and [C53A]DJ-1 were also cytoprotective and were incorporated into the ASK1 signalosome even in the basal state. Thus, DJ-1 may be activated by a complex mechanism, which depends on the redox center Cys-106 and is modulated by the peripheral cysteine residues. Impairments of oxidative DJ-1 activation might contribute to the pathogenesis of PD.     

Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: an ES- derived cell model of primary Parkinsonism

The hallmark of Parkinson's disease (PD) is the selective loss of dopamine neurons in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in rare cases, indicating a genetic factor. Furthermore, pathological analyses of PD substantia nigra have correlated cellular oxidative stress and altered proteasomal function with PD. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive Parkinsonism, one of which is a large deletion that is likely to lead to loss of function. Here we show that embryonic stem cells deficient in DJ-1 display increased sensitivity to oxidative stress and proteasomal inhibition. The accumulation of reactive oxygen species in toxin-treated DJ-1-deficient cells initially appears normal, but these cells are unable to cope with the consequent damage that ultimately leads to apoptotic death. Furthermore, we find that dopamine neurons derived from in vitro–differentiated DJ-1-deficient embryonic stem cells display decreased survival and increased sensitivity to oxidative stress. These data are consistent with a protective role for DJ-1, and demonstrate the utility of genetically modified embryonic stem cell–derived neurons as cellular models of neuronal disorders.     

中国哺乳动物形态、生活史和生态学特征数据集

物种特征是生物对生存环境适应和响应的表现, 反映了物种的生态位、适合度和生态功能。特征数据库的建立和共享是研究生物多样性维持与丧失、物种进化与适应、生态过程与生态系统功能、物种对气候变化和人类干扰响应、种内与种间关系等的基础。中国是世界哺乳动物物种数最多的国家之一, 然而目前中国还没有包含哺乳动物形态、生活史、生态学和地理分布等物种特征的数据库。我们系统查阅了文献和各种数据资料, 共收集整理出中国有分布记录的754种哺乳动物(包括近些年野外绝灭种、分布存疑种)的体重、脑容量、体长、尾长、前臂长(翼手目)、后足长、耳长、性成熟时间、妊娠期、窝崽数、年窝数、世代长度、食性、活动模式、是否特有种、濒危等级、海拔范围、栖息地类型、栖息地宽度、动物地理界、生物群系、分布型、动物地理区划和分布省份或水域等24个生态特征数据。在这些特征中, 除了分布省份或水域及是否特有种外, 其余特征数据均存在不同程度的缺失, 数据的完整度为30%‒100%。本数据库收录的哺乳动物种数为目前中国哺乳动物种数的上限, 为中国哺乳动物研究提供了基础数据, 推进中国哺乳动物多样性信息共享和深度挖掘。     

Transducing oxidative stress to death signals in neurons

Accumulation of reactive oxygen species (ROS) has been associated with aging and neurodegenerative diseases. Nevertheless, how elevated ROS levels cause neurodegeneration is unclear. In this issue, Wakatsuki et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506102) delineate how oxidative stress is transduced into death signals, leading to neuronal apoptosis and axonal degeneration.The human brain consumes ∼20% of the body’s energy in the resting state. Oxygen metabolism produces reactive oxygen species (ROS) as a byproduct. Under normal conditions, ROS regulate redox homeostasis and serve as important messengers in cell signaling. However, when environmental stressors exacerbate ROS generation or when detoxification mechanisms fail to remove excessive ROS, the imbalance results in abnormally high levels of ROS that become toxic to cells (referred to as oxidative stress). Neurons have high energy–demanding activities, which cause significant challenges for ROS detoxification, especially in highly specialized cellular compartments such as branchy dendrites and lengthy axons. In addition, because neurons are postmitotic cells and have limited capacity to regenerate in the adult central nervous system, they are especially prone to oxidative stress and its consequences (Mattson and Magnus, 2006).Oxidative stress has long been associated with human neurological disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Friedreich ataxia (Andersen, 2004; Calabrese et al., 2006). For example, mutations of known Parkinson’s disease–associated genes, including PAR7, PINK1, PARK2, SNCA (encoding α-synuclein), and LRRK2, can directly or indirectly impair mitochondrial functions and lead to increased ROS levels as well as vulnerability to oxidative stress (Dias et al., 2013). Similarly, mutations in the gene encoding superoxide dismutase 1 (SOD1), a key antioxidative enzyme in the cell, account for ∼20% of familial amyotrophic lateral sclerosis cases (Barber and Shaw, 2010). Oxidative stress can exert cytotoxic effects through the generation of peroxides and free radicals that damage DNA, proteins, and lipids. However, the pathogenic mechanisms and the exact signaling pathways by which oxidative stress causes neurodegeneration are elusive. In this issue, Wakatsuki et al. show that the E3 ubiquitin ligase zinc and ring finger 1 (ZNRF1) plays a critical role in mediating oxidative stress–induced neuronal cell death and axonal degeneration.In a previous study by the Araki group, Wakatsuki et al. (2011) demonstrated that ZNRF1 targets AKT for ubiquitin proteasome system (UPS)–dependent degradation in Wallerian degeneration, the progressive degeneration of the distal axonal segment that is separated from the neuronal cell body in nerve injury. Removal of AKT releases phosphorylation suppression on glycogen synthase kinase 3β (GSK3β), which subsequently phosphorylates and induces collapsin response mediator protein 2 (CRMP2) degradation. CRMP2 is required for microtubule stabilization. Thus, the ZNRF1–AKT–GSK3β–CRMP2 pathway mediates axon destruction in Wallerian degeneration (Wakatsuki et al., 2011). As ZNRF1 functions as a key mediator for axonal degeneration, it is reasonable to hypothesize that this pathway may also participate in another major form of neurodegeneration—neuronal cell death. With a particular interest in oxidative stress–induced neurodegeneration, in this study, the authors first used a mouse model of focal cerebral ischemia to demonstrate that CRMP2 phosphorylation is increased in ischemic neurons. They then applied 6-hydroxydopamine (6OHDA) and H₂O₂ (both are frequently used to induce cellular oxidative stress) in primary cultured cortical neurons. They found that oxidative stress triggers AKT ubiquitination and degradation, which is prevented by overexpressing a dominant-negative form of ZNRF1 or by RNAi knockdown of endogenous ZNRF1. These results indicated that the ZNRF1–AKT–GSK3β–CRMP2 pathway is indeed activated in neurons upon oxidative stress.How then is ZNRF1 activated by oxidative stress? An important clue came from the observation that ZNRF1 is highly phosphorylated in SH-SY5Y neuroblastoma cells when treated with 6OHDA (Wakatsuki et al., 2015). A web-based program predicted that ZNRF1 may be phosphorylated at the tyrosine residue Y103 by EGF receptor (EGFR) tyrosine kinase. Using a combination of primary neuronal culture, in vivo mouse models of cerebral ischemia and 6OHDA-induced brain lesions, and in vitro kinase assay, Wakatsuki et al. (2015) demonstrated that ZNRF1 is specifically phosphorylated at Y103 by EGFR in response to oxidative stress. This activation of ZNRF1 resulted in neuronal apoptosis, as evidenced by increases in caspase 3 cleavage, annexin V–positive staining, and lactate dehydrogenase release. Moreover, the authors found that application of antioxidants such as N-acetyl-l-cysteine and curcumin, down-regulation of EGFR activity by siRNA or via the EGFR inhibitor C56, and expression of the dominant-negative form ZNRF1 C184A or the phosphorylation-resistant mutant ZNRF1 Y103F all prevented 6OHDA-induced neuronal apoptosis. Furthermore, Wakatsuki et al. (2015) characterized the cellular signaling downstream of ZNRF1 and showed that EGFR-dependent ZNRF1 phosphorylation stimulated AKT ubiquitination and degradation. Expression of either a constitutively active form of AKT or a kinase-dead form of GSK3β potently suppressed 6OHDA-induced neuronal cell death (Wakatsuki et al., 2015). Together, these results demonstrate that EGFR-dependent phosphorylation of ZNRF1 at Y103 promotes degradation of AKT and resultant activation of GSK3β, which mediates oxidative stress–induced neuronal apoptosis (Fig. 1).Open in a separate windowFigure 1.The ZNRF1 signaling pathway mediates oxidative stress–induced neuronal apoptosis and axonal degeneration. Oxidative stress induced by treatment with 6OHDA or H₂O₂ or generated by NADPH oxidases in axons upon traumatic injury activates EGFR tyrosine kinase activity and leads to ZNRF1 phosphorylation at Y103. This stimulates the E3 ubiquitin ligase activity of ZNRF1, which ubiquitinates and targets AKT for degradation via the UPS. Degradation of AKT relieves the inhibitory phosphorylation of GSK3β, which then phosphorylates CRMP2 and subjects it to degradation. In axons, CRMP2 is required for microtubule stabilization, whose disassembly results in axonal degeneration. In soma, oxidative stress activates this same ZNRF1 signaling pathway, which causes cleavage and activation of caspase 3, leading to neuronal apoptosis.Does axonal degeneration also use oxidative stress–induced activation of ZNRF1 signaling? If so, where does oxidative stress come from in the first place? Another important finding of this study is that traumatic neural injury induces oxidative stress in axons by NADPH oxidases (Wakatsuki et al., 2015). Using an in vitro model of Wallerian degeneration by primary cultured dorsal root ganglion (DRG) neurons, Wakatsuki et al. (2015) showed that the levels of oxidative stress, the activity of the endogenous EGFR kinase, and the EGFR-dependent phosphorylation of ZNRF1 at Y103 in injured neurites are all robustly increased as early as 3 h after transection. In an attempt to identify the generators of oxidative stress in injured axons, Wakatsuki et al. (2015) examined the effects of inhibiting NADPH oxidase activity. They found that the NADPH oxidase inhibitors not only prevented injury-elicited elevation of oxidative stress, but also remarkably suppressed Wallerian degeneration. Knockdown of the NADPH oxidase catalytic subunits by RNAi in cultured DRG neurons confirmed these results and further pointed out that the NADPH oxidases NOX2, 3, 4, and DUOX2 may be particularly involved in this process (Wakatsuki et al., 2015). Consistent with their previous study (Wakatsuki et al., 2011) and similar to oxidative stress–induced neuronal apoptosis, interruption of the ZNRF1 signaling pathway at each step significantly delayed Wallerian degeneration of injured axons in cultured DRG neurons (Fig. 1). Finally, Wakatsuki et al. (2015) generated transgenic mice expressing the dominant-negative mutant ZNRF1 C184A and validated the hypothesis that blocking the ZNRF1 signaling cascade protects neurons from oxidative stress–induced cell death and axonal degeneration in vivo.Wakatsuki et al. (2015) used a combination of pharmacological, genetic, biochemical, immunohistological, and other approaches to unveil the involvement of the EGFR–ZNRF1–AKT–GSK3β–CRMP2 pathway in oxidative stress–induced neurodegeneration. This is a Herculean task, especially given that this is a multistep signaling cascade, and the authors made tremendous efforts to confirm their results in various experimental setups from both in vitro and in vivo models, which establish the oxidative stress–induced, EGFR-dependent activation of the ZNRF1 E3 ligase activity as a common signaling mechanism in both of the two major neurodegeneration forms—neuronal apoptosis and axonal degeneration (Fig. 1).The ubiquitin–proteasome-mediated degradation system plays an important role in regulating protein homeostasis and is involved in neurodegenerative diseases (McKinnon and Tabrizi, 2014; Zheng et al., 2014). In addition, emerging evidence has linked the UPS to Wallerian degeneration: inhibition of the UPS activity by both pharmacological and genetic methods remarkably suppressed axonal degeneration both in vitro and in vivo (Zhai et al., 2003). A recent study in Drosophila melanogaster revealed that highwire, an E3 ubiquitin ligase, promotes Wallerian degeneration by targeting the NAD⁺ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat) for degradation (Xiong et al., 2012). And now, the study by Wakatsuki et al. (2015) exemplifies a mechanism by which E3 ligase ZNRF1 acts as a mediator to transduce oxidative stress to death signals in neurons. It should be noted, however, that the ZNRF1 signaling pathway is unlikely to be the only mechanism promoting neurodegeneration. For example, inhibition of ZNRF1 signaling offers axon protection for up to 48 h, whereas expression of the neuroprotective Wld^S protein (Lunn et al., 1989; Coleman and Freeman, 2010; Fang and Bonini, 2012) or down-regulation of the prodegenerative SARM1 gene (Osterloh et al., 2012; Gerdts et al., 2015; Yang et al., 2015) protects injured axons for up to 72 h (Wakatsuki et al., 2015). These results strongly suggest that there are other mechanisms transducing neural injury and oxidative stress to degenerative signals in neurons.The exciting findings by Wakatsuki et al. (2015) have raised a number of further questions. One immediate question is how EGFR senses oxidative stress. EGFR exists on the cell surface and dimerizes upon binding to its specific ligands that activate its intrinsic intracellular protein–tyrosine kinase activity (Herbst, 2004). In the context of cellular oxidative stress, how is EGFR activated? A second question is, in addition to the ZNRF1–AKT–GSK3β–CRMP2 pathway, is any other signaling pathway downstream of EGFR also activated? A recent study reported that the MAPK cascade is activated in the early response of axon injury (Yang et al., 2015). MAPK pathway activation is another well-known outcome of EGFR signaling (Nguyen et al., 2013). Of note, although Wakatsuki et al. (2011, 2015) argue that AKT phosphorylates and inhibits GSK3β, which subsequently stabilizes CRMP2 and microtubules to prevent axonal degeneration, Yang et al. (2015) claim that AKT promotes axonal survival by phosphorylation of MKK4 at serine 78, which suppresses MKK4-mediated activation of prodegenerative JNK signaling. Further study of EGFR signaling in oxidative stress and neural injury is needed to provide a better understanding of the regulatory mechanisms at different stages of the degenerative process. Third, although NADPH oxidases are involved in the elevation of oxidative stress in injured axons (Wakatsuki et al., 2015), it remains unclear whether this is because axon injury promotes the activity of NADPH oxidases or because the ROS detoxification system is impaired in injured axons and the NADPH oxidase activity is merely required to maintain a steady-state level of ROS. Fourth, what is the relationship between the ZNRF1 signaling pathway, Wld^S/Nmnat, and SARM1 in axonal degeneration? Does Wld^S/Nmnat or loss of function of SARM1 manifest axonal protection by blocking a step in the EGFR–ZNRF1–AKT–GSK3β–CRMP2 axis? Finally, because EGFR has been successfully targeted in the development of antitumor drugs, identification of specific inhibitors of the EGFR–ZNRF1 signaling cascade holds a high hope for the development of effective therapeutics treating neuronal and axonal degeneration in diseases and traumatic injury. And the ultimate question is, when?     

Mitochondrial Quality Control Mediated by PINK1 and Parkin: Links to Parkinsonism

Mutations in Parkin or PINK1 are the most common cause of recessive familial parkinsonism. Recent studies suggest that PINK1 and Parkin form a mitochondria quality control pathway that identifies dysfunctional mitochondria, isolates them from the mitochondrial network, and promotes their degradation by autophagy. In this pathway the mitochondrial kinase PINK1 senses mitochondrial fidelity and recruits Parkin selectively to mitochondria that lose membrane potential. Parkin, an E3 ligase, subsequently ubiquitinates outer mitochondrial membrane proteins, notably the mitofusins and Miro, and induces autophagic elimination of the impaired organelles. Here we review the recent rapid progress in understanding the molecular mechanisms of PINK1- and Parkin-mediated mitophagy and the identification of Parkin substrates suggesting how mitochondrial fission and trafficking are involved. We also discuss how defects in mitophagy may be linked to Parkinson''s disease.Parkinson''s disease (PD) is the second most common neurodegenerative disorder and is characterized by cardinal motor symptoms: slowness of movement, rigidity, rest tremor, and postural instability (Ropper et al. 2009). Although these symptoms initially respond to drugs that modulate dopamine metabolism or surgeries that alter basal ganglia circuitry, the disease eventually progresses. With a modest exception (Olanow et al. 2009), no therapy has been shown to alter the disease course.The pathogenesis of sporadic Parkinson''s disease is likely complex involving altered metabolism of the protein α-synuclein, lysosomal dysfunction, and a dysregulated inflammatory response (reviewed in Shulman et al. 2011). Several lines of evidence also point to mitochondrial dysfunction as a central player in the pathogenesis of PD. Complex I dysfunction is associated with sporadic PD and is sufficient to induce parkinsonism (reviewed in Schapira 2008). The inhibitors of complex I, MPTP (Langston et al. 1983) and rotenone (Betarbet et al. 2000), replicate the symptoms of PD, and rotenone recapitulates key pathognomonic features of PD, such as the α-synuclein-rich inclusion bodies (Betarbet et al. 2000). The cause of mitochondrial dysfunction in sporadic PD is not entirely clear, but laser capture microdissection of substantia nigra neurons from patients with PD reveal a higher burden of mitochondrial DNA deletions relative to age-matched controls (Bender et al. 2006). That such deletions are sufficient to cause parkinsonism is suggested by the occurrence of parkinsonism in patients with rare mutations in their mtDNA replication machinery (e.g., the catalytic subunit of the mtDNA polymerase POLG [Luoma et al. 2004] or the mtDNA helicase Twinkle [Baloh et al. 2007]). The defective mtDNA replicative machinery generates high levels of mtDNA deletions throughout the body that are qualitatively similar to those observed in the substantia nigra in patients with sporadic PD (Reeve et al. 2008). Thus, mitochondrial dysfunction is both associated with sporadic PD and sufficient to cause the parkinsonian syndrome.As is discussed in this review, recent insights from certain genetic forms of PD—resulting from mutations in Parkin or PINK1—support the model that mitochondrial damage is a central driver of PD pathogenesis. Additionally, they provide a rationale for targeting mitochondrial quality control pathways in patients with PD.     

Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli

In nature, Escherichia coli are exposed to harsh and non-ideal growth environments—nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper (Leichert and Jakob 2004), which provide evidence that MetE is one of the proteins in E. coli most susceptible to oxidation. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. Our studies suggest a simpler mechanism is employed by E. coli to achieve the same effect.     

Traversing a wormhole to combat Parkinson’s disease

Human movement disorders represent a significant and unresolved societal burden. Among these, the most prevalent is Parkinson’s disease (PD), a disorder afflicting millions worldwide. Despite major advances, stemming primarily from human genetics, there remains a significant gap in our understanding of what factors underlie disease susceptibility, onset, and progression. Innovative strategies to discern specific intracellular targets for subsequent drug development are needed to more rapidly translate basic findings to the clinic. Here we briefly review the recent contributions of research using the nematode roundworm Caenorhabditis elegans as a model system for identifying and characterizing gene products associated with PD. As a microscopic but multicellular and genetically tractable animal with a well-defined nervous system and an experimentally tenable lifespan, C. elegans affords significant advantages to researchers attempting to determine causative and therapeutic factors that influence neuronal dysfunction and age-associated neurodegeneration. The rapidity with which traditional genetic, large-scale genomic, and pharmacological screening can be applied to C. elegans epitomizes the utility of this animal for disease research. Moreover, with mature bioinformatic and functional genomic data readily available, the nematode is well positioned to play an increasingly important role in PD-associated discoveries.Physicists and astronomers have long posited the concept of a ‘wormhole’ as a means of rapid interstellar travel by analogy with how a worm could eat a hole from one end of an apple, through the center to the other end, and create a shortcut through the intervening space. Unfortunately, bending the fabric of space and time is not typically considered a viable option to more rapidly explore cures for neurodegenerative diseases (and would likely result in a poorly received grant application). The biological equivalent of the space exploration program, the human genome project, has unleashed an age of genomic, proteomic, metabolomic, and bioinformatic analyses that has generated a wealth of datasets primed for subsequent discovery. This exponential growth demands the development of functional means for exploiting this treasure trove of biological information. In this regard, biomedical researchers are literally turning to a worm to accelerate the path toward therapeutic advances and get to the core of mechanisms underlying Parkinson’s disease (PD).While drugs to treat the symptoms of PD have been prescribed for decades (e.g. L-DOPA), an unmet need for innovative strategies to discern disease etiology and treatments that either halt or reverse progression remains. Application of a microscopic nematode roundworm toward gaining insights into a human neurodegenerative disorder may seem impractical; yet, C. elegans affords many advantages for such research, as it has a defined cell lineage, completed genome sequence, and lifespan of only 2 weeks. As opposed to the human brain, which is estimated to have over 100 billion nerve cells, this nematode contains precisely 302 neurons for which a defined neuronal connectivity map has been determined. In this regard, C. elegans is ideal for investigation of diseases associated with neuronal dysfunction and ageing, and represents a model system that is poised to bridge the gap between basic and translational research.Interpretation of disease-associated data obtained in invertebrate systems requires downstream validation in mammals prior to establishing the therapeutic significance of any findings. The likelihood of a positive outcome is significant, however, due to the evolutionary conservation of metazoan genomes. For example, human homologs have been identified for at least 50% of C. elegans genes. Remarkably, these include all orthologs of reported genes linked to familial forms of PD (Fig. 1), with the one exception being the gene encoding α-synuclein. Despite this difference, ectopic overexpression of both wildtype and mutant (A53T) human α-synuclein led to motor deficits or DA neuron loss in C. elegans (Lasko et al., 2003). Subsequent reports also demonstrated phenotypic changes associated with driving α-synuclein expression from a variety of pan-neuronal and specific neuronal promoters (Cao et al., 2005; Ved et al., 2005; Kuwahara et al., 2006). The translational utility of C. elegans is perhaps best demonstrated by the characterization of conserved neuroprotective genes that block α-synuclein-associated degeneration in nematodes (Cooper et al., 2006; Gitler et al., 2008; Hamamichi et al., 2008).Open in a separate windowFig. 1PD-associated gene products and their prospective sites of cellular functionPD is hypothesized to be a consequence of the dysfunction of intersecting and compensatory protein degradation components, including those associated with the lysosome and autophagy, as well as those associated with the ubiquitin proteasome system (UPS). Inefficient clearance and enhanced misfolding, or expression, of α-synuclein has been shown to block intracellular trafficking and increase cytotoxicity. Accordingly, mutations in Parkin (an E3 ubiquitin ligase), ubiquitin C-terminal hydrolase-1 (UCHL-1), as well as in a lysosomal ATPase termed ATP13A2 and the Gaucher’s disease-related protein glucocerebrosidase (GBA), have further implicated protein degradation pathways in PD. PD-associated mutations in genes linked with mitochondrial function, such as those encoding DJ-1 and the PTEN-induced kinase (PINK1), presumably affect the production of reactive oxygen species (ROS), which accelerate protein damage and neurodegeneration. While mutations in the leucine-rich repeat kinase 2 (LRRK2) protein now represent the most common heritable cause of PD, the function of this large, 2527 amino acid multidomain-containing protein remains undefined.The rich, 40-year history of C. elegans genetics provides a legacy of behavioral and neuroanatomical information that researchers draw upon to elucidate relationships between gene function and PD-associated mechanisms (Bargmann, 1998). Using traditional genetic suppressor and enhancer screening, investigators have begun to dissect genetic contributors to DA neuron development and function (Chase et al., 2004). A limbless simple invertebrate will certainly never recapitulate the phenotypic behaviors associated with the tremor and dyskinesia of PD, yet this nematode does afford opportunities for accurate quantification of factors that influence DA neuron survival. Loss of DA neurons in C. elegans is not lethal and leads only to a subtle behavioral deficit where affected animals display an inability to discriminate local mechanosensory cues (Sawin et al., 2000). Unfortunately, this behavior, while quantifiable, is not robust enough to be easily used in extensive screening paradigms.Mammalian and primate modeling approaches to PD have traditionally involved use of neurotoxins to induce DA neuron loss and evaluate the consequences of neurodegeneration. Among these toxins are 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), as well as the pesticides rotenone and paraquat. Worms are susceptible to these toxins (Nass et al., 2002; Braungart et al., 2004; Cao et al., 2005); moreover, the defined and transparent neuroanatomy of C. elegans includes precisely eight neurons that produce DA, thereby enabling an unparalleled level of quantitative analysis of neurodegeneration and protection by use of fluorescent protein labeling (Fig. 2). Furthermore, these animals can be evaluated in specific genetic or transgenic backgrounds to screen for factors that confer either neuroprotection or enhancement of degeneration. One neuroprotective gene product termed TOR-2, a human torsinA-related protein with molecular chaperone-like activity (Caldwell et al., 2008), was shown to deter multiple forms of toxic insults to DA neurons, including 6-OHDA, excess intracellular DA production, and α-synuclein overexpression (Cao et al., 2005). The neuroprotective capacity of torsin-like proteins renders them intriguing candidates for subsequent drug targeting and validation in mammalian systems, where human torsinA is endogenously expressed in DA neurons (Augood et al., 1998).Open in a separate windowFig. 2Anterior dopamine neurons of C. elegansCell bodies and processes of the six anterior DA neurons including two pairs of the CEP (cephalic) neurons and one pair of ADE (anterior deirid) neurons, as illuminated using GFP driven from the dopamine transporter promoter (P_dat-1::GFP). Two additional DA neurons (PDEs) are found in the posterior (not shown).Directed and comprehensive screens have been undertaken to define chemical and genetic effectors of worm DA neuron sensitivity to 6-OHDA (Nass et al., 2005; Maranova and Nichols, 2007). These studies identified a collection of intragenic mutations in the gene sequence encoding the key protein required for DA reuptake into presynaptic neurons, the DA transporter (DAT-1). Structural and functional relationships revealed through this work have implications not only for PD, but also for other disorders associated with DAT function (e.g. depression, ADHD). It has also been shown that worm dat-1 knockout mutants exhibit diminished α-synuclein-dependent degeneration, which indicates a possible role for DAT in maintaining an important balance of intraneuronal levels of DA, the dysregulation of which may contribute to cytotoxicity (Cao et al., 2005).Elegant studies in Drosophila have demonstrated the capacity for model systems to reveal unsuspected relationships between PD gene products, such as PINK1 and Parkin, and their impact on mitochondrial integrity and morphology (Clark et al., 2006; Park et al., 2006; Poole et al., 2008). Likewise, the ability of worm research to link genotype to phenotype in this manner is accelerating our understanding of the underlying nature of PD. Ved et al. investigated the functional consequences of rotenone-induced stress and reported that pan-neuronal overexpression of human αsynuclein (wild-type or A53T mutant), RNAi knockdown of a C. elegans DJ-1 ortholog (B0432.2), or a deletion mutant of the C. elegans parkin ortholog (pdr-1), all produced similar patterns of mitochondrial vulnerability in response to pharmacological challenges associated with complex I inhibition (Ved et al., 2005). Furthermore, the effect of rotenone, which led to mortality in these animals, was more severe in α-synuclein-expressing strains and the pdr-1 deletion mutant than in control animals of wild-type background. Initial work on worm PDR-1 revealed that this Parkin ortholog, an E3 ubiquitin ligase, cooperates with conserved degradation machinery to mediate ubiquitin conjugation (Springer et al., 2005). Co-expression of C. elegans pdr-1 and α-synuclein variants in human cell cultures showed that a truncated protein derived from an in-frame pdr-1 (lg103) deletion allele causes aggregation of α-synuclein, similar to Parkin variants isolated from PD patients. This altered gene product also resulted in proteotoxicity and hypersensitivity to ER stress (Springer et al., 2005). These examples demonstrate how mutant analysis in C. elegans offers a powerful strategy for uncovering functional relationships between gene products with key roles in PD pathogenesis in mammalian cells.C. elegans has been recently used to show a protective role for human LRRK2 (leucine-rich repeat kinase 2) against mitochondrial toxicity induced by rotenone, an effect potentiated by knockdown of an endogenous worm ortholog, lrk-1 (Wolozin et al., 2008). These data are surprising when considering a PD-associated G2019S mutant version of LRRK2 that exhibits increased kinase activity was also found to be protective; kinase activity has been suggested to contribute to the pathogenesis associated with LRRK2 neurotoxicity (West et al., 2007). An initial report on lrk-1 function in C. elegans indicated a role for the product of this gene in establishing neuronal polarity (Sakaguchi-Nakashima et al., 2007). As mutations in LRRK2 now represent the most common genetic cause of PD (Haugarvoll et al., 2008), further insights gleaned from C. elegans on this important, yet relatively uncharacterized, protein family will undoubtedly be informative.The capacity for genomic-scale screening has brought C. elegans to the forefront of modern functional analysis (Kamath and Ahringer, 2003). In contrast to mammals, where individual knockouts of mice are expensive and time consuming, worms are an efficient and economical alternative. Hamamichi et al. took a hypothesis-driven approach toward identification of putative genetic modifiers of PD via a multi-tiered screen for C. elegans genes that impact the misfolding of transgenic human α-synuclein, as well as neuroprotection, in vivo (Hamamichi et al., 2008). This study encompassed RNAi knockdown of nearly 900 bioinformatically prioritized gene targets, comprising components of cellular pathways implicated in protein folding or degradation, as well as gene products that are either co-expressed or interact with worm orthologs of familial PD genes. From this varied, but biased, ‘guilt by association’ list of targets, 20 candidate genes emerged as having the greatest propensity to influence α-synuclein misfolding. Internal validation was evident within this short list, as included were the worm homologs of two established recessive PD genes (DJ-1, PINK1), as well as a gene (ULK2) shown to be one of only six identified in a genome-wide association study of polymorphisms in PD patients (Fung et al., 2006). Importantly, overexpression of select cDNAs in C. elegans DA neurons revealed that five of seven gene products tested, chosen from the primary effectors of α-synuclein misfolding identified in the RNAi screen, exhibited significant protection from age- and dose-dependent neurodegeneration induced by α-synuclein (Hamamichi et al., 2008). Thus, application of C. elegans in this context uncovered functionally evaluated targets identified by the two primary clinical criteria associated with PD: α-synuclein accumulation and DA neurodegeneration.These genes, which included uncharacterized proteins, as well as regulators of autophagy, lysosomal function, cellular trafficking, and G-protein signaling, now represent outstanding candidates for strategically targeted drug development and validation in mammalian systems. A more recently conducted unbiased genome-wide RNAi screen for genetic factors that influence αsynuclein inclusion formation in C. elegans also yielded an over-representation of genes encoding components of vesicular trafficking, as well as specific age-associated genes (e.g. sir-2.1) (van Ham et al., 2008). Similar validation of the effect that these additional targets may have on DA neuron survival will likely reveal functionally significant relationships as well. Associations between PD and lysosomal degradation are of growing interest following the identification of a lysosomal ATPase, ATP13A2 (PARK9), as a gene product linked to hereditary early-onset PD with dementia (Ramirez et al., 2006), in addition to the discovery of a higher incidence of PD in patients with mutations in the Gaucher’s disease-associated gene, GBA, encoding the lysosomal storage-enzyme glucocerebrosidase. (Aharon-Peretz et al., 2004; Clark et al., 2007). It is easy to envision an expansion of such gene-specific data and the application of C. elegans toward investigating the functional consequences of single-nucleotide polymorphisms (SNPs) found in human populations, as these may potentially lead to genetic biomarkers of disease susceptibility.The burgeoning promise that a microscopic worm may contribute toward the goal of drug discovery for PD has become a tangible reality. Containing a rudimentary nervous system, unlike single-celled organisms, but more amenable to transgenic analysis and drug screening than mammals, C. elegans serves as an excellent intermediary bottleneck in translational research pipelines to characterize therapeutic gene and drug targets across animal models. Concerted efforts toward therapeutic development have been initiated that exploit the high-throughput screening capabilities of yeast cells to define numerous gene targets of interest, followed by subsequent evaluation of their neuroprotective capacity in worms, fruit flies and rat neuronal cultures (Cooper et al., 2006; Gitler et al., 2008). This approach has already revealed that overproduction of αsynuclein leads to a cytotoxic blockage in intracellular vesicular trafficking that can be alleviated by specific members of the Rab protein family (Cooper et al., 2006; Gitler et al., 2008). Thus, through the employment of a combination of powerful model systems, not only has a prospective cellular cause of PD been illuminated, but targeted screens for small molecules that protect against underlying functional aspects of neurodegeneration are now possible.When envisioning future directions whereby C. elegans will benefit PD research, several inescapable advantages of this model system should be considered. The most obvious of these is the well-established use of the worm for investigating mechanisms of aging (Kenyon, 2005). Indeed, the only definitive risk factor for PD is aging, where PD symptomatically affects over 2% of people above age 65. Extensive studies on worm longevity mutants (e.g. daf-2 or age-1) have demonstrated that evolutionarily conserved mechanisms are shared between invertebrates and mammals. Microarray and genomic-scale RNAi analyses conducted in age-extending backgrounds have yielded significant insights into gene sets that implicate dietary restriction and insulin-like signaling pathways as crucial mediators of lifespan (Murphy et al., 2003; Panowski et al., 2007). The vital, but poorly understood, interface between aging and age-associated neurodegenerative disease represents an exciting frontier that is readily explored using C. elegans.Furthermore, in the advent of the microRNA (miRNA) revolution, our nascent understanding of putative regulatory roles for miRNAs in neuron function will likely soon interface with our understanding of neurological disease and PD (Kosik, 2006; Kim et al., 2007). Considering the pioneering role worm research has played in defining miRNA function, a universe of possibilities exist, as worm researchers are well-poised to explore relationships between miRNAs, ageing and neuroprotective gene activity (Ibanez-Ventoso, 2007).

Advantages of C. elegans as a model for Parkinson’s disease

• All orthologs of genes linked to familial PD have been identified in C. elegans, except α-synuclein
• Ectopic overexpression of α-synuclein has neurotoxic effects in C. elegans, which are blocked by neuroprotective genes
• C. elegans has a simple nervous system (302 neurons compared with over 100 billion in humans) that is amenable to quantitative analysis of neurodegeneration
• C. elegans is susceptible to toxins commonly used to model neurodegeneration
• C. elegans studies predict relationships between cellular signaling, trafficking and protein degradation pathways, which are being tested for their susceptibility to targeted drug development for PD

Finally, although the primary advances in PD etiology have come through human genetics, the largely idiopathic nature of PD remains linked to inexplicable environmental causes. C. elegans research into innate immunity and cellular stress response has already provided mechanistic insights into organismal defenses and environmental influences on homeostasis (Kim et al., 2002; Mohri-Shiomi and Garsin, 2008). An expansion of neurotoxicity studies conducted in C. elegans is warranted and may yield a greater understanding of the interplay between genetic composition and environmental factors such as heavy metals, pesticides and other untested exposures.The worm is unquestionably a powerful system, yet it has its limitations: the anatomical and functional connectivity of the neuronal circuitry within this simple nematode cannot recapitulate the complex features of mammalian dopamine neurons or mimic the precise behavioral deficits associated with their loss. Likewise, as a cautionary caveat to inferring conservation of function from genetic interaction data, Tischler et al. demonstrated that synthetic lethal gene interactions between yeast and worm genes are not significantly conserved (Tischler et al., 2008). In this context, as the march toward systems biology proceeds and integrated analyses of gene or protein activity continue to lead to an increasing number of complex datasets, our ability to eventually define causes and cures will depend on functional strategies for wading through the emerging ‘information overload’. Science will benefit from the efficient manner by which C. elegans research can contribute to the quest for translational and personalized medical breakthroughs, by boldly going where no worm has gone before.