Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging.

DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. Here we present two-color fluorogenic DNA-PAINT, which uses improved imager probe and docking strand designs to solve these problems. These self-quenching single-stranded DNA probes are conjugated with a fluorophore and quencher at the terminals, which permits an increase in fluorescence by up to 57-fold upon binding and unquenching. In addition, the engineering of base pair mismatches between the fluorogenic imager probes and docking strands allowed us to achieve both high fluorogenicity and the fast binding kinetics required for fast imaging. We demonstrate a 26-fold increase in imaging speed over regular DNA-PAINT and show that our new implementation enables three-dimensional super-resolution DNA-PAINT imaging without optical sectioning.

The role of age-associated alpha-synuclein aggregation in a conditional transgenic mouse model of Parkinson's disease: Implications for Lewy body formation.

Parkinson's disease (PD) is a common neurodegenerative disorder that is affecting an increasing number of older adults. Although PD is mostly sporadic, genetic mutations have been found in cohorts of families with a history of familial PD (FPD). The first such mutation linked to FPD causes a point mutation (A53T) in α-synuclein (α-syn), a major component of Lewy bodies, which are a classical pathological hallmark of PD. These findings suggest that α-syn is an important contributor to the development of PD. In our previous study, we developed an adenoviral mouse model of PD and showed that the expression of wild-type (WT) α-syn or a mutant form with an increased propensity to aggregate, designated as WT-CL1 α-syn, could be used to study how α-syn aggregation contributes to PD. In this study, we established a transgenic mouse model that conditionally expresses WT or WT-CL1 α-syn in dopaminergic (DA) neurons and found that the expression of either WT or WT-CL1 α-syn was associated with an age-dependent degeneration of DA neurons and movement dysfunction. Using this model, we were able to monitor the process of α-syn aggregate formation and found a correlation between age and the number and sizes of α-syn aggregates formed. These results provide a potential mechanism by which age-dependent α-syn aggregation may lead to the formation of Lewy bodies in PD pathogenesis.

PICK1 inhibits the E3 ubiquitin ligase activity of Parkin and reduces its neuronal protective effect.

Parkin functions as a multipurpose E3 ubiquitin ligase, and Parkin loss of function is associated with both sporadic and familial Parkinson's disease (PD). We report that the Bin/Amphiphysin/Rvs (BAR) domain of protein interacting with PRKCA1 (PICK1) bound to the really interesting new gene 1 (RING1) domain of Parkin and potently inhibited the E3 ligase activity of Parkin by disrupting its interaction with UbcH7. Parkin translocated to damaged mitochondria and led to their degradation in neurons, whereas PICK1 robustly inhibited this process. PICK1 also impaired the protective function of Parkin against stresses in SH-SY5Y cells and neurons. The protein levels of several Parkin substrates were reduced in young PICK1-knockout mice, and these mice were resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated toxicity. Taken together, the results indicate that PICK1 is a potent inhibitor of Parkin, and the reduction of PICK1 enhances the protective effect of Parkin.

S-nitrosylation of XIAP at Cys 213 of BIR2 domain impairs XIAP's anti-caspase 3 activity and anti-apoptotic function.

X-linked inhibitor of apoptosis (XIAP) is a protein that possesses anti-apoptotic function and dysregulation of it has been linked to a number human disease such as cancers and neurodegenerative disorders. In our previous study, we have found that nitric oxide (NO) can modulate the anti-apoptotic function of XIAP and found that this can contribute to the pathogenesis of Parkinson's disease. Specifically, we found that modification of baculoviral IAP repeat 2 of XIAP by S-nitrosylation can compromise XIAP's anti-caspase 3 and anti-apoptotic function. In this study, we report that cysteine (Cys) 90, Cys 213 and Cys 327 can be specifically S-nitrosylated by NO. We found that mutations of Cys 90 and Cys 327 affect the normal structure of XIAP. More importantly, we found that S-nitrosylation of XIAP Cys 213 impairs the anti-caspase 3 and anti-apoptotic function of XIAP that we observed in our previous study.

Alpha-synuclein impairs normal dynamics of mitochondria in cell and animal models of Parkinson's disease.

Alpha-synuclein (α-syn) is a synaptic protein that mutations have been linked to Parkinson's disease (PD), a common neurodegenerative disorder that is caused by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). How α-syn can contribute to neurodegeneration in PD is not conclusive but it is agreed that mutations or excessive accumulation of α-syn can lead to the formation of α-syn oligomers or aggregates that interfere with normal cellular function and contribute to the degeneration of dopaminergic neurons. In this study, we found that α-syn can impair the normal dynamics of mitochondria and this effect is particular prominent in A53T α-syn mutant. In mice expressing A53T α-syn, age-dependent changes in both mitochondrial morphology and proteins that regulate mitochondrial fission and fusion were observed. In the cellular model of PD, we found that α-syn reduces the movement of mitochondria in both SH-SY5Y neuroblastoma and hippocampal neurons. Taken together, our study provides a new mechanism of how α-syn can contribute to PD through the impairment of normal dynamics of mitochondria.

S-nitrosylation of XIAP compromises neuronal survival in Parkinson's disease.

Inhibitors of apoptosis (IAPs) are a family of highly-conserved proteins that regulate cell survival through binding to caspases, the final executioners of apoptosis. X-linked IAP (XIAP) is the most widely expressed IAP and plays an important function in regulating cell survival. XIAP contains 3 baculoviral IAP repeats (BIRs) followed by a RING finger domain at the C terminal. The BIR domains of XIAP possess anticaspase activities, whereas the RING finger domain enables XIAP to function as an E3 ubiquitin ligase in the ubiquitin and proteasomal system. Our previous study showed that parkin, a protein that is important for the survival of dopaminergic neurons in Parkinson's disease (PD), is S-nitrosylated both in vitro and in vivo in PD patients. S-nitrosylation of parkin compromises its ubiquitin E3 ligase activity and its protective function, which suggests that nitrosative stress is an important factor in regulating neuronal survival during the pathogenesis of PD. In this study we show that XIAP is S-nitrosylated in vitro and in vivo in an animal model of PD and in PD patients. Nitric oxide modifies mainly cysteine residues within the BIR domains. In contrast to parkin, S-nitrosylation of XIAP does not affect its E3 ligase activity, but instead directly compromises its anticaspase-3 and antiapoptotic function. Our results confirm that nitrosative stress contributes to PD pathogenesis through the impairment of prosurvival proteins such as parkin and XIAP through different mechanisms, indicating that abnormal S-nitrosylation plays an important role in the process of neurodegeneration.

S-nitrosylation of cyclin-dependent kinase 5 (cdk5) regulates its kinase activity and dendrite growth during neuronal development.

Precise regulation of cyclin-dependent kinase 5 (Cdk5), a member of the cyclin-dependent kinase family, is critical for proper neuronal development and functions. Cdk5 is activated through its association with the neuron-specific activator p35 or p39. Nonetheless, how its kinase activity is regulated in neurons is not well understood. In this study, we found that Cdk5 activity is regulated by S-nitrosylation, a post-translational modification of protein that affects a plethora of neuronal functions. S-nitrosylation of Cdk5 occurs at Cys83, which is one of the critical amino acids within the ATP-binding pocket of the kinase. Upon S-nitrosylation, Cdk5 exhibits reduced kinase activity, whereas mutation of Cys83 to Ala on Cdk5 renders the kinase refractory to such inhibition. Importantly, S-nitrosylated Cdk5 can be detected in the mouse brain, and blocking the S-nitrosylation of Cdk5 in cultured hippocampal neurons enhances dendritic growth and branching. Together, our findings reveal an important role of S-nitrosylation in regulating Cdk5 kinase activity and dendrite growth in neurons during development.

New insights into the role of mitochondrial dysfunction and protein aggregation in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative movement disorder that affects increasing number of elderly in the world population. The disease is caused by a selective degeneration of dopaminergic neurons in the substantia nigra pars compacta with the molecular mechanism underlying this neurodegeneration still not fully understood. However, various studies have shown that mitochondrial dysfunction and abnormal protein aggregation are two of the major contributors for PD. In fact this notion has been supported by recent studies on genes that are linked to familial PD (FPD). For instance, FPD linked gene products such as PINK1 and parkin have been shown to play critical roles in the quality control of mitochondria, whereas α-synuclein has been found to be the major protein aggregates accumulated in PD patients. These findings suggest that further understanding of how dysfunction of these pathways in PD will help develop new approaches for the treatment of this neurodegenerative disorder.

Expression and functional properties of TRPM2 channels in dopaminergic neurons of the substantia nigra of the rat.

Transient receptor potential melastatin 2 (TRPM2) channels are sensitive to oxidative stress, and their activation can lead to cell death. Although these channels have been extensively studied in expression systems, their role in the brain, particularly in the substantia nigra pars compacta (SNc), remains unknown. In this study, we assessed the expression and functional properties of TRPM2 channels in rat dopaminergic SNc neurons, using acute brain slices. RT-PCR analysis revealed TRPM2 mRNA expression in the SNc region. Immunohistochemistry demonstrated expression of TRPM2 protein in tyrosine hydroxylase-positive neurons. Channel function was tested with whole cell patch-clamp recordings and calcium (fura-2) imaging. Intracellular application of ADP-ribose (50-400 µM) evoked a dose-dependent, desensitizing inward current and intracellular free calcium concentration ([Ca(2+)](i)) rise. These responses were strongly inhibited by the nonselective TRPM2 channel blockers clotrimazole and flufenamic acid. Exogenous application of H(2)O(2) (1-5 mM) evoked a rise in [Ca(2+)](i) and an outward current mainly due to activation of ATP-sensitive potassium (K(ATP)) channels. Inhibition of K(+) conductance with Cs(+) and tetraethylammonium unmasked an inward current. The inward current and/or [Ca(2+)](i) rise were partially blocked by clotrimazole and N-(p-amylcinnamoyl)anthranilic acid (ACA). The H(2)O(2)-induced [Ca(2+)](i) rise was abolished in "zero" extracellular Ca(2+) concentration and was enhanced at higher baseline [Ca(2+)](i), consistent with activation of TRPM2 channels in the cell membrane. These results provide evidence for the functional expression of TRPM2 channels in dopaminergic SNc neurons. Given the involvement of oxidative stress in degeneration of SNc neurons in Parkinson's disease, further studies are needed to determine the pathophysiological role of these channels in the disease process.

Oxidative and nitrosative stress in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder marked by movement impairment caused by a selective degeneration of dopaminergic neurons. The mechanism for dopaminergic neuronal degeneration in PD is not completely clear, but it is believed that oxidative and nitrosative stress plays an important role during the pathogenesis of PD. This notion is supported by various studies that several indices of oxidative and nitrosative stress are increased in PD patients. In recent years, different pathways that are known to be important for neuronal survival have been shown to be affected by oxidative and nitrosative stress. Apart from the well-known oxidative free radicals induced protein nitration, lipid peroxidation and DNA damage, increasing evidence also suggests that some neuroprotective pathways can be affected by nitric oxide through S-nitrosylation. In addition, the selective dopaminergic neurodegeneration suggests that generation of oxidative stress associated with the metabolism of dopamine is an important contributor. Thorough understanding of how oxidative stress can contribute to the pathogenesis of PD will help formulate potential therapy for the treatment of this neurodegenerative disorder in the future.

Modulation of pro-survival proteins by S-nitrosylation: implications for neurodegeneration.

Nitric oxide (NO) is a gaseous signaling molecule in the biological system. It mediates its function through the direct modification of various cellular targets, such as through S-nitrosylation. The process of S-nitrosylation involves the attachment of NO to the cysteine residues of proteins. Interestingly, an increasing number of cellular pathways are found to be regulated by S-nitrosylation, and it has been proposed that this redox signaling pathway is comparable to phosphorylation in cells. However, imbalance of NO metabolism has also been linked to a number of human diseases. For instance, NO is known to contribute to neurodegeneration by causing protein nitration, lipid peroxidation and DNA damage. Moreover, recent studies show that NO can also contribute to the process of neurodegeneration through the impairment of pro-survival proteins by S-nitrosylation. Thus, further understanding of how NO, through S-nitrosylation, can compromise neuronal survival will provide potential therapeutic targets for neurodegenerative diseases.

Emerging roles of nitric oxide in neurodegeneration.

Nitric oxide (NO) is a gaseous signaling molecule which has physiological and pathological roles in the cell. Under normal conditions, NO is produced by nitric oxide synthase (NOS) and can induce physiological responses such as vasodilation. However, over-activation of NOS has been linked to a number of human pathological conditions. For instance, most neurodegenerative disorders are marked by the presence of nitrated protein aggregates. How nitrosative stress leads to neurodegeneration is not clear, but various studies suggest that increased nitrosative stress causes protein nitration which then leads to protein aggregation. Protein aggregates are highly toxic to neurons and can promote neurodegeneration. In addition to inducing protein aggregation, recent studies show that nitrosative stress can also compromise a number of neuroprotective pathways by modifying activities of certain proteins through S-nitrosylation. These findings suggest that increased nitrosative stress can contribute to neurodegeneration through multiple pathways.

S-Nitrosylation of G protein-coupled receptor kinase 6 and Casein kinase 2 alpha modulates their kinase activity toward alpha-synuclein phosphorylation in an animal model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder which is mostly sporadic but familial-linked PD (FPD) cases have also been found. The first reported gene mutation that linked to PD is α-synuclein (α-syn). Studies have shown that mutations, increased expression or abnormal processing of α-syn can contribute to PD, but it is believed that multiple mechanisms are involved. One of the contributing factors is post-translational modification (PTM), such as phosphorylation of α-syn at serine 129 by G-protein-coupled receptor kinases (GRKs) and casein kinase 2α (CK2α). Another known important contributing factor to PD pathogenesis is oxidative and nitrosative stress. In this study, we found that GRK6 and CK2α can be S-nitrosylated by nitric oxide (NO) both in vitro and in vivo. S-nitrosylation of GRK6 and CK2α enhanced their kinase activity towards the phosphorylation of α-syn at S129. In an A53T α-syn transgenic mouse model of PD, we found that increased GRK6 and CK2α S-nitrosylation were observed in an age dependent manner and it was associated with an increased level of pSer129 α-syn. Treatment of A53T α-syn transgenic mice with Nω-Nitro-L-arginine (L-NNA) significantly reduced the S-nitrosylation of GRK6 and CK2α in the brain. Finally, deletion of neuronal nitric oxide synthase (nNOS) in A53T α-syn transgenic mice reduced the levels of pSer129 α-syn and α-syn in an age dependent manner. Our results provide a novel mechanism of how NO through S-nitrosylation of GRK6 and CK2α can enhance the phosphorylation of pSer129 α-syn in an animal model of PD.

Correction: S-Nitrosylation of G protein-coupled receptor kinase 6 and Casein kinase 2 alpha modulates their kinase activity toward alpha-synuclein phosphorylation in an animal model of Parkinson's disease.

[This corrects the article DOI: 10.1371/journal.pone.0232019.].

The role of ubiquitin linkages on alpha-synuclein induced-toxicity in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is a common movement disorder marked by the loss of dopaminergic (DA) neurons in the brain stem and the presence of intraneuronal inclusions designated as Lewy bodies (LB). The cause of neurodegeneration in PD is not clear, but it has been suggested that protein misfolding and aggregation contribute significantly to the development of the disease. Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and autophagy lysosomal pathway (ALP). Recent studies suggested that different types of ubiquitin linkages can modulate these two pathways in the process of protein degradation. In this study, we found that co-expression of ubiquitin can rescue neurons from alpha-syn-induced neurotoxicity in a Drosophila model of PD. This neuroprotection is dependent on the formation of lysine 48 polyubiquitin linkage which is known to target protein degradation via the proteasome. Consistent with our results that we observed in vivo, we found that ubiquitin co-expression in the cell can facilitate cellular protein degradation by the proteasome in a lysine 48 polyubiquitin-dependent manner. Taken together, these results suggest that facilitation of proteasomal protein degradation can be a potential therapeutic approach for PD.

Acute action of rotenone on nigral dopaminergic neurons--involvement of reactive oxygen species and disruption of Ca2+ homeostasis.

Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05-1 microm) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 +/- 15 pA) associated with increases in intracellular [Ca(2+)] ([Ca(2+)](i)) (73.8 +/- 7.7 nm) and intracellular [Na(+)] (3.1 +/- 0.6 mm) (all with 1 microm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca(2+)](i) rise was abolished by removing extracellular Ca(2+), and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N-(p-amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca(2+)](i) rise resulted in a large (46.6 +/- 25.3 nm) Ca(2+) response when baseline [Ca(2+)](i) was increased by a 'priming' protocol that activated voltage-gated Ca(2+) channels. There was also a positive correlation between 'naturally' occurring variations in baseline [Ca(2+)](i) and the rotenone-induced [Ca(2+)](i) rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K(+) channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca(2+)](i) rise by a small increase in baseline [Ca(2+)](i).

Parkin and Hsp70 sacked by BAG5.

Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, are the major cause of early-onset Parkinson's disease (PD). In this issue of Neuron, Kalia et al. show that the bcl-2-associated athanogene 5 (BAG5) enhances dopamine neuron death in an in vivo model of PD through inhibiting the E3 ligase activity of parkin and the chaperone activity of Hsp70.

Novel enhancement mechanism of tyrosine hydroxylase enzymatic activity by nitric oxide through S-nitrosylation.

Tyrosine hydroxylase (TH) is a rate-limiting step enzyme in the synthesis of catecholamines. Catecholamines function both as hormone and neurotransmitters in the peripheral and central nervous systems, therefore TH's expression and enzymatic activity is tightly regulated by various mechanisms. Several post-translational modifications have been shown to regulate TH's enzymatic activity such as phosphorylation, nitration and S-glutathionylation. While phosphorylation at N-terminal of TH can activate its enzymatic activity, nitration and S-glutathionylation can inactivate TH. In this study, we found that TH can also be S-nitrosylated by nitric oxide (NO). S-nitrosylation is a reversible modification of cysteine (cys) residue in protein and is known to be an emerging signaling mechanism mediated by NO. We found that TH can be S-nitrosylated at cys 279 and TH S-nitrosylation enhances its enzymatic activity both in vitro and in vivo. These results provide a novel mechanism of how NO can modulate TH's enzymatic activity through S-nitrosylation.

Parkin mediates nonclassical, proteasomal-independent ubiquitination of synphilin-1: implications for Lewy body formation.

It is widely accepted that the familial Parkinson's disease (PD)-linked gene product, parkin, functions as a ubiquitin ligase involved in protein turnover via the ubiquitin-proteasome system. Substrates ubiquitinated by parkin are hence thought to be destined for proteasomal degradation. Because we demonstrated previously that parkin interacts with and ubiquitinates synphilin-1, we initially expected synphilin-1 degradation to be enhanced in the presence of parkin. Contrary to our expectation, we found that synphilin-1 is normally ubiquitinated by parkin in a nonclassical, proteasomal-independent manner that involves lysine 63 (K63)-linked polyubiquitin chain formation. Parkin-mediated degradation of synphilin-1 occurs appreciably only at an unusually high parkin to synphilin-1 expression ratio or when primed for lysine 48 (K48)-linked ubiquitination. In addition we found that parkin-mediated ubiquitination of proteins within Lewy-body-like inclusions formed by the coexpression of synphilin-1, alpha-synuclein, and parkin occurs predominantly via K63 linkages and that the formation of these inclusions is enhanced by K63-linked ubiquitination. Our results suggest that parkin is a dual-function ubiquitin ligase and that K63-linked ubiquitination of synphilin-1 by parkin may be involved in the formation of Lewy body inclusions associated with PD.

Accumulation of the authentic parkin substrate aminoacyl-tRNA synthetase cofactor, p38/JTV-1, leads to catecholaminergic cell death.

Autosomal-recessive juvenile parkinsonism (AR-JP) is caused by loss-of-function mutations of the parkin gene. Parkin, a RING-type E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of substrate proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). Accordingly, the abnormal accumulation of neurotoxic parkin substrates attributable to loss of parkin function may be the cause of neurodegeneration in parkin-related parkinsonism. We evaluated the known parkin substrates identified to date in parkin null mice to determine whether the absence of parkin results in accumulation of these substrates. Here we show that only the aminoacyl-tRNA synthetase cofactor p38 is upregulated in the ventral midbrain/hindbrain of both young and old parkin null mice. Consistent with upregulation in parkin knock-out mice, brains of AR-JP and idiopathic PD and diffuse Lewy body disease also exhibit increased level of p38. In addition, p38 interacts with parkin and parkin ubiquitinates and targets p38 for degradation. Furthermore, overexpression of p38 induces cell death that increases with tumor necrosis factor-alpha treatment and parkin blocks the pro-cell death effect of p38, whereas the R42P, familial-linked mutant of parkin, fails to rescue cell death. We further show that adenovirus-mediated overexpression of p38 in the substantia nigra in mice leads to loss of dopaminergic neurons. Together, our study represents a major advance in our understanding of parkin function, because it clearly identifies p38 as an important authentic pathophysiologic substrate of parkin. Moreover, these results have important implications for understanding the molecular mechanisms of neurodegeneration in PD.