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1.  A53T Human α-Synuclein Overexpression in Transgenic Mice Induces Pervasive Mitochondria Macroautophagy Defects Preceding Dopamine Neuron Degeneration 
The Journal of Neuroscience  2015;35(3):890-905.
In vitro evidence suggests that the inefficient removal of damaged mitochondria by macroautophagy contributes to Parkinson's disease (PD). Using a tissue-specific gene amplification strategy, we generated a transgenic mouse line with human α-synuclein A53T overexpression specifically in dopamine (DA) neurons. Transgenic mice showed profound early-onset mitochondria abnormalities, characterized by macroautophagy marker-positive cytoplasmic inclusions containing mainly mitochondrial remnants, which preceded the degeneration of DA neurons. Genetic deletion of either parkin or PINK1 in these transgenic mice significantly worsened mitochondrial pathologies, including drastically enlarged inclusions and loss of total mitochondria contents. These data suggest that mitochondria are the main targets of α-synuclein and their defective autophagic clearance plays a significant role during pathogenesis. Moreover, endogenous PINK1 or parkin is indispensable for the proper autophagic removal of damaged mitochondria. Our data for the first time establish an essential link between mitochondria macroautophagy impairments and DA neuron degeneration in an in vivo model based on known PD genetics. The model, its well-defined pathologies, and the demonstration of a main pathogenesis pathway in the present study have set the stage and direction of emphasis for future studies.
PMCID: PMC4300331  PMID: 25609609
alpha-synuclein; mitophagy; parkin; PINK1; positive feedback; transgenic model
2.  Drp1 inhibition attenuates neurotoxicity and dopamine release deficits in vivo 
Nature communications  2014;5:5244.
Mitochondrial dysfunction has been reported in both familial and sporadic Parkinson’s disease (PD). However, effective therapy targeting this pathway is currently inadequate. Recent studies suggest that manipulating the processes of mitochondrial fission and fusion has considerable potential for treating human diseases. To determine the therapeutic impact of targeting these pathways on PD, we used two complementary mouse models of mitochondrial impairments as seen in PD. We show here that blocking mitochondrial fission is neuroprotective in the PTEN-induced putative kinase-1 deletion (PINK1−/−) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models. Specifically, we show that inhibition of the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) using gene-based and small molecule approaches attenuates neurotoxicity and restores pre-existing striatal dopamine release deficits in these animal models. These results suggest Drp1 inhibition as a potential treatment for PD.
PMCID: PMC4223875  PMID: 25370169
3.  Drp1 inhibition attenuates neurotoxicity and dopamine release deficits in vivo 
Nature Communications  2014;5:5244.
Mitochondrial dysfunction has been reported in both familial and sporadic Parkinson’s disease (PD). However, effective therapy targeting this pathway is currently inadequate. Recent studies suggest that manipulating the processes of mitochondrial fission and fusion has considerable potential for treating human diseases. To determine the therapeutic impact of targeting these pathways on PD, we used two complementary mouse models of mitochondrial impairments as seen in PD. We show here that blocking mitochondrial fission is neuroprotective in the PTEN-induced putative kinase-1 deletion (PINK1−/−) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse models. Specifically, we show that inhibition of the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) using gene-based and small-molecule approaches attenuates neurotoxicity and restores pre-existing striatal dopamine release deficits in these animal models. These results suggest Drp1 inhibition as a potential treatment for PD.
Mitochondrial dysfunction has been associated with Parkinson’s disease but effective therapies targeting this pathway are yet to be developed. Here the authors show that inhibition of the mitochondrial fission protein Drp-1 using genetic or small-molecule approaches in mouse models of the disease, leads to improvements in the pathology.
PMCID: PMC4223875  PMID: 25370169
4.  Cyclic AMP and Afferent Activity Govern Bidirectional Synaptic Plasticity in Striatopallidal Neurons 
The Journal of Neuroscience  2014;34(19):6692-6699.
Recent experimental evidence suggests that the low dopamine conditions in Parkinson's disease (PD) cause motor impairment through aberrant motor learning. Those data, along with computational models, suggest that this aberrant learning results from maladaptive corticostriatal plasticity and learned motor inhibition. Dopaminergic modulation of both corticostriatal long-term depression (LTD) and long-term potentiation (LTP) is proposed to be critical for these processes; however, the regulatory mechanisms underlying bidirectional corticostriatal plasticity are not fully understood. Previously, we demonstrated a key role for cAMP signaling in corticostriatal LTD. In this study, mouse brain slices were used to perform a parametric experiment that tested the impact of varying both intracellular cAMP levels and the strength of excitatory inputs on corticostriatal plasticity. Using slice electrophysiology in the dorsolateral striatum, we demonstrate that both LTP and LTD can be sequentially induced in the same D2-expressing neuron and that LTP was strongest with high intracellular cAMP and LFS, whereas LTD required low intracellular cAMP and high-frequency stimulation. Our results provide a molecular and cellular basis for regulating bidirectional corticostriatal synaptic plasticity and may help to identify novel therapeutic targets for blocking or reversing the aberrant synaptic plasticity that likely contributes to motor deficits in PD.
PMCID: PMC4012319  PMID: 24806695
cAMP; dorsolateral striatum; LTD; LTP; motor learning; synaptic plasticity
5.  Loss of PINK1 Attenuates HIF-1α Induction by Preventing 4E-BP1-Dependent Switch in Protein Translation under Hypoxia 
The Journal of Neuroscience  2014;34(8):3079-3089.
Parkinson's disease (PD) has multiple proposed etiologies with implication of abnormalities in cellular homeostasis ranging from proteostasis to mitochondrial dynamics to energy metabolism. PINK1 mutations are associated with familial PD and here we discover a novel PINK1 mechanism in cellular stress response. Using hypoxia as a physiological trigger of oxidative stress and disruption in energy metabolism, we demonstrate that PINK1−/− mouse cells exhibited significantly reduced induction of HIF-1α protein, HIF-1α transcriptional activity, and hypoxia-responsive gene upregulation. Loss of PINK1 impairs both hypoxia-induced 4E-BP1 dephosphorylation and increase in the ratio of internal ribosomal entry site (IRES)-dependent to cap-dependent translation. These data suggest that PINK1 mediates adaptive responses by activating IRES-dependent translation, and the impairments in translation and the HIF-1α pathway may contribute to PINK1-associated PD pathogenesis that manifests under cellular stress.
PMCID: PMC3931509  PMID: 24553947
4EBP; hypoxia; IRES; Parkinson's disease; PINK1; translation
6.  A Nutritional Conditional Lethal Mutant Due to Pyridoxine 5′-Phosphate Oxidase Deficiency in Drosophila melanogaster 
G3: Genes|Genomes|Genetics  2014;4(6):1147-1154.
The concept of auxotrophic complementation has been proposed as an approach to identify genes in essential metabolic pathways in Drosophila melanogaster. However, it has achieved limited success to date, possibly due to the low probability of finding mutations fit with the chemically defined profile. Instead of using the chemically defined culture media lacking specific nutrients, we used bare minimum culture medium, i.e., 4% sucrose, for adult Drosophila. We identified a nutritional conditional lethal mutant and localized a c.95C > A mutation in the Drosophila pyridoxine 5′-phosphate oxidase gene [dPNPO or sugarlethal (sgll)] using meiotic recombination mapping, deficiency mapping, and whole genome sequencing. PNPO converts dietary vitamin B6 such as pyridoxine to its active form pyridoxal 5′-phosphate (PLP). The missense mutation (sgll95) results in the substitution of alanine to aspartate (p.Ala32Asp). The sgll95 flies survive well on complete medium but all die within 6 d on 4% sucrose only diet, which can be rescued by pyridoxine or PLP supplement, suggesting that the mutation does not cause the complete loss of PNPO activity. The sgll knockdown further confirms its function as the Drosophila PNPO. Because better tools for positional cloning and cheaper whole genome sequencing have made the identification of point mutations much easier than before, alleviating the necessity to pinpoint specific metabolic pathways before gene identification, we propose that nutritional conditional screens based on bare minimum growth media like ours represent promising approaches for discovering important genes and mutations in metabolic pathways, thereby accelerating the establishment of in vivo models that recapitulate human metabolic diseases.
PMCID: PMC4065258  PMID: 24739647
nutritional conditional lethal; auxotrophs; pyridoxine 5′-phosphate oxidase; vitamin B6; congenital metabolic diseases
The European journal of neuroscience  2012;36(4):2533-2546.
Recent findings suggest the reward system encodes metabolic value independent of taste, provoking speculation that the hedonic value of taste could be derived from nutritional value as a secondary appetitive property. We therefore dissociated and compared the impact of nutrition and taste on appetitive behavior in several paradigms. Though taste alone induces preference and increased consumption, in the absence of nutritional value its reinforcing properties are greatly diminished and it does not, like sucrose, induce increased responding over time. In agreement with behavioral data, saccharin- but not sucrose- evoked dopamine release is greatly attenuated following pre-exposure, suggesting that nutritional value is critical for dopamine mediated reward and reinforcement. Further supporting the primacy of nutrition over taste, genetically increased dopaminergic tone enhances incentive associated with nutritional value with minimal impact on taste-based, hedonic incentive. Overall, we suggest that the sensory-hedonic incentive value associated with taste functions as a conditioned stimulus that requires nutritional value to sustainably organize appetitive behavior.
PMCID: PMC3424366  PMID: 22712685
dopamine; reinforcement; feeding; obesity; palatability; reward; cyclic voltammetry
8.  A Role for Dopamine-Mediated Learning in the Pathophysiology and Treatment of Parkinson’s Disease 
Cell reports  2012;2(6):1747-1761.
Dopamine contributes to corticostriatal plasticity and motor learning. Dopamine denervation profoundly alters motor performance, as in Parkinson’s disease; however, the extent to which these symptoms reflect impaired motor learning is unknown. Here we demonstrate a D2 receptor blockade induced aberrant learning that impedes future motor performance when dopamine signaling is restored, an effect diminished by co-administration of adenosine antagonists during blockade. We hypothesize that an inappropriate corticostriatal potentiation in striatopallidal cells of the indirect pathway underlies aberrant learning. Here, we demonstrate synaptic potentiation in striatopallidal neurons induced by D2 blockade and diminished by application of an adenosine antagonist, consistent with behaviaoral observations. A neurocomputational model of the basal ganglia recapitulates the behavioral pattern and further links aberrant learning to plasticity in the indirect pathway. Thus, D2-mediated aberrant learning may contribute to motor deficits in PD and suggest new avenues for the development of therapeutics.
PMCID: PMC3538862  PMID: 23246005
9.  Dopaminergic enhancement of local food seeking is under global homeostatic control 
Recent work has implicated dopaminergic mechanisms in overeating and obesity with some researchers suggesting parallels between the dopamine dysregulation associated with addiction and an analogous dysregulation in obesity. The precise role of dopamine in mediating reward and reinforcement, however, remains controversial. In contrast to drugs of abuse, pursuit of a natural reward, such as food, is regulated by homeostatic processes that putatively maintain a stable energy balance keeping unrestrained consumption and reward pursuit in check. Understanding how the reward system is constrained by or escapes homeostatic regulation is a critical question. The widespread use of food restriction to motivate animal subjects in behavior paradigms precludes investigation of this relationship as the homeostatic system is locked into deficit mode. In the present study, we examine the role of dopamine in modulating adaptive feeding behavior in semi-naturalistic home cage paradigms where mice earn all their food from lever pressing. We compared consumption and meal patterning between hyperdopaminergic dopamine transporter knock-down mice (DATkd) with wild-type (WT) in two paradigms that introduce escalating costs for procuring food. We found that hyperdopaminergic mice exhibited similar demand elasticity, weight loss and energy balance in response to cost. However, the DATkd show clear differences in meal patterning. Consistent with expectations of enhanced motivation, elevated dopamine increased meal size and reduced intrameal cost sensitivity. Nonetheless, this did not alter overall energy balance. We conclude that elevated dopamine enhances incentive or willingness to work locally within meals without shifting energy balance, enhancing global food-seeking or generating an energy surplus.
PMCID: PMC3251719  PMID: 22118191
mouse; dopamine; feeding; homeostatic regulation; semi-naturalistic homecage operant; dopamine transporter knockdown
10.  Putting desire on a budget: dopamine and energy expenditure, reconciling reward and resources 
Accumulating evidence indicates integration of dopamine function with metabolic signals, highlighting a potential role for dopamine in energy balance, frequently construed as modulating reward in response to homeostatic state. Though its precise role remains controversial, the reward perspective of dopamine has dominated investigation of motivational disorders, including obesity. In the hypothesis outlined here, we suggest instead that the primary role of dopamine in behavior is to modulate activity to adapt behavioral energy expenditure to the prevailing environmental energy conditions, with the role of dopamine in reward and motivated behaviors derived from its primary role in energy balance. Dopamine has long been known to modulate activity, exemplified by psychostimulants that act via dopamine. More recently, there has been nascent investigation into the role of dopamine in modulating voluntary activity, with some investigators suggesting that dopamine may serve as a final common pathway that couples energy sensing to regulated voluntary energy expenditure. We suggest that interposed between input from both the internal and external world, dopamine modulates behavioral energy expenditure along two axes: a conserve-expend axis that regulates generalized activity and an explore-exploit axes that regulates the degree to which reward value biases the distribution of activity. In this view, increased dopamine does not promote consumption of tasty food. Instead increased dopamine promotes energy expenditure and exploration while decreased dopamine favors energy conservation and exploitation. This hypothesis provides a mechanistic interpretation to an apparent paradox: the well-established role of dopamine in food seeking and the findings that low dopaminergic functions are associated with obesity. Our hypothesis provides an alternative perspective on the role of dopamine in obesity and reinterprets the “reward deficiency hypothesis” as a perceived energy deficit. We propose that dopamine, by facilitating energy expenditure, should be protective against obesity. We suggest the apparent failure of this protective mechanism in Western societies with high prevalence of obesity arises as a consequence of sedentary lifestyles that thwart energy expenditure.
PMCID: PMC3400936  PMID: 22833718
reward; energy management; dopamine; basal ganglia; incentive-salience; cost sensitivity; effort; explore-exploit
11.  Dopamine-Dependent Motor Learning Insight into Levodopa’s Long-Duration Response 
Annals of neurology  2010;67(5):639-647.
Dopamine (DA) is critical for motor performance, motor learning, and corticostriatal plasticity. The relationship between motor performance and learning, and the role of DA in the mediation of them, however, remain unclear.
To examine this question, we took advantage of PITx3-deficient mice (aphakia mice), in which DA in the dorsal striatum is reduced by 90%. PITx3-deficient mice do not display obvious motor deficits in their home cage, but are impaired in motor tasks that require new motor skills. We used the accelerating rotarod as a motor learning task.
We show that the deficiency in motor skill learning in PITx3(−/−) is dramatic and can be rescued with levodopa treatment. In addition, cessation of levodopa treatment after acquisition of the motor skill does not result in an immediate drop in performance. Instead, there is a gradual decline of performance that lasts for a few days, which is not related to levodopa pharmacokinetics. We show that this gradual decline is dependent on the retesting experience.
This observation resembles the long-duration response to levodopa therapy in its slow buildup of improvement after the initiation of therapy and gradual degradation. We hypothesize that motor learning may play a significant, underappreciated role in the symptomatology of Parkinson disease as well as in the therapeutic effects of levodopa. We suggest that the important, yet enigmatic long-duration response to chronic levodopa treatment is a manifestation of rescued motor learning.
PMCID: PMC3129617  PMID: 20437561
12.  DJ-1 mRNA anatomical localization and cell type identification in the mouse brain 
Neuroscience letters  2009;465(3):214-219.
Mutations in DJ-1 cause familial Parkinson’s disease (PD). The expression pattern of DJ-1 in the brain remains controversial. In the present study, we used DJ-1 deficient mice as negative controls and examined DJ-1 mRNA expression in mouse brains. By sequential double labeling on the same sections, in situ hybridization of DJ-1 mRNA was followed by immunofluorescence detection for cell type markers. We found that DJ-1 mRNA was expressed in the majority of neurons in all brain areas examined. In particular, all dopamine neurons in the ventral midbrain expressed DJ-1 mRNA. Interestingly, the choroid plexus and ependymal cells lining the ventricles were the only non-neuronal regions strongly expressing DJ-1 mRNA. However, DJ-1 mRNA was not detected in astrocytes. DJ-1 mRNA expression in all nigra dopamine neurons but not in astrocytes suggests that its potential neuroprotective role could be cell-autonomous. The fact that DJ-1 expression is not restricted to substantia nigra dopamine neurons suggests that DJ-1 mutations may collaborate with other predisposing factors to cause the relatively selective dopamine neuron degeneration in Parkinson’s disease.
PMCID: PMC2852142  PMID: 19766170
13.  A reverse translational study of dysfunctional exploration in psychiatric disorders: from mice to men 
Archives of general psychiatry  2009;66(10):1072-1080.
Bipolar mania and schizophrenia are recognized as separate disorders but share many commonalities, raising the question of whether they are in fact the same disorder on different ends of a continuum. The lack of distinct endophenotypes of bipolar mania and schizophrenia has complicated the development of animal models that are specific to these disorders. Exploration is fundamental to survival and is dysregulated in these two disorders. Although exploratory behavior in rodents has been widely studied, surprisingly little work has examined this critical function in humans.
We used a novel human open field paradigm, the human Behavioral Pattern Monitor (BPM), to quantify exploratory behavior of individuals with bipolar mania and schizophrenia and to identify distinctive phenotypes of these illnesses.
Static group comparison.
Psychiatric hospital.
15 bipolar mania and 16 schizophrenia subjects were compared to 26 healthy volunteers in the human BPM. The effects of amphetamine, the selective dopamine transporter (DAT) inhibitor GBR12909, and genetic knockdown of the DAT were compared to controls in the mouse BPM.
The amount of motor activity, spatial patterns of activity, and exploration of novel stimuli were quantified in both the human and mouse BPMs.
Bipolar manic subjects demonstrated a unique exploratory pattern, characterized by high motor activity and increased object exploration. Schizophrenia subjects did not show the expected habituation of motor activity. Selective genetic or pharmacological inhibition of the DAT matched the mania phenotype better than the “gold standard” model of mania (amphetamine).
These findings validate the human open field paradigm and identify defining characteristics of bipolar mania that are distinct from schizophrenia. This cross-species study of exploration calls into question an accepted animal model of mania and should help to develop more accurate human and animal models, which are essential to identify neurobiological underpinnings of neuropsychiatric disorders.
PMCID: PMC2897252  PMID: 19805697
Dopamine (DA)-dependent corticostriatal plasticity is thought to underlie incremental procedural learning. A primary effector of striatal DA signaling is cAMP, yet its role in corticostriatal plasticity and striatum-dependent learning remains unclear. Here, we show that genetic deletion of a striatum-enriched isoform of adenylyl cyclase, AC5 (AC5KO), impairs two forms of striatum-dependent learning and corticostriatal synaptic plasticity. AC5KO mice were severely impaired in acquisition of a response strategy in the cross maze, a striatum dependent task requiring a correct body turn to find a goal arm. In addition, AC5KO mice were impaired in acquisition of a motor skill, as assessed by the accelerated rotarod. Slice electrophysiology revealed a deficit in corticostriatal LTD following high frequency stimulation of tissue from AC5KO mice. LTD was rescued by activation of either presynaptic cannabinoid type 1 (CB1) receptors, or postsynaptic metabotropic glutamate receptors (mGluRs), suggesting a postsynaptic role of AC5-cAMP, upstream of endocannabinoid release. In striatopallidal projecting medium spiny neurons (MSNs), DA D2 receptors are negatively coupled to cAMP production and activation of these receptors is required for endocannabinoid release and corticostriatal LTD. Recordings from striatopallidal neurons indicated that this is mediated by AC5, as co-activation of D2 and mGluR receptors could induce LTD in WT, but not in AC5KO neurons. To further examine the role of cAMP in corticostriatal plasticity, we elevated cAMP in striatal neurons of wild-type mice via the recording electrode. Under these conditions corticostriatal LTD was eliminated. Together, these data suggest an AC5-cAMP-endocannabinoid-CB1 signaling pathway in corticostriatal plasticity and striatum-dependent learning.
PMCID: PMC2782774  PMID: 19793969
Adenylyl cyclase; striatum; motor learning; plasticity; dopamine; LTD
15.  Tonic Dopamine Modulates Exploitation of Reward Learning 
The impact of dopamine on adaptive behavior in a naturalistic environment is largely unexamined. Experimental work suggests that phasic dopamine is central to reinforcement learning whereas tonic dopamine may modulate performance without altering learning per se; however, this idea has not been developed formally or integrated with computational models of dopamine function. We quantitatively evaluate the role of tonic dopamine in these functions by studying the behavior of hyperdopaminergic DAT knockdown mice in an instrumental task in a semi-naturalistic homecage environment. In this “closed economy” paradigm, subjects earn all of their food by pressing either of two levers, but the relative cost for food on each lever shifts frequently. Compared to wild-type mice, hyperdopaminergic mice allocate more lever presses on high-cost levers, thus working harder to earn a given amount of food and maintain their body weight. However, both groups show a similarly quick reaction to shifts in lever cost, suggesting that the hyperdominergic mice are not slower at detecting changes, as with a learning deficit. We fit the lever choice data using reinforcement learning models to assess the distinction between acquisition and expression the models formalize. In these analyses, hyperdopaminergic mice displayed normal learning from recent reward history but diminished capacity to exploit this learning: a reduced coupling between choice and reward history. These data suggest that dopamine modulates the degree to which prior learning biases action selection and consequently alters the expression of learned, motivated behavior.
PMCID: PMC2991243  PMID: 21120145
dopamine; reinforcement learning; DAT knock-down; explore-exploit; behavioral flexibility; environmental adaptation
16.  Loss of Cocaine Locomotor Response in Pitx3-Deficient Mice Lacking a Nigrostriatal Pathway 
Both the dorsal and ventral striatum have been demonstrated to have a critical role in reinforcement learning and addiction. Dissecting the specific function of these striatal compartments and their associated nigrostriatal and mesoaccumbens dopamine pathways, however, has proved difficult. Previous studies using lesions to isolate the contribution of nigrostriatal and mesoaccumbens dopamine in mediating the locomotor and reinforcing effects of psychostimulant drugs have yielded inconsistent and inconclusive results. Using a naturally occurring mutant mouse line, aphakia, that lacks a nigrostriatal dopamine pathway but retains an intact mesoaccumbens pathway, we show that the locomotor activating effects of cocaine, including locomotor sensitization, are dependent on an intact nigrostriatal dopamine projection. In contrast, cocaine reinforcement, as measured by conditioned place preference and cocaine sensitization of sucrose preference, is intact in these mice. In light of the well-established role of the nucleus accumbens in mediating the effects of psychostimulants, these data suggest that the nigrostriatal pathway can act as a critical effector mechanism for the nucleus accumbens highlighting the importance of intrastriatal connectivity and providing insight into the functional architecture of the striatum.
PMCID: PMC2752723  PMID: 18704092
cocaine; conditioned place preference; Pitx3; locomotor sensitization; dorsal striatum; nucleus accumbens
17.  Decline in NRF2-regulated Antioxidants in Chronic Obstructive Pulmonary Disease Lungs Due to Loss of Its Positive Regulator, DJ-1 
Rationale: Oxidative stress is a key contributor in chronic obstructive pulmonary disease (COPD) pathogenesis caused by cigarette smoking. NRF2, a redox-sensitive transcription factor, dissociates from its inhibitor, KEAP1, to induce antioxidant expression that inhibits oxidative stress.
Objectives: To determine the link between severity of COPD, oxidative stress, and NRF2-dependent antioxidant levels in the peripheral lung tissue of patients with COPD.
Methods: We assessed the expression of NRF2, NRF2-dependent antioxidants, regulators of NRF2 activity, and oxidative damage in non-COPD (smokers and former smokers) and smoker COPD lungs (mild and advanced). Cigarette smoke–exposed human lung epithelial cells (Beas2B) and mice were used to understand the mechanisms.
Measurements and Main Results: When compared with non-COPD lungs, the COPD patient lungs showed (1) marked decline in NRF2-dependent antioxidants and glutathione levels, (2) increased oxidative stress markers, (3) significant decrease in NRF2 protein with no change in NRF2 mRNA levels, and (4) similar KEAP1 but significantly decreased DJ-1 levels (a protein that stabilizes NRF2 protein by impairing KEAP1-dependent proteasomal degradation of NRF2). Exposure of Bea2B cells to cigarette smoke caused oxidative modification and enhanced proteasomal degradation of DJ-1 protein. Disruption of DJ-1 in mouse lungs, mouse embryonic fibroblasts, and Beas2B cells lowered NRF2 protein stability and impaired antioxidant induction in response to cigarette smoke. Interestingly, targeting KEAP1 by siRNA or the small-molecule activator sulforaphane restored induction of NRF2-dependent antioxidants in DJ-1–disrupted cells in response to cigarette smoke.
Conclusions: NRF2-dependent antioxidants and DJ-1 expression was negatively associated with severity of COPD. Therapy directed toward enhancing NRF2-regulated antioxidants may be a novel strategy for attenuating the effects of oxidative stress in the pathogenesis of COPD.
PMCID: PMC2542433  PMID: 18556627
chronic obstructive pulmonary disease; NRF2; DJ-1; oxidative stress; antioxidants
18.  A cAMP pathway underlying reward prediction in associative learning 
In associative learning, animals learn to associate external cues or their own actions with appetitive or aversive outcomes. Although the dopamine (DA) system and the striatum/nucleus accumbens have been implicated in both the Pavlovian and instrumental form of associative learning, whether specific neuronal signaling mechanisms underlie one form or the other is unknown. Here we report that the striatum-enriched isoform of adenylyl cyclase (AC), AC5, is selectively required for appetitive Pavlovian learning. Mice with genetic deletion of AC5 (AC5KO) acquired instrumental responding, yet were unable to use cues that predicted reward delivery. The specificity of this deficit was confirmed by an inability of AC5KO mice to learn a simple appetitive Pavlovian conditioning task. Conversely, AC5KO mice showed intact aversive Pavlovian learning, suggesting the deficit was specific for learning about appetitive outcomes. Our results suggest that AC5 is a critical component of DA dependent strengthening of stimulus-reward contingencies.
PMCID: PMC2610269  PMID: 18971482
Pavlovian conditioning; dopamine; instrumental conditioning; striatum; adenylyl cyclase; reward prediction
19.  Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation 
Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock–induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis.
PMCID: PMC2648688  PMID: 19229105
20.  Sucrose Exposure in Early Life Alters Adult Motivation and Weight Gain 
PLoS ONE  2008;3(9):e3221.
The cause of the current increase in obesity in westernized nations is poorly understood but is frequently attributed to a ‘thrifty genotype,’ an evolutionary predisposition to store calories in times of plenty to protect against future scarcity. In modern, industrialized environments that provide a ready, uninterrupted supply of energy-rich foods at low cost, this genetic predisposition is hypothesized to lead to obesity. Children are also exposed to this ‘obesogenic’ environment; however, whether such early dietary experience has developmental effects and contributes to adult vulnerability to obesity is unknown. Using mice, we tested the hypothesis that dietary experience during childhood and adolescence affects adult obesity risk. We gave mice unlimited or no access to sucrose for a short period post-weaning and measured sucrose-seeking, food consumption, and weight gain in adulthood. Unlimited access to sucrose early in life reduced sucrose-seeking when work was required to obtain it. When high-sugar/high-fat dietary options were made freely-available, however, the sucrose-exposed mice gained more weight than mice without early sucrose exposure. These results suggest that early, unlimited exposure to sucrose reduces motivation to acquire sucrose but promotes weight gain in adulthood when the cost of acquiring palatable, energy dense foods is low. This study demonstrates that early post-weaning experience can modify the expression of a ‘thrifty genotype’ and alter an adult animal's response to its environment, a finding consistent with evidence of pre- and peri-natal programming of adult obesity risk by maternal nutritional status. Our findings suggest the window for developmental effects of diet may extend into childhood, an observation with potentially important implications for both research and public policy in addressing the rising incidence of obesity.
PMCID: PMC2529404  PMID: 18797507
21.  Cocaine reward and locomotion stimulation in mice with reduced dopamine transporter expression 
BMC Neuroscience  2007;8:42.
The dopamine transporter (DAT) plays a critical role in regulating dopamine neurotransmission. Variations in DAT or changes in basal dopaminergic tone have been shown to alter behavior and drug responses. DAT is one of the three known high affinity targets for cocaine, a powerful psychostimulant that produces reward and stimulates locomotor activity in humans and animals. We have shown that cocaine no longer produces reward in knock-in mice with a cocaine insensitive mutant DAT (DAT-CI), suggesting that cocaine inhibition of DAT is critical for its rewarding effect. However, in DAT-CI mice, the mutant DAT has significantly reduced uptake activity resulting in elevated basal dopaminergic tone, which might cause adaptive changes that alter responses to cocaine. Therefore, the objective of this study is to determine how elevated dopaminergic tone affects how mice respond to cocaine.
We examined the cocaine induced behavior of DAT knockdown mice that have DAT expression reduced by 90% when compared to the wild type mice. Despite a dramatic reduction of DAT expression and marked elevation in basal dopamine tone, cocaine produced reward, as measured by conditioned place preference, and stimulated locomotor activity in these mice.
A reduction in DAT expression and elevation of dopaminergic tone do not lead to adaptive changes that abolish the rewarding and stimulating effects of cocaine. Therefore, the lack of reward to cocaine observed in DAT-CI mice is unlikely to have resulted from the reduced DAT activity but instead is likely due to the inability of cocaine to block the mutated DAT and increase extracellular dopamine. This study supports the conclusion that the blockade of DAT is required for cocaine reward and locomotor stimulation.
PMCID: PMC1914080  PMID: 17584943
22.  Absence of Ret Signaling in Mice Causes Progressive and Late Degeneration of the Nigrostriatal System 
PLoS Biology  2007;5(3):e39.
Support of ageing neurons by endogenous neurotrophic factors such as glial cell line–derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) may determine whether the neurons resist or succumb to neurodegeneration. GDNF has been tested in clinical trials for the treatment of Parkinson disease (PD), a common neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons. BDNF modulates nigrostriatal functions and rescues DA neurons in PD animal models. The physiological roles of GDNF and BDNF signaling in the adult nigrostriatal DA system are unknown. We generated mice with regionally selective ablations of the genes encoding the receptors for GDNF (Ret) and BDNF (TrkB). We find that Ret, but not TrkB, ablation causes progressive and adult-onset loss of DA neurons specifically in the substantia nigra pars compacta, degeneration of DA nerve terminals in striatum, and pronounced glial activation. These findings establish Ret as a critical regulator of long-term maintenance of the nigrostriatal DA system and suggest conditional Ret mutants as useful tools for gaining insights into the molecular mechanisms involved in the development of PD.
Author Summary
What does a neuron need to survive? Our body produces its own survival factors for neurons, so-called neurotrophic factors, which have additional roles in neuron differentiation, growth, and function. Declining production of a neurotrophic factor or impaired signal transduction in ageing neurons may contribute to pathological neurodegeneration in humans. Glial cell line–derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) have been suggested as survival factors for midbrain dopaminergic neurons, a group of neurons primarily affected in Parkinson disease.
To investigate the physiological requirements for GDNF and BDNF to establish and maintain an important output pathway of these neurons—the nigrostriatal pathway—in the intact brain, we generated mutant mice with regionally selective ablations of the receptors for these survival factors, Ret (receptor of GDNF and related family members) or TrkB (BDNF receptor). Surprisingly, these mice survive to adulthood and show normal development and maturation of the nigrostriatal system. However, in ageing mice, ablation of Ret leads to a progressive and cell-type–specific loss of substantia nigra pars compacta neurons and their projections into the striatum. Our findings establish Ret and subsequent downstream effectors as critical regulators of long-term maintenance of the nigrostriatal system.
Ret, a receptor for glial cell line-derived neurotrophic factor, selectively regulates long-term maintenance of the nigrostriatal dopaminergic system.
PMCID: PMC1808500  PMID: 17298183
23.  Sequential super-stereotypy of an instinctive fixed action pattern in hyper-dopaminergic mutant mice: a model of obsessive compulsive disorder and Tourette's 
BMC Biology  2005;3:4.
Excessive sequential stereotypy of behavioral patterns (sequential super-stereotypy) in Tourette's syndrome and obsessive compulsive disorder (OCD) is thought to involve dysfunction in nigrostriatal dopamine systems. In sequential super-stereotypy, patients become trapped in overly rigid sequential patterns of action, language, or thought. Some instinctive behavioral patterns of animals, such as the syntactic grooming chain pattern of rodents, have sufficiently complex and stereotyped serial structure to detect potential production of overly-rigid sequential patterns. A syntactic grooming chain is a fixed action pattern that serially links up to 25 grooming movements into 4 predictable phases that follow 1 syntactic rule. New mutant mouse models allow gene-based manipulation of brain function relevant to sequential patterns, but no current animal model of spontaneous OCD-like behaviors has so far been reported to exhibit sequential super-stereotypy in the sense of a whole complex serial pattern that becomes stronger and excessively rigid. Here we used a hyper-dopaminergic mutant mouse to examine whether an OCD-like behavioral sequence in animals shows sequential super-stereotypy. Knockdown mutation of the dopamine transporter gene (DAT) causes extracellular dopamine levels in the neostriatum of these adult mutant mice to rise to 170% of wild-type control levels.
We found that the serial pattern of this instinctive behavioral sequence becomes strengthened as an entire entity in hyper-dopaminergic mutants, and more resistant to interruption. Hyper-dopaminergic mutant mice have stronger and more rigid syntactic grooming chain patterns than wild-type control mice. Mutants showed sequential super-stereotypy in the sense of having more stereotyped and predictable syntactic grooming sequences, and were also more likely to resist disruption of the pattern en route, by returning after a disruption to complete the pattern from the appropriate point in the sequence. By contrast, wild-type mice exhibited weaker forms of the fixed action pattern, and often failed to complete the full sequence.
Sequential super-stereotypy occurs in the complex fixed action patterns of hyper-dopaminergic mutant mice. Elucidation of the basis for sequential super-stereotypy of instinctive behavior in DAT knockdown mutant mice may offer insights into neural mechanisms of overly-rigid sequences of action or thought in human patients with disorders such as Tourette's or OCD.
PMCID: PMC552313  PMID: 15710042
24.  PINK1 Deficiency Attenuates Astrocyte Proliferation Through Mitochondrial Dysfunction, Reduced AKT and Increased p38 MAPK Activation, and Downregulation of EGFR 
Glia  2013;61(5):800-812.
PINK1 (PTEN induced putative kinase 1), a familial Parkinson's disease (PD)-related gene, is expressed in astrocytes, but little is known about its role in this cell type. Here, we found that astrocytes cultured from PINK1-knockout (KO) mice exhibit defective proliferative responses to epidermal growth factor (EGF) and fetal bovine serum. In PINK1-KO astrocytes, basal and EGF-induced p38 activation (phosphorylation) were increased whereas EGF receptor (EGFR) expression and AKT activation were decreased. p38 inhibition (SB203580) or knockdown with small interfering RNA (siRNA) rescued EGFR expression and AKT activation in PINK1-KO astrocytes. Proliferation defects in PINK1-KO astrocytes appeared to be linked to mitochondrial defects, manifesting as decreased mitochondrial mass and membrane potential, increased intracellular reactive oxygen species level, decreased glucose-uptake capacity, and decreased ATP production. Mitochondrial toxin (oligomycin) and a glucose-uptake inhibitor (phloretin) mimicked the PINK1-deficiency phenotype, decreasing astrocyte proliferation, EGFR expression and AKT activation, and increasing p38 activation. In addition, the proliferation defect in PINK1-KO astrocytes resulted in a delay in the wound healing process. Taken together, these results suggest that PINK1 deficiency causes astrocytes dysfunction, which may contribute to the development of PD due to delayed astrocytes-mediated repair of microenvironment in the brain.
PMCID: PMC3657120  PMID: 23440919
PINK1; astrocyte; proliferation; Parkinson's disease

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