Characterizing dopaminergic neuron vulnerability using genome-wide analysis

Parkinson’s disease (PD) is primarily characterized by the loss of dopaminergic (DA) neurons in the brain. However, little is known about why DA neurons are selectively vulnerable to PD. To identify genes that are associated with DA neuron loss, we screened through 201 wild-caught populations of Drosophila melanogaster as part of the Drosophila Genetic Reference Panel. Here, we identify the top-associated genes containing single-nucleotide polymorphisms that render DA neurons vulnerable. These genes were further analyzed by using mutant analysis and tissue-specific knockdown for functional validation. We found that this loss of DA neurons caused progressive locomotor dysfunction in mutants and gene knockdown analysis. The identification of genes associated with the progressive loss of DA neurons should help to uncover factors that render these neurons vulnerable in PD, and possibly develop strategies to make these neurons more resilient.     

IDH2 deficiency promotes mitochondrial dysfunction and dopaminergic neurotoxicity: implications for Parkinson’s disease

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and its pathogenesis is under intense investigation. Substantial evidence indicates that mitochondrial dysfunction and oxidative stress play central roles in the pathophysiology of PD, through activation of mitochondria-dependent apoptotic molecular pathways. Several mitochondrial internal regulating factors act to maintain mitochondrial function. However, the mechanism by which these internal regulating factors contribute to mitochondrial dysfunction in PD remains elusive. One of these factors, mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2), has been implicated in the regulation of mitochondrial redox balance and reduction of oxidative stress-induced cell injury. Here we report that IDH2 regulates mitochondrial dysfunction and cell death in MPP⁺/MPTP-induced DA neuronal cells, and in a mouse model of PD. Down-regulation of IDH2 increased DA neuron sensitivity to MPP⁺; lowered IDH2 levels facilitated induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Deficient IDH2 also promoted loss of DA SNpc neurons in an MPTP mouse model of PD. Interestingly, Mito-TEMPO, a mitochondrial ROS-specific scavenger, protected degeneration of SNpc DA neurons in the MPTP model of PD. These findings demonstrate that IDH2 contributes to degeneration of the DA neuron in the neurotoxin model of PD and establish IDH2 as a molecular target of potential therapeutic significance for this disabling neurological illness.     

Drosophila DJ-1 mutants are sensitive to oxidative stress and show reduced lifespan and motor deficits

Parkinson's disease (PD) is a progressive movement disorder caused by the selective and massive loss of dopaminergic neurons (DA) in the substantia nigra pars compacta (SNc). DJ-1 loss-of-function mutations are involved in inherited early-onset PD forms and result in dysfunction of the oxidative stress response. In mice models, DJ-1 loss provokes sensitivity to oxidative insults but does not produce neurodegeneration. Similar results have been found when analyzing Drosophila mutants for the DJ-1 orthologous genes, DJ-1 and DJ-1β. Here, we report the analysis of two new mutations for the Drosophila DJ-1 genes. Both ubiquitous induction of DJ-1 knockdown by RNAi and loss of function of DJ-1β caused by an insertional mutation result in increased sensitivity to paraquat insults, reduced lifespan and motor impairments. However these mutations do not lead to DA neuron loss. Besides, we find that targeted inhibition of DJ-1 function in DA neuron results in certain DA neurodegeneration. Our results, together with findings in other Drosophila DJ-1 mutants, indicate that both Drosophila DJ-1 genes are implicated in the protection against the chemical induced oxidative stress response, but also in fly survival. The differences observed in DA neurodegeneration suggest that the motor impairments exhibited by the mutants could be caused by different pathways.     

The Divalent Metal Transporter Homologues SMF-1/2 Mediate Dopamine Neuron Sensitivity in Caenorhabditis elegans Models of Manganism and Parkinson Disease

Parkinson disease (PD) and manganism are characterized by motor deficits and a loss of dopamine (DA) neurons in the substantia nigra pars compacta. Epidemiological studies indicate significant correlations between manganese exposure and the propensity to develop PD. The vertebrate divalent metal transporter-1 (DMT-1) contributes to maintaining cellular Mn²⁺ homeostasis and has recently been implicated in Fe²⁺-mediated neurodegeneration in PD. In this study we describe a novel model for manganism that incorporates the genetically tractable nematode Caenorhabditis elegans. We show that a brief exposure to Mn²⁺ increases reactive oxygen species and glutathione production, decreases oxygen consumption and head mitochondria membrane potential, and confers DA neuronal death. DA neurodegeneration is partially dependent on a putative homologue to DMT-1, SMF-1, as genetic knockdown or deletion partially inhibits the neuronal death. Mn²⁺ also amplifies the DA neurotoxicity of the PD-associated protein α-synuclein. Furthermore, both SMF-1 and SMF-2 are expressed in DA neurons and contribute to PD-associated neurotoxicant-induced DA neuron death. These studies describe a C. elegans model for manganism and show that DMT-1 homologues contribute to Mn²⁺- and PD-associated DA neuron vulnerability.     

Zebrafish Tyrosine Hydroxylase 2 Gene Encodes Tryptophan Hydroxylase

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.     

Non-Motor and Motor Features in LRRK2 Transgenic Mice

Background

Non-motor symptoms are increasingly recognized as important features of Parkinson’s disease (PD). LRRK2 mutations are common causes of familial and sporadic PD. Non-motor features have not been yet comprehensively evaluated in LRRK2 transgenic mouse models.

Objective

Using a transgenic mouse model overexpressing the R1441G mutation of the human LRRK2 gene, we have investigated the longitudinal correlation between motor and non-motor symptoms and determined if specific non-motor phenotypes precede motor symptoms.

Methodology

We investigated the onset of motor and non-motor phenotypes on the LRRK2^R1441G BAC transgenic mice and their littermate controls from 4 to 21 month-old using a battery of behavioral tests. The transgenic mutant mice displayed mild hypokinesia in the open field from 16 months old, with gastrointestinal dysfunctions beginning at 6 months old. Non-motor features such as depression and anxiety-like behaviors, sensorial functions (pain sensitivity and olfaction), and learning and memory abilities in the passive avoidance test were similar in the transgenic animals compared to littermate controls.

Conclusions

LRRK2^R1441G BAC transgenic mice displayed gastrointestinal dysfunction at an early stage but did not have abnormalities in fine behaviors, olfaction, pain sensitivity, mood disorders and learning and memory compared to non-transgenic littermate controls. The observations on olfaction and gastrointestinal dysfunction in this model validate findings in human carriers. These mice did recapitulate mild Parkinsonian motor features at late stages but compensatory mechanisms modulating the progression of PD in these models should be further evaluated.     

Experimental Strategy to Identify Genes Susceptible to Oxidative Stress in Nigral Dopaminergic Neurons

Neuropathological evidence from both human and experimental models of Parkinson's disease (PD) firmly supports a significant role for oxidative stress (OS) in the death of dopaminergic (DA) neurons in substantia nigra. Largely unknown are the genes underlying selective susceptibility of nigral DA neuron to OS and how they effect nigral DA cell death. The major barriers to high-throughput identification of candidate genes are the paucity of nigral DA neurons as well as the dilution effect of non-DA cells both in primary cultures and brain tissues. To overcome these barriers, we have developed a DA cell line model, SN4741, appropriate for cDNA microarray analysis. Candidate genes were selected from both the microarray analysis and the molecular implication of their pathological mechanisms (i.e., decreased mitochondrial complex I activity and proteasomal dysfunction) of PD. Subsequent secondary validation tests were devised to characterize genes including clone #45 that may underlie selective vulnerability of nigral DA neuron to OS.     

Motor neuronal protection by l-arginine prolongs survival of mutant SOD1 (G93A) ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that l-arginine protects cultured motor neurons from excitotoxic injury. We also found that l-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, l-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that l-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.     

Targeted Overexpression of α-Synuclein by rAAV2/1 Vectors Induces Progressive Nigrostriatal Degeneration and Increases Vulnerability to MPTP in Mouse

Mutations, duplication and triplication of α-synuclein genes are linked to familial Parkinson’s disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.     

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson''s disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.     

VPS35 D620N knockin mice recapitulate cardinal features of Parkinson’s disease

D620N mutation in the vacuolar protein sorting 35 ortholog (VPS35) gene causes late‐onset, autosomal dominant familial Parkinson''s disease (PD) and contributes to idiopathic PD. However, how D620N mutation leads to PD‐related deficits in vivo remains unclear. In the present study, we thoroughly characterized the biochemical, pathological, and behavioral changes of a VPS35 D620N knockin (KI) mouse model with chronic aging. We reported that this VPS35 D620N KI model recapitulated a spectrum of cardinal features of PD at 14 months of age which included age‐dependent progressive motor deficits, significant changes in the levels of dopamine (DA) and DA metabolites in the striatum, and robust neurodegeneration of the DA neurons in the SNpc and DA terminals in the striatum, accompanied by increased neuroinflammation, and accumulation and aggregation of α‐synuclein in DA neurons. Mechanistically, D620N mutation induced mitochondrial fragmentation and dysfunction in aged mice likely through enhanced VPS35‐DLP1 interaction and increased turnover of mitochondrial DLP1 complexes in vivo. Finally, the VPS35 D620N KI mice displayed greater susceptibility to MPTP‐mediated degeneration of nigrostriatal pathway, indicating that VPS35 D620N mutation increased vulnerability of DA neurons to environmental toxins. Overall, this VPS35 D620N KI mouse model provides a powerful tool for future disease modeling and pharmacological studies of PD. Our data support the involvement of VPS35 in the development of α‐synuclein pathology in vivo and revealed the important role of mitochondrial fragmentation/dysfunction in the pathogenesis of VPS35 D620N mutation‐associated PD in vivo.     

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson''s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.     

Neuroprotective Effects of Protocatechuic Aldehyde against Neurotoxin-Induced Cellular and Animal Models of Parkinson’s Disease

Protocatechuic aldehyde (PAL) has been reported to bind to DJ-1, a key protein involved in Parkinson’s disease (PD), and exerts potential neuroprotective effects via DJ-1 in SH-SY5Y cells. In this study, we investigated the neuroprotective pharmacological effects of PAL against neurotoxin-induced cell and animal models of PD. In cellular models of PD, PAL markedly increased cell viability rates, mitochondrial oxidation-reduction activity and mitochondrial membrane potential, and reduced intracellular ROS levels to prevent neurotoxicity in PC12 cells. In animal models of PD, PAL reduced the apomorphine injection, caused turning in 6-OHDA treated rats, and increased the motor coordination and stride decreases in MPTP treated mice. Meanwhile, in an MPTP mouse model, PAL prevented a decrease of the contents of dopamine (DA) and its metabolites in the striatum and TH-positive dopaminergic neuron loss in the substantia nigra (SN). In addition, PAL increased the protein expression of DJ-1 and reduced the level of α-synuclein in the SN of MPTP lesioned mice. PAL also increased the spine density in hippocampal CA1 neurons. The current study demonstrates that PAL can efficiently protect dopaminergic neurons against neurotoxin injury in vitro and in vivo, and that the potential mechanisms may be related to its effects in increasing DJ-1, decreasing α-synuclein and its growth-promoting effect on spine density.     

The role of dopamine oxidation in mitochondrial dysfunction: implications for Parkinson’s disease

The etiology of sporadic Parkinson’s disease (PD) is unknown, although mitochondrial dysfunction and oxidative stress have been implicated in the mechanisms associated with PD pathogenesis. Dopamine (DA) neurons of the substantia nigra pars compacta have been shown to degenerate to a greater extent in PD than other neurons suggesting the possibility that DA itself may be contributing to the neurodegenerative process. This review discusses our work on the effects of DA oxidation and reactive DA quinones on mitochondrial function and protein modification and the potential for exacerbating toxicity associated with mitochondrial dysfunction in PD.     

Presymptomatic biochemical changes in hindlimb muscle of G93A human Cu/Zn superoxide dismutase 1 transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is primarily a motor neuron disorder. Intriguingly, early muscle denervation preceding motor neuron loss is observed in mouse models of ALS. Enhanced muscle vulnerability to denervation process has been suggested by accelerated muscle deterioration following peripheral nerve injury in an ALS mouse model. Here we provide evidence of biochemical changes in the hindlimb muscle of young, presymptomatic G93A hSOD1 transgenic mice. In this report, we demonstrate that cdk5 activity is reduced in hindlimb muscle of 27-day-old G93A hSOD1 transgenic mice. In vitro analysis revealed mutant hSOD1-mediated suppression of cdk5 activity. Furthermore, the decrease in muscle cdk5 activity was accompanied by a significant reduction in MyoD and cyclin D1 levels. These early muscle changes raise the possibility that the progressive deterioration of muscle function is potentiated by altered muscle biochemistry in these mice at a very young, presymptomatic age.     

Neuroprotective effects of erythropoietin on 6-hydroxydopamine-treated ventral mesencephalic dopamine-rich cultures

Parkinson's disease (PD) is a neurodegenerative disorder with motor symptoms caused by the loss of dopaminergic (DA) cells and consequently dopamine release in the nigrostriatal system. In vivo and in vitro 6-hydroxydopamine (6-OHDA) PD models are widely used to study the effect of striatal dopamine depletion as well as novel neuroprotective or restorative therapeutic strategies for PD. In the present study, we investigated in vitro the toxicity of 6-OHDA on DA neurons derived from E14 rat ventral mesencephalon (VM) and the neuroprotective efficiency of erythropoietin (Epo) on VM-derived cell cultures against 6-OHDA toxicity. Using E14 VM-derived DA-rich primary cultures, we could demonstrate that 6-OHDA toxicity works in a time-and concentration-dependent way, and leads to cell death not only in DA cells but also in non-DA cells in direct relation to concentration and incubation times. In addition, we found that 6-OHDA toxicity induces caspase-3 activation and an increment of intracellular reactive oxygen species (ROS) in VM-derived cultures. When 6-OHDA-treated VMs were cultured in the presence of the anti-apoptotic protein erythropoietin (Epo), the total neuronal population, including the DA neurons, was protected. However, untreated VM cultures exposed to Epo showed an increase in the total neuronal population, but not an additional increase in DA neuron cell number.These findings suggest that 6-OHDA toxicity is time and concentration-dependent and does not exclusively affect DA neurons. In high concentration and long incubation times, 6-OHDA influences the survival of other neuronal and non-neuronal cell populations derived from the VM cultures. 6-OHDA toxicity induces caspase-3 activation, indicating cell death via the apoptotic pathway which could be restricted or even prevented by pre-exposure to Epo, known to interact via the apoptotic pathway. Our results support and expand on previous findings showing that Epo is an interesting candidate molecule to mediate neuroprotective effects on DA neurons in PD. Furthermore, it could be used in promoting the survival of DA neurons after transplantation in clinical trials.