The specific NQO2 inhibitor, S29434, only marginally improves the survival of dopamine neurons in MPTP-intoxicated mice.

Over the years, evidence has accumulated on a possible contributive role of the cytosolic quinone reductase NQO2 in models of dopamine neuron degeneration induced by parkinsonian toxin, but most of the data have been obtained in vitro. For this reason, we asked the question whether NQO2 is involved in the in vivo toxicity of MPTP, a neurotoxin classically used to model Parkinson disease-induced neurodegeneration. First, we show that NQO2 is expressed in mouse substantia nigra dopaminergic cell bodies and in human dopaminergic SH-SY5Y cells as well. A highly specific NQO2 inhibitor, S29434, was able to reduce MPTP-induced cell death in a co-culture system of SH-SY5Y cells with astrocytoma U373 cells but was inactive in SH-SY5Y monocultures. We found that S29434 only marginally prevents substantia nigra tyrosine hydroxylase+ cell loss after MPTP intoxication in vivo. The compound produced a slight increase of dopaminergic cell survival at day 7 and 21 following MPTP treatment, especially with 1.5 and 3 mg/kg dosage regimen. The rescue effect did not reach statistical significance (except for one experiment at day 7) and tended to decrease with the 4.5 mg/kg dose, at the latest time point. Despite the lack of robust protective activity of the inhibitor of NQO2 in the mouse MPTP model, we cannot rule out a possible role of the enzyme in parkinsonian degeneration, particularly because it is substantially expressed in dopaminergic neurons.

Genes critical for development and differentiation of dopaminergic neurons are downregulated in Parkinson's disease.

We performed transcriptome analysis using RNA sequencing on substantia nigra pars compacta (SNpc) from mice after acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment and from Parkinson's disease (PD) patients. Acute and chronic exposure to MPTP resulted in decreased expression of genes involved in sodium channel regulation. However, upregulation of pro-inflammatory pathways was seen after single dose but not after chronic MPTP treatment. Dopamine biosynthesis and synaptic vesicle recycling pathways were downregulated in PD patients and after chronic MPTP treatment in mice. Genes essential for midbrain development and determination of dopaminergic phenotype such as, LMX1B, FOXA1, RSPO2, KLHL1, EBF3, PITX3, RGS4, ALDH1A1, RET, FOXA2, EN1, DLK1, GFRA1, LMX1A, NR4A2, GAP43, SNCA, PBX1, and GRB10 were downregulated in human PD and overexpression of GFP tagged LMX1B rescued MPP+ induced death in SH-SY5Y neurons. Downregulation of gene ensemble involved in development and differentiation of dopaminergic neurons indicate their potential involvement in pathogenesis and progression of human PD.

Ten Unsolved Questions About Neuroinflammation in Parkinson's Disease.

Parkinson's disease is a progressive and debilitating disorder that has so far eluded attempts to develop disease-modifying treatment. Both epidemiological and genetic studies support a role of neuroinflammation in the pathophysiology of Parkinson's disease. Postmortem studies and experimental analyses suggest the involvement of both innate and adaptive immunity in the degenerative process. There is also some circumstantial evidence for effects of immune therapies on the disease. In the present article, we review 10 unanswered questions related to neuroinflammatory processes in Parkinson's disease with the goal of stimulating research in the field and accelerating the clinical development of neuroprotective therapies based on anti-inflammatory strategies. © 2020 International Parkinson and Movement Disorder Society.

Seven Solutions for Neuroprotection in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra and accumulation of iron and alpha-synuclein; it follows a characteristic pattern throughout the nervous system. Despite decades of successful preclinical neuroprotective studies, no drug has then shown efficacy in clinical trials. Considering this dilemma, we have reviewed and organized solutions of varying importance that can be exclusive or additive, and we outline approaches to help generate successful development of neuroprotective drugs for PD: (1) select patients in which the targeted mechanism is involved in the pathological process associated with the monitoring of target engagement, (2) combine treatments that target multiple pathways, (3) establish earliest interventions and develop better prodromal biomarkers, (4) adopt rigorous methodology and specific disease-relevant designs for disease-modifying clinical trials, (5) customize drug with better brain biodistribution, (6) prioritize repurposed drugs as a first line approach, and (7) adapt preclinical models to the targeted mechanisms with translational biomarkers to increase their predictive value. © 2020 International Parkinson and Movement Disorder Society.

Glutaredoxin 1 Downregulation in the Substantia Nigra Leads to Dopaminergic Degeneration in Mice.

BACKGROUND: Parkinson's disease (PD) is characterized by a severe loss of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Perturbation of protein thiol redox homeostasis has been shown to play a role in the dysregulation of cell death and cell survival signaling pathways in these neurons. Glutaredoxin 1 (Grx1) is a thiol/disulfide oxidoreductase that catalyzes the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis. OBJECTIVES: We evaluated if the downregulation of Grx1 could lead to dopaminergic degeneration and PD-relevant motor deficits in mice. METHODS: Grx1 was downregulated unilaterally through viral vector-mediated transduction of short hairpin RNA against Grx1 into the SNpc. Behavioral assessment was performed through rotarod and elevated body swing test. Stereological analysis of tyrosine hydroxylase-positive and Nissl-positive neurons was carried out to evaluate neurodegeneration. RESULTS: Downregulation of Grx1 resulted in contralateral bias of elevated body swing and reduced latency to fall off, accelerating rotarod. This was accompanied by a loss of tyrosine hydroxylase-positive neurons in the SNpc and their DA projections in the striatum. Furthermore, there was a loss Nissl-positive neurons in the SNpc, indicating cell death. This was selective to the SNpc neurons because DA neurons in the ventral tegmental area were unaffected akin to that seen in human PD. Furthermore, Grx1 mRNA expression was substantially decreased in the SNpc from PD patients. CONCLUSIONS: Our study indicates that Grx1 is critical for the survival of SNpc DA neurons and that it is downregulated in human PD. © 2020 International Parkinson and Movement Disorder Society.

Neuroprotection of dopamine neurons by xenon against low-level excitotoxic insults is not reproduced by other noble gases.

Using midbrain cultures, we previously demonstrated that the noble gas xenon is robustly protective for dopamine (DA) neurons exposed to L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate uptake used to generate sustained, low-level excitotoxic insults. DA cell rescue was observed in conditions where the control atmosphere for cell culture was substituted with a gas mix, comprising the same amount of oxygen (20%) and carbon dioxide (5%) but 75% of xenon instead of nitrogen. In the present study, we first aimed to determine whether DA cell rescue against PDC remains detectable when concentrations of xenon are progressively reduced in the cell culture atmosphere. Besides, we also sought to compare the effect of xenon to that of other noble gases, including helium, neon and krypton. Our results show that the protective effect of xenon for DA neurons was concentration-dependent with an IC50 estimated at about 44%. We also established that none of the other noble gases tested in this study protected DA neurons from PDC-mediated insults. Xenon's effectiveness was most probably due to its unique capacity to block NMDA glutamate receptors. Besides, mathematical modeling of gas diffusion in the culture medium revealed that the concentration reached by xenon at the cell layer level is the highest of all noble gases when neurodegeneration is underway. Altogether, our data suggest that xenon may be of potential therapeutic value in Parkinson disease, a chronic neurodegenerative condition where DA neurons appear vulnerable to slow excitotoxicity.

Long-term outcome in neuroZika: When biological diagnosis matters.

OBJECTIVE: To characterize the full spectrum, relative frequency, and prognosis of the neurologic manifestations in Zika virus (ZIKV) postnatal infection. METHODS: We conducted an observational study in consecutive ZIKV-infected patients presenting with neurologic manifestations during the French West Indies 2016 outbreak. RESULTS: Eighty-seven patients, including 6 children, were enrolled. Ninety-five percent of all cases required hospitalization. Guillain-Barré syndrome was the most frequent manifestation (46.0%) followed by encephalitis or encephalomyelitis (20.7%), isolated single or multiple cranial nerve palsies (9.2%), other peripheral manifestations (6.9%), and stroke (1.1%). Fourteen patients (16.1%), including one child, developed a mixed disorder involving both the central and peripheral nervous system. Mechanical ventilation was required in 21 cases, all of whom had ZIKV RNA in at least one biological fluid. Two adult patients died due to neuroZika. Clinical follow-up (median 14 months; interquartile range, 13-17 months) was available for 76 patients. Residual disability (modified Rankin Scale score ≥2) was identified in 19 (25.0%) patients; in 6 cases (7.9%), disability was severe (modified Rankin Scale score ≥4). Among patients with ZIKV RNA detected in one biological fluid, the risk of residual disability or death was higher (odds ratio 9.19; confidence interval 1.12-75.22; p = 0.039). CONCLUSIONS: NeuroZika spectrum represents a heterogeneous group of clinical neurologic manifestations. During an outbreak, clinicians should consider neuroZika in patients presenting with cranial nerve palsies and a mixed neurologic disorder. Long-term sequelae are frequent in NeuroZika. ZIKV reverse-transcription PCR status at admission can inform prognosis and should therefore be taken into consideration in the management of hospitalized patients.

Glucocorticoid receptor in astrocytes regulates midbrain dopamine neurodegeneration through connexin hemichannel activity.

The precise contribution of astrocytes in neuroinflammatory process occurring in Parkinson's disease (PD) is not well characterized. In this study, using GRCx30CreERT2 mice that are conditionally inactivated for glucocorticoid receptor (GR) in astrocytes, we have examined the actions of astrocytic GR during dopamine neuron (DN) degeneration triggered by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results show significantly augmented DN loss in GRCx30CreERT2 mutant mice in substantia nigra (SN) compared to controls. Hypertrophy of microglia but not of astrocytes was greatly enhanced in SN of these astrocytic GR mutants intoxicated with MPTP, indicating heightened microglial reactivity compared to similarly-treated control mice. In the SN of GR astrocyte mutants, specific inflammation-associated transcripts ICAM-1, TNF-α and Il-1ß as well as TNF-α protein levels were significantly elevated after MPTP neurotoxicity compared to controls. Interestingly, this paralleled increased connexin hemichannel activity and elevated intracellular calcium levels in astrocytes examined in acute midbrain slices from control and mutant mice treated with MPP+ . The increased connexin-43 hemichannel activity was found in vivo in MPTP-intoxicated mice. Importantly, treatment of MPTP-injected GRCx30CreERT2 mutant mice with TAT-Gap19 peptide, a specific connexin-43 hemichannel blocker, reverted both DN loss and microglial activation; in wild-type mice there was partial but significant survival effect. In the SN of post-mortem PD patients, a significant decrease in the number of astrocytes expressing nuclear GR was observed, suggesting the participation of astrocytic GR deregulation of inflammatory process in PD. Overall, these data provide mechanistic insights into GR-modulated processes in vivo, specifically in astrocytes, that contribute to a pro-inflammatory state and dopamine neurodegeneration in PD pathology.

S29434, a Quinone Reductase 2 Inhibitor: Main Biochemical and Cellular Characterization.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.

The Oxygen Paradox, the French Paradox, and age-related diseases.

A paradox is a seemingly absurd or impossible concept, proposition, or theory that is often difficult to understand or explain, sometimes apparently self-contradictory, and yet ultimately correct or true. How is it possible, for example, that oxygen "a toxic environmental poison" could be also indispensable for life (Beckman and Ames Physiol Rev 78(2):547-81, 1998; Stadtman and Berlett Chem Res Toxicol 10(5):485-94, 1997)?: the so-called Oxygen Paradox (Davies and Ursini 1995; Davies Biochem Soc Symp 61:1-31, 1995). How can French people apparently disregard the rule that high dietary intakes of cholesterol and saturated fats (e.g., cheese and paté) will result in an early death from cardiovascular diseases (Renaud and de Lorgeril Lancet 339(8808):1523-6, 1992; Catalgol et al. Front Pharmacol 3:141, 2012; Eisenberg et al. Nat Med 22(12):1428-1438, 2016)?: the so-called, French Paradox. Doubtless, the truth is not a duality and epistemological bias probably generates apparently self-contradictory conclusions. Perhaps nowhere in biology are there so many apparently contradictory views, and even experimental results, affecting human physiology and pathology as in the fields of free radicals and oxidative stress, antioxidants, foods and drinks, and dietary recommendations; this is particularly true when issues such as disease-susceptibility or avoidance, "healthspan," "lifespan," and ageing are involved. Consider, for example, the apparently paradoxical observation that treatment with low doses of a substance that is toxic at high concentrations may actually induce transient adaptations that protect against a subsequent exposure to the same (or similar) toxin. This particular paradox is now mechanistically explained as "Adaptive Homeostasis" (Davies Mol Asp Med 49:1-7, 2016; Pomatto et al. 2017a; Lomeli et al. Clin Sci (Lond) 131(21):2573-2599, 2017; Pomatto and Davies 2017); the non-damaging process by which an apparent toxicant can activate biological signal transduction pathways to increase expression of protective genes, by mechanisms that are completely different from those by which the same agent induces toxicity at high concentrations. In this review, we explore the influences and effects of paradoxes such as the Oxygen Paradox and the French Paradox on the etiology, progression, and outcomes of many of the major human age-related diseases, as well as the basic biological phenomenon of ageing itself.

The noble gas xenon provides protection and trophic stimulation to midbrain dopamine neurons.

Despite its low chemical reactivity, the noble gas xenon possesses a remarkable spectrum of biological effects. In particular, xenon is a strong neuroprotectant in preclinical models of hypoxic-ischemic brain injury. In this study, we wished to determine whether xenon retained its neuroprotective potential in experimental settings that model the progressive loss of midbrain dopamine (DA) neurons in Parkinson's disease. Using rat midbrain cultures, we established that xenon was partially protective for DA neurons through either direct or indirect effects on these neurons. So, when DA neurons were exposed to l-trans-pyrrolidine-2,4-dicarboxylic acid so as to increase ambient glutamate levels and generate slow and sustained excitotoxicity, the effect of xenon on DA neurons was direct. The vitamin E analog Trolox also partially rescued DA neurons in this setting and enhanced neuroprotection by xenon. However, in the situation where DA cell death was spontaneous, the protection of DA neurons by xenon appeared indirect as it occurred through the repression of a mechanism mediated by proliferating glial cells, presumably astrocytes and their precursor cells. Xenon also exerted trophic effects for DA neurons in this paradigm. The effects of xenon were mimicked and improved by the N-methyl-d-aspartate glutamate receptor antagonist memantine and xenon itself appeared to work by antagonizing N-methyl-d-aspartate receptors. Note that another noble gas argon could not reproduce xenon effects. Overall, present data indicate that xenon can provide protection and trophic support to DA neurons that are vulnerable in Parkinson's disease. This suggests that xenon might have some therapeutic value for this disorder.

Dysfunction of mitochondrial Lon protease and identification of oxidized protein in mouse brain following exposure to MPTP: Implications for Parkinson disease.

Compelling evidence suggests that mitochondrial dysfunction leading to reactive oxygen species (ROS) production and protein oxidation could represent a critical event in the pathogenesis of Parkinson's disease (PD). Pioneering studies have shown that the mitochondrial matrix contains the Lon protease, which degrades oxidized, dysfunctional, and misfolded protein. Using the PD animal model of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication in mice, we showed that Lon protease expression increased in the ventral mesencephalon of intoxicated animals, concomitantly with the appearance of oxidized proteins and dopaminergic cell loss. In addition, we report that Lon is inactivated by ROS. Moreover, proteomic experiments provide evidence of carbonylation in α-ketoglutarate dehydrogenase (KGDH), aconitase or subunits of respiratory chain complexes. Lon protease inactivation upon MPTP treatment in mice raises the possibility that Lon protease dysfunction is an early event in the pathogenesis of PD.

Hepcidin attenuates amyloid beta-induced inflammatory and pro-oxidant responses in astrocytes and microglia.

Alzheimer's disease (AD) is characterized by extracellular senile plaques, intracellular neurofibrillary tangles, and neuronal death. Aggregated amyloid-ß (Aß) induces inflammation and oxidative stress, which have pivotal roles in the pathogenesis of AD. Hepcidin is a key regulator of systemic iron homeostasis. Recently, an anti-inflammatory response to hepcidin was reported in macrophages. Under the hypothesis that hepcidin mediates anti-inflammatory response in the brain, in this study, we evaluated the putative anti-inflammatory role of hepcidin on Aß-activated astrocytes and microglia. Primary culture of astrocytes and microglia were treated with Aß, with or without hepcidin, and cytokine levels were then evaluated. In addition, the toxicity of Aß-treated astrocyte- or microglia-conditioned media was tested on neurons, evaluating cellular death and oxidative stress generation. Finally, mice were injected in the right lateral ventricle with Aß, with or without hepcidin, and hippocampus glial activation and oxidative stress were evaluated. Pre-treatment with hepcidin reduced the expression and secretion of TNF-α and IL-6 in astrocytes and microglia treated with Aß. Hepcidin also reduced neurotoxicity and oxidative damage triggered by conditioned media obtained from astrocytes and microglia treated with Aß. Stereotaxic intracerebral injection of hepcidin reduced glial activation and oxidative damage triggered by Aß injection in mice. Overall, these results are consistent with the hypothesis that in astrocytes and microglia hepcidin down-regulates the inflammatory and pro-oxidant processes induced by Aß, thus protecting neighboring neurons. This is a newly described property of hepcidin in the central nervous system, which may be relevant for the development of strategies to prevent the neurodegenerative process associated with AD.

Analysis of monocyte infiltration in MPTP mice reveals that microglial CX3CR1 protects against neurotoxic over-induction of monocyte-attracting CCL2 by astrocytes.

BACKGROUND: Evidence from mice suggests that brain infiltrating immune cells contribute to neurodegeneration, and we previously identified a deleterious lymphocyte infiltration in Parkinson's disease mice. However, this remains controversial for monocytes, due to artifact-prone techniques used to distinguish them from microglia. Our aim was to reassess this open question, by taking advantage of the recent recognition that chemokine receptors CCR2 and CX3CR1 can differentiate between inflammatory monocytes and microglia, enabling to test whether CCR2+ monocytes infiltrate the brain during dopaminergic (DA) neurodegeneration and whether they contribute to neuronal death. This revealed unexpected insights into possible regulation of monocyte-attracting CCL2 induction. METHODS: We used acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice and assessed monocyte infiltration by combining laser microdissection-guided chemokine RNA profiling of the substantia nigra (SN) with immunohistochemistry and CCR2-GFP reporter mice. To determine contribution to neuronal loss, we used CCR2-deletion and CCL2-overexpression, to reduce and increase CCR2+ monocyte infiltration, and CX3CR1-deletion to assess a potential implication in CCL2 regulation. RESULTS: Nigral chemokine profiling revealed early CCL2/7/12-CCR2 axis induction, suggesting monocyte infiltration in MPTP mice. CCL2 protein showed early peak induction in nigral astrocytes, while CCR2-GFP mice revealed early but limited nigral monocyte infiltration. However, blocking infiltration by CCR2 deletion did not influence DA neuronal loss. In contrast, transgenic astrocytic CCL2 over-induction increased CCR2+ monocyte infiltration and DA neuronal loss in MPTP mice. Surprisingly, CCL2 over-induction was also detected in MPTP intoxicated CX3CR1-deleted mice, which are known to present increased DA neuronal loss. Importantly, CX3CR1/CCL2 double-deletion suggested that increased neurotoxicity was driven by astrocytic CCL2 over-induction. CONCLUSIONS: We show that CCR2+ monocytes infiltrate the affected CNS, but at the level observed in acute MPTP mice, this does not contribute to DA neuronal loss. In contrast, the underlying astrocytic CCL2 induction seemed to be tightly controled, as already moderate CCL2 over-induction led to increased neurotoxicity in MPTP mice, likely due to the increased CCR2+ monocyte infiltration. Importantly, we found evidence suggesting that during DA neurodegeneration, this control was mediated by microglial CX3CR1 signaling, which protects against such neurotoxic CCL2 over-induction by astrocytes, thus hinting at an endogenous mechanism to limit neurotoxic effects of the CCL2-CCR2 axis.

Pedunculopontine Nucleus Region Deep Brain Stimulation in Parkinson Disease: Surgical Anatomy and Terminology.

Several lines of evidence over the last few years have been important in ascertaining that the pedunculopontine nucleus (PPN) region could be considered as a potential target for deep brain stimulation (DBS) to treat freezing and other problems as part of a spectrum of gait disorders in Parkinson disease and other akinetic movement disorders. Since the introduction of PPN DBS, a variety of clinical studies have been published. Most indicate improvements in freezing and falls in patients who are severely affected by these problems. The results across patients, however, have been variable, perhaps reflecting patient selection, heterogeneity in target selection and differences in surgical methodology and stimulation settings. Here we outline both the accumulated knowledge and the domains of uncertainty in surgical anatomy and terminology. Specific topics were assigned to groups of experts, and this work was accumulated and reviewed by the executive committee of the working group. Areas of disagreement were discussed and modified accordingly until a consensus could be reached. We demonstrate that both the anatomy and the functional role of the PPN region need further study. The borders of the PPN and of adjacent nuclei differ when different brainstem atlases and atlas slices are compared. It is difficult to delineate precisely the PPN pars dissipata from the nucleus cuneiformis, as these structures partially overlap. This lack of clarity contributes to the difficulty in targeting and determining the exact localization of the electrodes implanted in patients with akinetic gait disorders. Future clinical studies need to consider these issues.

Pedunculopontine Nucleus Region Deep Brain Stimulation in Parkinson Disease: Surgical Techniques, Side Effects, and Postoperative Imaging.

The pedunculopontine nucleus (PPN) region has received considerable attention in clinical studies as a target for deep brain stimulation (DBS) in Parkinson disease. These studies have yielded variable results with an overall impression of improvement in falls and freezing in many but not all patients treated. We evaluated the available data on the surgical anatomy and terminology of the PPN region in a companion paper. Here we focus on issues concerning surgical technique, imaging, and early side effects of surgery. The aim of this paper was to gain more insight into the reasoning for choosing specific techniques and to discuss shortcomings of available studies. Our data demonstrate the wide range in almost all fields which were investigated. There are a number of important challenges to be resolved, such as identification of the optimal target, the choice of the surgical approach to optimize electrode placement, the impact on the outcome of specific surgical techniques, the reliability of intraoperative confirmation of the target, and methodological differences in postoperative validation of the electrode position. There is considerable variability both within and across groups, the overall experience with PPN DBS is still limited, and there is a lack of controlled trials. Despite these challenges, the procedure seems to provide benefit to selected patients and appears to be relatively safe. One important limitation in comparing studies from different centers and analyzing outcomes is the great variability in targeting and surgical techniques, as shown in our paper. The challenges we identified will be of relevance when designing future studies to better address several controversial issues. We hope that the data we accumulated may facilitate the development of surgical protocols for PPN DBS.