Inhibition of Autophagy In Vivo Extends Methamphetamine Toxicity to Mesencephalic Cell Bodies

Methamphetamine (METH) is a widely abused psychostimulant and a stress-inducing compound, which leads to neurotoxicity for nigrostriatal dopamine (DA) terminals in rodents and primates including humans. In vitro studies indicate that autophagy is a strong modulator of METH toxicity. In detail, suppressing autophagy increases METH toxicity, while stimulating autophagy prevents METH-induced toxicity in cell cultures. In the present study, the role of autophagy was investigated in vivo. In the whole brain, METH alone destroys meso-striatal DA axon terminals, while fairly sparing DA cell bodies within substantia nigra pars compacta (SNpc). No damage to either cell bodies or axons from ventral tegmental area (VTA) is currently documented. According to the hypothesis that ongoing autophagy prevents METH-induced DA toxicity, we tested whether systemic injection of autophagy inhibitors such as asparagine (ASN, 1000 mg/Kg) or glutamine (GLN, 1000 mg/Kg), may extend METH toxicity to DA cell bodies, both within SNpc and VTA, where autophagy was found to be inhibited. When METH (5 mg/Kg × 4, 2 h apart) was administered to C57Bl/6 mice following ASN or GLN, a frank loss of cell bodies takes place within SNpc and a loss of both axons and cell bodies of VTA neurons is documented. These data indicate that, ongoing autophagy protects DA neurons and determines the refractoriness of cell bodies to METH-induced toxicity.

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Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.682067 ◽

2021 ◽

Vol 9 ◽

Author(s):

Verónica Company ◽

Abraham Andreu-Cervera ◽

M. Pilar Madrigal ◽

Belén Andrés ◽

Francisca Almagro-García ◽

...

Keyword(s):

Substantia Nigra ◽

Ventral Tegmental Area ◽

Dopaminergic Neurons ◽

Functional Organization ◽

Substantia Nigra Pars Compacta ◽

Fasciculus Retroflexus ◽

Ventral Tegmental

The fasciculus retroflexus is an important fascicle that mediates reward-related behaviors and is associated with different psychiatric diseases. It is the main habenular efference and constitutes a link between forebrain regions, the midbrain, and the rostral hindbrain. The proper functional organization of habenular circuitry requires complex molecular programs to control the wiring of the habenula during development. However, the mechanisms guiding the habenular axons toward their targets remain mostly unknown. Here, we demonstrate the role of the mesodiencephalic dopaminergic neurons (substantia nigra pars compacta and ventral tegmental area) as an intermediate target for the correct medial habenular axons navigation along the anteroposterior axis. These neuronal populations are distributed along the anteroposterior trajectory of these axons in the mesodiencephalic basal plate. Using in vitro and in vivo experiments, we determined that this navigation is the result of netrin 1 attraction generated by the mesodiencephalic dopaminergic neurons. This attraction is mediated by the receptor deleted in colorectal cancer (DCC), which is strongly expressed in the medial habenular axons. The increment in our knowledge on the fasciculus retroflexus trajectory guidance mechanisms opens the possibility of analyzing if its alteration in mental health patients could account for some of their symptoms.

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DNAJB6b is Downregulated in Synucleinopathies

Journal of Parkinson s Disease ◽

10.3233/jpd-202512 ◽

2021 ◽

pp. 1-13

Author(s):

Jonas Folke ◽

Sertan Arkan ◽

Isak Martinsson ◽

Susana Aznar ◽

Gunnar Gouras ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Substantia Nigra Pars Compacta ◽

Endogenous Protein ◽

Highly Sensitive ◽

Brain Material ◽

Health And Disease ◽

Isoform B

Background: α-synuclein (α-syn) aggregation contributes to the progression of multiple neurodegenerative diseases. We recently found that the isoform b of the co-chaperone DNAJB6 is a strong suppressor of a-syn aggregation in vivo and in vitro. However, nothing is known about the role of the endogenous isoform b of DNAJB6 (DNAJB6b) in health and disease, due to lack of specific antibodies. Objective: Here we generated a novel anti-DNAJB6b antibody to analyze the localization and expression this isoform in cells, in tissue and in clinical material. Methods: To address this we used immunocytochemistry, immunohistochemistry, as well as a novel quantitative DNAJB6 specific ELISA method. Results: The endogenous protein is mainly expressed in the cytoplasm and in neurites in vitro, where it is found more in dendrites than in axons. We further verified in vivo that DNAJB6b is expressed in the dopaminergic neurons of the substantia nigra pars compacta (SNpc), which is a neuronal subpopulation highly sensitive to α-syn aggregation, that degenerate to a large extend in patients with Parkinson’s disease (PD) and multiple system atrophy (MSA). When we analyzed the expression levels of DNAJB6b in brain material from PD and MSA patients, we found a downregulation of DNAJB6b by use of ELISA based quantification. Interestingly, this was also true when analyzing tissue from patients with progressive supranuclear palsy, a taupathic atypical parkinsonian disorder. However, the total level of DNAJB6 was upregulated in these three diseases, which may indicate an upregulation of the other major isoform of DNAJB6, DNAJB6a. Conclusion: This study shows that DNAJB6b is downregulated in several different neurodegenerative diseases, which makes it an interesting target to further investigate in relation to amyloid protein aggregation and disease progression.

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Characteristics of Electrically Evoked Somatodendritic Dopamine Release in Substantia Nigra and Ventral Tegmental Area In Vitro

Journal of Neurophysiology ◽

10.1152/jn.1997.77.2.853 ◽

1997 ◽

Vol 77 (2) ◽

pp. 853-862 ◽

Cited By ~ 100

Author(s):

M. E. Rice ◽

S. J. Cragg ◽

S. A. Greenfield

Keyword(s):

Electrical Stimulation ◽

Substantia Nigra ◽

Ventral Tegmental Area ◽

Dopamine Release ◽

Substantia Nigra Pars Compacta ◽

Frequency Dependent ◽

Body Regions ◽

Ventral Tegmental ◽

Da Release

Rice, M. E., S. J. Cragg, and S. A. Greenfield. Characteristics of electrically evoked somatodendritic dopamine release in substantia nigra and ventral tegmental area in vitro. J. Neurophysiol. 77: 853–862, 1997. Somatodendritic dopamine (DA) release from neurons of the midbrain represents a nonclassical form of neuronal signaling. We assessed characteristics of DA release during electrical stimulation of the substantia nigra pars compacta (SNc) in guinea pig midbrain slices. With the use of parameters optimized for this region, we compared stimulus-induced increases in extracellular DA concentration ([DA]o) in medial and lateral SNc, ventral tegmental area (VTA), and dorsal striatum in vitro. DA release was monitored directly with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Detection of DA in SNc was confirmed by electrochemical, pharmacological, and anatomic criteria. Voltammograms of the released substance had the same peak potentials as those of DA obtained during in vitro calibration, but different from those of the indoleamine 5-hydroxytryptamine. Similar voltammograms were also obtained in the DA-rich striatum during local electrical stimulation. Contribution from the DA metabolite 3,4-dihydroxyphenylacetic acid to somatodendritic release was negligible, as indicated by the lack of effect of the monoamine oxidase inhibitor pargyline (20 μM) on the signal. Lastly, DA voltammograms could only be elicited in regions that were subsequently determined to be positive for tyrosine hydroxylase immunoreactivity (TH-ir). The frequency dependence of stimulated DA release in SNc was determined over a range of 1–50 Hz, with a constant duration of 10 s. Release was frequency dependent up to 10 Hz, with no further increase at higher frequencies. Stimulation at 10 Hz was used in all subsequent experiments. With this paradigm, DA release in SNc was tetrodotoxin insensitive, but strongly Ca2+ dependent. Stimulated [DA]o in the midbrain was also site specific. At the midcaudal level examined, DA efflux was significantly greater in VTA (1.04 ± 0.05 μM, mean ± SE) than in medial SNc (0.52 ± 0.05 μM), which in turn was higher than in lateral SNc (0.35 ± 0.03 μM). This pattern followed the apparent density of TH-ir, which was also VTA > medial SNc > lateral SNc. This report has introduced a new paradigm for the study of somatodendritic DA release. Voltammetric recording with electrodes of 2–4 μm tip diameter permitted highly localized, direct detection of endogenous DA. The Ca2+ dependence of stimulated release indicated that the process was physiologically relevant. Moreover, the findings that somatodendritic release was frequency dependent across a range characteristic of DA cell firing rates and that stimulated [DA]o varied markedly among DA cell body regions have important implications for how dendritically released DA may function in the physiology and pathophysiology of substantia nigra and VTA.

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Pedunculopontine glutamatergic neurons control spike patterning in substantia nigra dopaminergic neurons

eLife ◽

10.7554/elife.30352 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 13

Author(s):

Daniel J Galtieri ◽

Chad M Estep ◽

David L Wokosin ◽

Stephen Traynelis ◽

D James Surmeier

Keyword(s):

Substantia Nigra ◽

Dopaminergic Neurons ◽

Substantia Nigra Pars Compacta ◽

Glutamatergic Synapses ◽

Glutamatergic Neurons ◽

Oscillatory Mechanisms ◽

Goal Directed Behavior ◽

Stimulation Of

Burst spiking in substantia nigra pars compacta (SNc) dopaminergic neurons is a key signaling event in the circuitry controlling goal-directed behavior. It is widely believed that this spiking mode depends upon an interaction between synaptic activation of N-methyl-D-aspartate receptors (NMDARs) and intrinsic oscillatory mechanisms. However, the role of specific neural networks in burst generation has not been defined. To begin filling this gap, SNc glutamatergic synapses arising from pedunculopotine nucleus (PPN) neurons were characterized using optical and electrophysiological approaches. These synapses were localized exclusively on the soma and proximal dendrites, placing them in a good location to influence spike generation. Indeed, optogenetic stimulation of PPN axons reliably evoked spiking in SNc dopaminergic neurons. Moreover, burst stimulation of PPN axons was faithfully followed, even in the presence of NMDAR antagonists. Thus, PPN-evoked burst spiking of SNc dopaminergic neurons in vivo may not only be extrinsically triggered, but extrinsically patterned as well.

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Role of the gut proteinases from mosquito larvae in the mechanism of action and the specificity of the Bacillus sphaericus toxin

Canadian Journal of Microbiology ◽

10.1139/m90-138 ◽

1990 ◽

Vol 36 (11) ◽

pp. 804-807 ◽

Cited By ~ 22

Author(s):

Luc Nicolas ◽

Anne Lecroisey ◽

Jean-François Charles

Keyword(s):

Cell Cultures ◽

Spodoptera Littoralis ◽

Mosquito Species ◽

Bacillus Sphaericus ◽

Cellular Level ◽

Mosquito Cell ◽

Mosquito Larvae

Gut proteinases from larvae of mosquito species both susceptible and not susceptible to Bacillus sphaericus converted the 43-kDa toxin to a 40-kDa polypeptide exhibiting enhanced cytotoxicity to mosquito cell cultures. The toxin was also activated by gut proteinases from the nonsusceptible Lepidoptera Spodoptera littoralis in vitro and in vivo. Therefore, the specificity of Bacillus sphaericus toxin does not seem to be determined by gut proteinase action. However, susceptibility of mosquito cell cultures did not reflect the specificity of the toxin, which must now be investigated at the cellular level in the larvae. Key words: mosquitoes, Bacillus sphaericus, bacterial toxins, proteinases, specificity.

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Estrogen receptors and estetrol-dependent neuroprotective actions: a pilot study

Journal of Endocrinology ◽

10.1530/joe-16-0434 ◽

2017 ◽

Vol 232 (1) ◽

pp. 85-95 ◽

Cited By ~ 8

Author(s):

Ekaterine Tskitishvili ◽

Christel Pequeux ◽

Carine Munaut ◽

Renaud Viellevoye ◽

Michelle Nisolle ◽

...

Keyword(s):

Estrogen Receptors ◽

Cell Survival ◽

Cell Cultures ◽

Damage Model ◽

Neuronal Cell ◽

Concomitant Treatment ◽

Ldh Activity

Estetrol (E4) has strong antioxidative, neurogenic and angiogenic effects in neural system resulting in the attenuation of neonatal hypoxic–ischemic encephalopathy. We aimed to define the role of estrogen receptors in E4-dependent actions in neuronal cell cultures and prove the promyelinating effect of E4. In vitro the antioxidative and cell survival/proliferating effects of E4 on H2O2-induced oxidative stress in primary hippocampal cell cultures were studied using different combinations of specific inhibitors for ERα (MPP dihydrochloride), ERβ (PHTTP), GPR30 (G15) and palmytoilation (2-BR). LDH activity and cell survival assays were performed. In vivo the promyelinating role of different concentrations of E4 (1 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 50 mg/kg/day) was investigated using the hypoxic–ischemic brain damage model in the 7-day-old immature rats before/after the induction of hypoxic–ischemic insult. Myelin basic protein (MBP) immunostaining was performed on brain coronal sections. Our results show that LDH activity is significantly upregulated in cell cultures where the E4’s effect was completely blocked by concomitant treatment either with ERα and ERβ inhibitors (MPP and PHTPP, respectively), or ERα and ERβ inhibitors combined with 2-BR. Cell survival is significantly downregulated in cell cultures where the effect of E4 was blocked by ERβ inhibitor (PHTTP) alone. The blockage of GRP30 receptor did affect neither LDH activity nor cell survival. MBP immunostaining is significantly upregulated in E4-pretreated groups at a concentration of 5 mg/kg/day and 50 mg/kg/day E4, whereas the MBP-positive area OD ratio is significantly increased in all the E4-treated groups. E4’s antioxidative actions mostly depend on ERα and ERβ, whereas neurogenesis and possibly promyelinating activities might be realized through ERβ.

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Characterization of substantia nigra neurogenesis in homeostasis and dopaminergic degeneration: beneficial effects of the microneurotrophin BNN-20

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02398-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Theodora Mourtzi ◽

Dimitrios Dimitrakopoulos ◽

Dimitrios Kakogiannis ◽

Charalampos Salodimitris ◽

Konstantinos Botsakis ◽

...

Keyword(s):

Stem Cell ◽

Substantia Nigra ◽

Mouse Model ◽

Dopaminergic Neurons ◽

Stem Cell Niche ◽

Substantia Nigra Pars Compacta ◽

Dopaminergic Cell ◽

Cell Niche

Abstract Background Loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) underlines much of the pathology of Parkinson’s disease (PD), but the existence of an endogenous neurogenic system that could be targeted as a therapeutic strategy has been controversial. BNN-20 is a synthetic, BDNF-mimicking, microneurotrophin that we previously showed to exhibit a pleiotropic neuroprotective effect on the dopaminergic neurons of the SNpc in the “weaver” mouse model of PD. Here, we assessed its potential effects on neurogenesis. Methods We quantified total numbers of dopaminergic neurons in the SNpc of wild-type and “weaver” mice, with or without administration of BNN-20, and we employed BrdU labelling and intracerebroventricular injections of DiI to evaluate the existence of dopaminergic neurogenesis in the SNpc and to assess the origin of newborn dopaminergic neurons. The in vivo experiments were complemented by in vitro proliferation/differentiation assays of adult neural stem cells (NSCs) isolated from the substantia nigra and the subependymal zone (SEZ) stem cell niche to further characterize the effects of BNN-20. Results Our analysis revealed the existence of a low-rate turnover of dopaminergic neurons in the normal SNpc and showed, using three independent lines of experiments (stereologic cell counts, BrdU and DiI tracing), that the administration of BNN-20 leads to increased neurogenesis in the SNpc and to partial reversal of dopaminergic cell loss. The newly born dopaminergic neurons, that are partially originated from the SEZ, follow the typical nigral maturation pathway, expressing the transcription factor FoxA2. Importantly, the pro-cytogenic effects of BNN-20 were very strong in the SNpc, but were absent in other brain areas such as the cortex or the stem cell niche of the hippocampus. Moreover, although the in vitro assays showed that BNN-20 enhances the differentiation of NSCs towards glia and neurons, its in vivo administration stimulated only neurogenesis. Conclusions Our results demonstrate the existence of a neurogenic system in the SNpc that can be manipulated in order to regenerate the depleted dopaminergic cell population in the “weaver” PD mouse model. Microneurotrophin BNN-20 emerges as an excellent candidate for future PD cell replacement therapies, due to its area-specific, pro-neurogenic effects.

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LRRK2 interacts with the vacuolar-type H+-ATPase pump a1 subunit to regulate lysosomal function

Human Molecular Genetics ◽

10.1093/hmg/ddz088 ◽

2019 ◽

Vol 28 (16) ◽

pp. 2696-2710 ◽

Cited By ~ 24

Author(s):

Rebecca Wallings ◽

Natalie Connor-Robson ◽

Richard Wade-Martins

Keyword(s):

Critical Role ◽

Substantia Nigra Pars Compacta ◽

Cellular Mechanisms ◽

Genomic Locus ◽

Lysosomal Dysfunction ◽

Lrrk2 G2019s ◽

Lrrk2 Kinase

Abstract Lysosomal dysfunction lies at the centre of the cellular mechanisms underlying Parkinson’s disease although the precise underlying mechanisms remain unknown. We investigated the role of leucine-rich repeat kinase 2 (LRRK2) on lysosome biology and the autophagy pathway in primary neurons expressing the human LRRK2-G2019S or LRKK2-R1441C mutant or the human wild-type (hWT-LRRK2) genomic locus. The expression of LRRK2-G2019S or hWT-LRRK2 inhibited autophagosome production, whereas LRRK2-R1441C induced a decrease in autophagosome/lysosome fusion and increased lysosomal pH. In vivo data from the cortex and substantia nigra pars compacta of aged LRRK2 transgenic animals revealed alterations in autophagosome puncta number reflecting those phenotypes seen in vitro. Using the two selective and potent LRRK2 kinase inhibitors, MLi-2 and PF-06447475, we demonstrated that the LRRK2-R1441C-mediated decrease in autolysosome maturation is not dependent on LRRK2 kinase activity. We showed that hWT-LRRK2 and LRRK2-G2019S bind to the a1 subunit of vATPase, which is abolished by the LRRK2-R1441C mutation, leading to a decrease in a1 protein and cellular mislocalization. Modulation of lysosomal zinc increased vATPase a1 protein levels and rescued the LRRK2-R1441C-mediated cellular phenotypes. Our work defines a novel interaction between the LRRK2 protein and the vATPase a1 subunit and demonstrates a mode of action by which drugs may rescue lysosomal dysfunction. These results demonstrate the importance of LRRK2 in lysosomal biology, as well as the critical role of the lysosome in PD.

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MPTP-induced Parkinsonism in genetically modified mice

Nauchno-prakticheskii zhurnal «Patogenez» ◽

10.25557/2310-0435.2021.02.12-23 ◽

2021 ◽

pp. 12-23

Author(s):

В.В. Голоборщева ◽

Н.А. Воронина ◽

Р.К. Овчинников ◽

В.Г. Кучеряну ◽

С.Г. Морозов

Keyword(s):

Animal Models ◽

Substantia Nigra ◽

Lewy Bodies ◽

In Vitro Models ◽

Model System ◽

Substantia Nigra Pars Compacta ◽

Motor Disorders ◽

Progressive Loss

Использование экспериментальных моделей на животных является чрезвычайно ценным методом изучения патогенеза заболеваний человека и используется для разработки их эффективной терапии. В отличие от изолированных моделей in vitro, использование животных в качестве модельной системы оказалось более продуктивным для изучения мультифакториальных заболеваний или болезней, влияющих на несколько систем организма. Данный метод особенно актуален в отношении использования моделей in vivo для понимания патофизиологических механизмов расстройств центральной нервной системы, включая эпилепсию, рассеянный склероз и нейродегенеративные заболевания. Одним из ключевых среди нейродегенеративных расстройств является болезнь Паркинсона (БП) - хроническое прогрессирующее заболевание, вызванное недостатком дофамина в стриатуме головного мозга в результате гибели дофаминергических (ДА) нейронов чёрной субстанции (ЧС). В настоящее время для воспроизведения у животных основных характеристик БП, включая двигательные нарушения, прогрессирующую потерю ДА-ергических нейронов в компактной части черной субстанции и образование телец Леви, широко используют соединение 1-метил-4-фенил-1,2,3,6-тетрагидропиридин (МФТП). В этом обзоре будут охарактеризованы популярные экспериментальные модели для изучения БП, в которых в рамках исследований вводили нейротоксин МФТП, с целью систематизировать уже имеющийся научный опыт, а также способствовать развитию моделирования паркинсонического синдрома. The use of experimental animal models to understand human diseases and the potential treatments has been extremely valuable and has led to many significant advances in medicine. In contrast to isolated in vitro models, the use of animals as a model system has proven to be more productive for studying of multi-factorial diseases or diseases that affect several human systems. This method is particularly relevant for the use of animal models to understand the pathophysiology of central nervous system disorders such as meningitis, multiple sclerosis, and neurodegenerative disorders. Parkinson’s disease (PD), a chronic progressive illness, caused by a lack of dopamine (DA) in the striatum due to the death of DAergic neurons in the substantia nigra (SN), is one of the neurodegenerative key disorders. Nowadays, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a widely used compound to reproduce the main characteristics of PD in animals, which include motor disorders, progressive loss of DA-neurons in the substantia nigra pars compacta and the formation of Lewy bodies. In this review, we observe MPTP-treated popular mouse models for the PD studying in order to systematize the existing scientific experience, as well as to promote the development of parkinsonian syndrome modeling.

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