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Journal of Proteomics Dec 2013This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low... (Clinical Trial)
Clinical Trial
UNLABELLED
This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (≥0.15g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson's disease and dementia with Lewy bodies compared to appropriate controls.
BIOLOGICAL SIGNIFICANCE
Key features of Parkinson's disease are the degeneration of dopaminergic neurons in the substantia nigra pars compacta, an associated loss of the brain pigment neuromelanin and a resulting impairment of the neuronal network. The accumulation of iron binding neuromelanin granules is age- and disease-dependent and disease specific alterations could affect the neuronal iron homeostasis leading to oxidative stress induced cell death. The focus of the described method is the analysis of neuromelanin granules as well as axonal cell-endings of nerve cells (synaptosomes) of individual donors (control and diseased). It is the basis for the identification of disease-relevant changes in the iron homeostasis and the generation of new insight into altered protein compositions or regulations which might lead to disturbed communications between nerve cells resulting in pathogenic processes.
Topics: Centrifugation, Density Gradient; Cytoplasmic Granules; Female; Humans; Male; Melanins; Neurodegenerative Diseases; Proteomics; Substantia Nigra; Synaptosomes
PubMed: 23917253
DOI: 10.1016/j.jprot.2013.07.015 -
Molecular Brain Mar 2020Pathogenic variants in the gene encoding the small GTPase Ras analogue in Brain 39b (RAB39B) are associated with early-onset parkinsonism. In this study we investigated...
Pathogenic variants in the gene encoding the small GTPase Ras analogue in Brain 39b (RAB39B) are associated with early-onset parkinsonism. In this study we investigated the expression and localization of RAB39B (RNA and protein) in mouse brain tissue to gain a better understanding of its normal physiological function(s) and role in disease.We developed novel resources, including monoclonal antibodies directed against RAB39B and mice with Rab39b knockout, and performed real-time PCR and western blot analysis on whole brain lysates. To determine the spatial localization of Rab39b RNA and protein, we performed in-situ hybridization and immunohistochemistry on fresh frozen and fixed brain tissue. Our results show that RAB39B is localized throughout the cortex, hippocampus and substantia nigra of mice throughout postnatal life. We found high levels of RAB39B within MAP2 positive cortical and hippocampal neurons, and TH positive dopaminergic neurons in the substantia nigra pars compacta.Our studies support and extend current knowledge of the localization of RAB39B. We validate RAB39B as a neuron-enriched protein and demonstrate that it is present throughout the mouse cortex and hippocampus. Further, we observe high levels in the substantia nigra pars compacta, the brain region most affected in Parkinson's disease pathology. The distribution of Rab39b is consistent with human disease associations with parkinsonism and cognitive impairment. We also describe and validate novel resources, including monoclonal antibodies directed against RAB39B and mice with Rab39b knockout, both of which are valuable tools for future studies of the molecular function of RAB39B.
Topics: Animals; Antibodies, Monoclonal; Brain; Mice, Knockout; Parkinson Disease; Time Factors; rab GTP-Binding Proteins
PubMed: 32228644
DOI: 10.1186/s13041-020-00584-7 -
Antidepressant and Neuroprotective Effects of 3-Hydroxy Paroxetine, an Analog of Paroxetine in Rats.The International Journal of... Mar 2023Paroxetine (PX) is a widely used antidepressant with side effects such as weakness, dizziness, and trouble sleeping. In search of novel compounds with better efficacy...
BACKGROUND
Paroxetine (PX) is a widely used antidepressant with side effects such as weakness, dizziness, and trouble sleeping. In search of novel compounds with better efficacy and fewer side effects, we synthesized 3HPX, a hydroxylated analog of PX, and compared the 2 in silico for their pharmacokinetic and binding properties and in vivo for their antidepressant and potential neuroprotective effects.
METHODS
In silico studies compared pharmacological properties as well as interactions of PX and 3HPX with the serotonin transporter. In vivo studies utilized an animal model of comorbid depression-Parkinson disease. Adult male Wistar rats were injected (sterotaxically) with lipopolysaccharide in the striatum (unilaterally), followed by 14 days of once-daily injections (i.p.) of 10 mg/kg PX or 3HPX. Animals were tested for motor asymmetry and locomotor activity as well as indices of anhedonia and helplessness using sucrose preference and forced swim tests, respectively. Brains of these animals were collected after the last test, and tyrosine hydroxylase-positive neurons in substantia nigra pars compacta and Iba-1-positive stained microglia in ipsilateral striatum were measured.
RESULTS
In silico findings indicated that 3HPX could bind stronger to serotonin transporter and also have a better clearance and hence less toxicity compared with PX. In vivo results revealed a more effective reversal of immobility in the swim test, substantial increase in tyrosine hydroxylase-positive cells in the substantia nigra pars compacta, and more ramified Iba-1+ cells by 3HPX compared with PX.
CONCLUSION
The findings suggest superior effectiveness of 3HPX as an antidepressant and neuroprotectant compared with PX and hence potential utility in Parkinson disease depression co-morbidity.
Topics: Rats; Male; Animals; Paroxetine; Parkinson Disease; Neuroprotective Agents; Rats, Wistar; Substantia Nigra; Tyrosine 3-Monooxygenase; Serotonin Plasma Membrane Transport Proteins; Antidepressive Agents; Disease Models, Animal
PubMed: 36433759
DOI: 10.1093/ijnp/pyac077 -
Frontiers in Cell and Developmental... 2021The fasciculus retroflexus is an important fascicle that mediates reward-related behaviors and is associated with different psychiatric diseases. It is the main...
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 and experiments, we determined that this navigation is the result of 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.
PubMed: 34169076
DOI: 10.3389/fcell.2021.682067 -
RGS Proteins as Critical Regulators of Motor Function and Their Implications in Parkinson's Disease.Molecular Pharmacology Dec 2020Parkinson disease (PD) is a devastating, largely nonfamilial, age-related disorder caused by the progressive loss of dopamine (DA) neurons in the substantia nigra pars... (Review)
Review
Parkinson disease (PD) is a devastating, largely nonfamilial, age-related disorder caused by the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Release of DA from these neurons into the dorsal striatum is crucial for regulating movement and their loss causes PD. Unfortunately, the mechanisms underlying SNc neurodegeneration remain unclear, and currently there is no cure for PD, only symptomatic treatments. Recently, several regulator of G protein signaling (RGS) proteins have emerged as critical modulators of PD pathogenesis and/or motor dysfunction and dyskinesia: RGSs 4, 6, 9, and 10. Striatal RGS4 has been shown to exacerbate motor symptoms of DA loss by suppressing M-autoreceptor-G signaling in striatal cholinergic interneurons. RGS6 and RGS9 are key regulators of DR-G signaling in SNc DA neurons and striatal medium spiny neurons, respectively. RGS6, expressed in human and mouse SNc DA neurons, suppresses characteristic PD hallmarks in aged mice, including SNc DA neuron loss, motor deficits, and -synuclein accumulation. After DA depletion, RGS9 (through its inhibition of medium spiny neuron DR signaling) suppresses motor dysfunction induced by L-DOPA or DR-selective agonists. RGS10 is highly expressed in microglia, the brain's resident immune cells. Within the SNc, RGS10 may promote DA neuron survival through the upregulation of prosurvival genes and inhibition of microglial inflammatory factor expression. Thus, RGSs 4, 6, 9, and 10 are critical modulators of cell signaling pathways that promote SNc DA neuron survival and/or proper motor control. Accordingly, these RGS proteins represent novel therapeutic targets for the treatment of PD pathology. SIGNIFICANCE STATEMENT: Parkinson disease (PD), the most common movement disorder, is a progressive neurodegenerative disease characterized by substantia nigra pars compacta (SNc) dopamine (DA) neuron loss and subsequent motor deficits. Current PD therapies only target disease motor symptomology and are fraught with side effects. Therefore, researchers have begun to explore alternative therapeutic options. Regulator of G protein signaling (RGS) proteins, whether primarily expressed in SNc DA neurons (RGS6), striatal neurons (RGSs 4 and 9), or microglia (RGS10), modulate key signaling pathways important for SNc DA neuron survival and/or proper motor control. As such, RGS proteins represent novel therapeutic targets in PD.
Topics: Animals; Corpus Striatum; Disease Models, Animal; Dopamine; Dopaminergic Neurons; GTP-Binding Proteins; Humans; Mice; Microglia; Motor Activity; Nerve Degeneration; Parkinson Disease; Pars Compacta; RGS Proteins; Signal Transduction
PubMed: 32015009
DOI: 10.1124/mol.119.118836 -
Human Brain Mapping May 2017The objective of this study was to measure neuromelanin-sensitive MRI contrast changes in the lateral-ventral tier of substantia nigra pars compacta in Parkinson's...
The objective of this study was to measure neuromelanin-sensitive MRI contrast changes in the lateral-ventral tier of substantia nigra pars compacta in Parkinson's disease (PD). Histopathological studies of PD have demonstrated both massive loss of melanized dopamine neurons and iron accumulation in the substantia nigra pars compacta. Neurodegeneration is most profound in the lateral-ventral tier of this structure. We have previously shown in both healthy controls and individuals with PD that neuromelanin-sensitive MRI and iron-sensitive MRI contrast regions in substantia nigra overlap. This overlap region is located in the lateral-ventral tier. Exploiting this area of contrast overlap for region of interest selection, we developed a semi-automated image processing approach to characterize the lateral-ventral tier in MRI data. Here we apply this approach to measure magnetization transfer contrast, which corresponds to local neuromelanin density, in both the lateral-ventral tier and the entire pars compacta in 22 PD patients and 19 controls. Significant contrast reductions were seen in PD in both the entire pars compacta (P = 0.009) and in its lateral-ventral tier (P = 0.0002); in PD contrast was significantly lower in the lateral-ventral tier than in the entire pars compacta (P = 0.0008). These findings are the first in vivo evidence of the selective vulnerability of this nigral subregion in PD, and this approach may be developed for high impact biomarker applications. Hum Brain Mapp 38:2627-2634, 2017. © 2017 Wiley Periodicals, Inc.
Topics: Aged; Analysis of Variance; Cohort Studies; Female; Humans; Image Processing, Computer-Assisted; Magnetic Resonance Imaging; Male; Middle Aged; Neurodegenerative Diseases; Parkinson Disease; ROC Curve; Severity of Illness Index; Substantia Nigra; Surveys and Questionnaires
PubMed: 28240402
DOI: 10.1002/hbm.23547 -
BioRxiv : the Preprint Server For... Feb 2024Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as...
Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of these neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter VMAT2, vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44%), VGAT+ (37%) and VGLUT2+ (41%) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54%), fewer were VGAT+ (42%), and VGLUT2+ neurons were least abundant (16%). Moreover, 20% of VTA neurons and 10% of SNc neurons expressed more than one vesicular transporter, including 45% of VGLUT2 neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.
PubMed: 38464250
DOI: 10.1101/2024.02.28.582356 -
Brain Sciences Dec 2016Glucocorticoid hormones (GCs) released from the fetal/maternal glands during late gestation are required for normal development of mammalian organs and tissues.... (Review)
Review
Glucocorticoid hormones (GCs) released from the fetal/maternal glands during late gestation are required for normal development of mammalian organs and tissues. Accordingly, synthetic glucocorticoids have proven to be invaluable in perinatal medicine where they are widely used to accelerate fetal lung maturation when there is risk of pre-term birth and to promote infant survival. However, clinical and pre-clinical studies have demonstrated that inappropriate exposure of the developing brain to elevated levels of GCs, either as a result of clinical over-use or after stress-induced activation of the fetal/maternal adrenal cortex, is linked with significant effects on brain structure, neurological function and behaviour in later life. In order to understand the underlying neural processes, particular interest has focused on the midbrain dopaminergic systems, which are critical regulators of normal adaptive behaviours, cognitive and sensorimotor functions. Specifically, using a rodent model of GC exposure in late gestation (approximating human brain development at late second/early third trimester), we demonstrated enduring effects on the shape and volume of the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) (origins of the mesocorticolimbic and nigrostriatal dopaminergic pathways) on the topographical organisation and size of the dopaminergic neuronal populations and astrocytes within these nuclei and on target innervation density and neurochemical markers of dopaminergic transmission (receptors, transporters, basal and amphetamine-stimulated dopamine release at striatal and prefrontal cortical sites) that impact on the adult brain. The effects of antenatal GC treatment (AGT) were both profound and sexually-dimorphic, not only in terms of quantitative change but also qualitatively, with several parameters affected in the opposite direction in males and females. Although such substantial neurobiological changes might presage marked behavioural effects, in utero GC exposure had only a modest or no effect, depending on sex, on a range of conditioned and unconditioned behaviours known to depend on midbrain dopaminergic transmission. Collectively, these findings suggest that apparent behavioural normality in certain tests, but not others, arises from AGT-induced adaptations or compensatory mechanisms within the midbrain dopaminergic systems, which preserve some, but not all functions. Furthermore, the capacities for molecular adaptations to early environmental challenge are different, even opponent, in males and females, which may account for their differential resilience or failure to perform adequately in behavioural tests. Behavioural "normality" is thus achieved by the midbrain dopaminergic network operating outside its normal limits (in a state of allostasis), rendering it at greater risk to malfunction when challenged in later life. Sex-specific neurobiological programming of midbrain dopaminergic systems may, therefore, have psychopathological relevance for the sex bias commonly found in brain disorders associated with these systems, and which have a neurodevelopmental component, including schizophrenia, ADHD (attention/deficit hyperactivity disorders), autism, depression and substance abuse.
PubMed: 28042822
DOI: 10.3390/brainsci7010005 -
Neural Regeneration Research Dec 2022Parkinson's disease is the second most common neurodegenerative disease and has currently no effective treatment, one that would be able to stop or reverse the loss of... (Review)
Review
Parkinson's disease is the second most common neurodegenerative disease and has currently no effective treatment, one that would be able to stop or reverse the loss of dopaminergic neurons in the substantia nigra pars compacta. In addition, Parkinson's disease diagnosis is typically done when a significant percentage of the dopaminergic neurons is already lost. In neurodegenerative disorders, some therapeutic strategies could be effective only at inhibiting further degeneration; on the other hand, cell replacement therapies aim at replacing lost neurons, an approach that would be ideal for the treatment of Parkinson's disease. Many cell replacement therapies have been tested since the 1970s in the field of Parkinson's disease; however, there are still significant limitations prohibiting a successful clinical application. From the first fetal midbrain intrastriatal graft to the most recent conversion of astrocytes into dopaminergic neurons, we have gained equally, significant insights and questions still looking for an answer. This review aims to summarize the main milestones in cell replacement approaches against Parkinson's disease. By focusing on achievements and failures, as well as on the additional research steps needed, we aim to provide perspective on how future cell replacement therapies treats Parkinson's disease.
PubMed: 35662194
DOI: 10.4103/1673-5374.336137 -
FEBS Letters Dec 2015The homeoprotein Engrailed (Engrailed-1/Engrailed-2, collectively En1/2) is not only a survival factor for mesencephalic dopaminergic (mDA) neurons during development,... (Review)
Review
The homeoprotein Engrailed (Engrailed-1/Engrailed-2, collectively En1/2) is not only a survival factor for mesencephalic dopaminergic (mDA) neurons during development, but continues to exert neuroprotective and physiological functions in adult mDA neurons. Loss of one En1 allele in the mouse leads to progressive demise of mDA neurons in the ventral midbrain starting from 6 weeks of age. These mice also develop Parkinson disease-like motor and non-motor symptoms. The characterization of En1 heterozygous mice have revealed striking parallels to central mechanisms of Parkinson disease pathogenesis, mainly related to mitochondrial dysfunction and retrograde degeneration. Thanks to the ability of homeoproteins to transduce cells, En1/2 proteins have also been used to protect mDA neurons in various experimental models of Parkinson disease. This neuroprotection is partly linked to the ability of En1/2 to regulate the translation of certain nuclear-encoded mitochondrial mRNAs for complex I subunits. Other transcription factors that govern mDA neuron development (e.g. Foxa1/2, Lmx1a/b, Nurr1, Otx2, Pitx3) also continue to function for the survival and maintenance of mDA neurons in the adult and act through partially overlapping but also diverse mechanisms.
Topics: Animals; Dopaminergic Neurons; Homeodomain Proteins; Humans; Mice, Transgenic; Mitochondria; Nerve Tissue Proteins; Parkinson Disease
PubMed: 26459030
DOI: 10.1016/j.febslet.2015.10.002