Association Between Decreased Srpk3 Expression And An Increase In Substantia Nigra Alpha-Synuclein Levels In An MPTP-Induced Parkinson’s Disease Mouse Model

Parkinson’s disease (PD) is known as the second most common neurodegenerative disease, which is caused by destruction of dopaminergic neurons in the substantia nigra (SN) of the brain; however, the reason for the death of dopaminergic neurons remains unclear. An increase in α-synuclein (α-syn) is considered an important factor in the pathogenesis of PD. In the current study, we investigated the association between PD and serine/arginine-rich protein specific kinase 3 (Srpk3) in MPTP-induced parkinsonism mice model and in SH-SY5Y cells treated with MPP+. Srpk3 expression was significantly downregulated, while tyrosine hydroxylase (TH) decreased and α-synuclein (α-syn) increased after 4 weeks of MPTP intoxication treatment. Dopaminergic cell reduction and α-syn increase were demonstrated by inhibiting Srpk3 expression by siRNA in SH-SY5Y cells. Moreover, a decrease in Srpk3 expression upon siRNA treatment promoted dopaminergic cell reduction and α-syn increase in SH-SY5Y cells treated with MPP+. These results suggest that the decrease in Srpk3 expression due to Srpk3 siRNA caused both a decrease in TH and an increase in α-syn. This raises new possibilities for studying how Srpk3 controls dopaminergic cells and α-syn expression, which may be related to the pathogenesis of PD. Our results provide an avenue for understanding the role of Srpk3 during dopaminergic cell loss and α-syn increase in the SN. Furthermore, this study could support a therapeutic possibility for PD in that the maintenance of Srpk3 expression inhibited dopaminergic cell reduction.

The Sphingolipid Asset Is Altered in the Nigrostriatal System of Mice Models of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disease incurable due to late diagnosis and treatment. Therefore, one of the priorities of neurology is to study the mechanisms of PD pathogenesis at the preclinical and early clinical stages. Given the important role of sphingolipids in the pathogenesis of neurodegenerative diseases, we aimed to analyze the gene expression of key sphingolipid metabolism enzymes (ASAH1, ASAH2, CERS1, CERS3, CERS5, GBA1, SMPD1, SMPD2, UGCG) and the content of 32 sphingolipids (subspecies of ceramides, sphingomyelins, monohexosylceramides and sphinganine, sphingosine, and sphingosine-1-phosphate) in the nigrostriatal system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models of the preclinical and clinical stages of PD. It has been shown that in PD models, the expression of five of the nine studied genes (CERS1, CERS5, ASAH1, ASAH2, and GBA1) increases but only in the substantia nigra (SN) containing dopaminergic cell bodies. Changes in the expression of enzyme genes were accompanied by an increase in the content of 7 of the 32 studied sphingolipids. Such findings suggest these genes as attractive candidates for diagnostic purposes for preclinical and clinical stages of PD.

Identification by proximity labeling of novel lipidic and proteinaceous potential partners of the dopamine transporter

Dopamine (DA) transporters (DATs) are regulated by trafficking and modulatory processes that probably rely on stable and transient interactions with neighboring proteins and lipids. Using proximity-dependent biotin identification (BioID), we found novel potential partners for DAT, including several membrane proteins, such as the transmembrane chaperone 4F2hc, the proteolipid M6a and a potential membrane receptor for progesterone (PGRMC2). We also detected two cytoplasmic proteins: a component of the Cullin1-dependent ubiquitination machinery termed F-box/LRR-repeat protein 2 (FBXL2), and the enzyme inositol 5-phosphatase 2 (SHIP2). Immunoprecipitation (IP) and immunofluorescence studies confirmed either a physical association or a close spatial proximity between these proteins and DAT. M6a, SHIP2 and the Cullin1 system were shown to increase DAT activity in coexpression experiments, suggesting a functional role for their association. Deeper analysis revealed that M6a, which is enriched in neuronal protrusions (filopodia or dendritic spines), colocalized with DAT in these structures. In addition, the product of SHIP2 enzymatic activity (phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]) was tightly associated with DAT, as shown by co-IP and by colocalization of mCherry-DAT with a specific biosensor for this phospholipid. PI(3,4)P2 strongly stimulated transport activity in electrophysiological recordings, and conversely, inhibition of SHIP2 reduced DA uptake in several experimental systems including striatal synaptosomes and the dopaminergic cell line SH-SY5Y. In summary, here we report several potential new partners for DAT and a novel regulatory lipid, which may represent new pharmacological targets for DAT, a pivotal protein in dopaminergic function of the brain.

Cerebroventricular Microinjections of MPTP on Adult Zebrafish Induces Dopaminergic Neuronal Death, Mitochondrial Fragmentation, and Sensorimotor Impairments

Mitochondria are dynamic organelles that mediate the energetic supply to cells and mitigate oxidative stress through the intricate balance of fission and fusion. Mitochondrial dysfunction is a prominent feature within Parkinson disease (PD) etiologies. To date, there have been conflicting studies of neurotoxin impact on dopaminergic cell death, mitochondrial function and behavioral impairment using adult zebrafish. Here, we performed cerebroventricular microinjections (CVMIs) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on adult transgenic zebrafish that resulted in significant reductions in dopaminergic neurons within the telencephalon and olfactory bulbs (OB) of Tg(dat:eGFP) fish. Visualization of mCherry and mitochondrial gene expression analysis in Tg(dat:tom20 MLS:mCherry) fish reveal that MPTP induces mitochondrial fragmentation in dopaminergic neurons and the activation of the pink1/parkin pathway involved mitophagy. Moreover, the loss of dopaminergic neurons translated into a transient locomotor and olfactory phenotype. Taken together, these data can contribute to a better understanding of the mitochondrial impact on dopaminergic survivability.

Disrupted basal ganglia output during movement preparation in hemi-parkinsonian mice is consistent with behavioral deficits

Parkinsonian motor deficits are associated with elevated inhibitory output from the basal ganglia (BG). However, several features of Parkinson's disease (PD) have not been accounted for by this simple "classical rate model" framework, including the observation in PD patients that movements guided by external stimuli are less impaired than otherwise-identical movements generated based on internal goals. Is this difference due to divergent processing within the BG itself, or to the recruitment of extra-BG pathways by sensory processing? In addition, surprisingly little is known about precisely when, in the sequence from selecting to executing movements, BG output is altered by PD. Here, we address these questions by recording activity in the SNr, a key BG output nucleus, in hemiparkinsonian mice performing a well-controlled behavioral task requiring stimulus-guided and internally-specified directional movements. We found that hemiparkinsonian mice exhibited a bias ipsilateral to the side of dopaminergic cell loss that was stronger when movements were internally specified rather than stimulus guided, consistent with clinical observations in parkinsonian patients. We further found that changes in parkinsonian SNr activity during movement preparation were consistent with the ipsilateral behavioral bias, as well as its greater magnitude for internally-specified movements. While these findings are inconsistent with some aspects of the classical rate model, they are accounted for by a related "directional rate model" positing that SNr output phasically over-inhibits motor output in a direction-specific manner. These results suggest that parkinsonian changes in BG output underlying movement preparation contribute to the greater deficit in internally-specified than stimulus-guided movements.

The Reassessed Impact of Nicotine against Neurotoxicity in Mesencephalic Dopaminergic Cell Cultures and Neuroblastoma N18TG2 Cells

Neuroprotective effects of nicotine are still under debate, so further studies on its effectiveness against Parkinsonʼs disease are required. In our present study, we used primary dopaminergic cell cultures and N18TG2 neuroblastoma cells to investigate the effect of nicotine and its neuroprotective potential against rotenone toxicity. Nicotine protected dopaminergic (tyrosine hydroxylase immunoreactive) neurons against rotenone. This effect was not nAChR receptor-dependent. Moreover, the alkaloid at a concentration of 5 µM caused an increase in neurite length, and at a concentration of 500 µM, it caused an increase in neurite count in dopaminergic cells exposed to rotenone. Nicotine alone was not toxic in either cell culture model, while the highest tested concentration of nicotine (500 µM) caused growth inhibition of N18TG2 neuroblastoma cells. Nicotine alone increased the level of glutathione in both cell cultures and also in rotenone-treated neuroblastoma cells. The obtained results may be helpful to explain the potential neuroprotective action of nicotine on neural cell cultures.

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

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.

The complete cell lineage and MAPK- and Otx-dependent specification of the dopaminergic cells in the Ciona brain

The Ciona larva has served as a unique model for understanding the development of dopaminergic cells at single-cell resolution due to the exceptionally small number of neurons in its brain and its fixed cell lineage during embryogenesis. A recent study suggested that the transcription factors Fer2 and Meis directly regulate the dopamine synthesis genes in Ciona, but the dopaminergic cell lineage and the gene regulatory networks that control the development of dopaminergic cells have not been fully elucidated. Here, we reveal that the dopaminergic cells in Ciona are derived from a bilateral pair of cells called a9.37 cells at the center of the neural plate. The a9.37 cells divide along the anterior-posterior axis, and all of the descendants of the posterior daughter cells differentiate into the dopaminergic cells. We show that the MAPK pathway and the transcription factor Otx are required for the expression of Fer2 in the dopaminergic cell lineage. Our findings establish the cellular and molecular framework for fully understanding the commitment to dopaminergic cells in the simple chordate brain.

Interoceptive perception of optogenetic brain stimulation

How do animals experience brain manipulations? Optogenetics has allowed us to manipulate selectively and interrogate neural circuits underlying brain function in health and disease. However, in addition to their evoked physiological functions, it is currently unknown whether mice could perceive arbitrary optogenetic stimulations. To address this issue, mice were trained to report optogenetic stimulations to obtain rewards and avoid punishments. It was found that mice could perceive optogenetic manipulations regardless of the brain area modulated, their rewarding effects, or the stimulation of glutamatergic, GABAergic, and dopaminergic cell types. We named this phenomenon optoception. Our findings reveal that mice’s brains are capable of “monitoring” their self-activity via interoception, opening a new way to introduce information to the brain and control brain-computer interfaces.One Sentence SummaryBrain manipulations are perceived