An Easy-to-Implement Protocol for Preparing Postnatal Ventral Mesencephalic Cultures.

Postnatally derived cultures of ventral mesencephalic neurons offer several crucial advantages over embryonic ventral mesencephalic cultures, including a higher content of TH-positive cells and the ability to derive cells from the substantia nigra, which contains the neurons most vulnerable to Parkinson's disease. On the other hand, these cultures are more challenging to produce consistently. Here, we provide an easy-to-implement protocol for culturing postnatal ventral mesencephalic cells from the substantia nigra (SN) and the ventral tegmental area using commercially available media, dishes, and general lab equipment, avoiding extensive material and equipment purchases. The protocol can be completed in about 5 h and provides ventral midbrain neuron cultures on cortex glia feeder layers in three weeks' time. The protocol uses an optimized protease digestion, tissue storage in Hibernate A during dissection and purification of neurons on an OptiPrep density gradient

Application of AA-PSP to hypersonic flows: the double ramp model

Anodized Aluminium Pressure Sensitive Paint (AA-PSP) is known for its rapid response characteristics, making it a highly desirable technique when studying high-speed phenomenon on a global scale. The current study examines the efficacy of the AA-PSP technique, which is prepared with a more practical approach than that reported in literature, in analysing the flow characteristics of a double ramp model placed in hypersonic flow of M = 5. Three different flow angles of 0°, −2°, and −4° are studied. Two-dimensional colour schlieren visualisation, using a colour wheel, is employed alongside high sensitivity Kulite pressure tap data to corroborate the AA-PSP findings. The AA-PSP results show good correlation between the qualitative schlieren and ±8.9% discrepency with the quantitative pressure tap data. The more practical AA-PSP preparation proposed in the current study, which uses aluminium alloy 6-series rather than pure aluminium, is proved to have the response time and the accuracy to be applied to unsteady high-speed flows

Investigation of the double ramp in hypersonic flow using luminescent measurement systems

Compression ramp flows in supersonic and hypersonic environments present unique flow patterns for shock wave-boundary layer interaction studies. They also represent the generic geometry of two-dimensional inlets and deflected control surfaces for re-entry vehicles. Therefore, a detailed knowledge of the flow behaviour created by such geometries is critical for optimum design. The flow is made more complicated due to the presence of separation regions and streamwise Görtler vortices. The objective of the current research is to study the behaviour and characteristics of the flow over the double ramp model placed in hypersonic flow at freestream Mach number of 5. Three different incidence angles of 0°, −2°, and −4° are studied using colour Schlieren and luminescent paints consisting of anodized aluminium pressure-sensitive paint (AA-PSP) and the temperature-sensitive paint (TSP) technique. The colour Schlieren provides description of the external flow while the global surface pressure and temperature distribution is obtained through the AA-PSP and TSP methods. The TSP technique also proves that it is very effective in identifying the location and properties of the Görtler vortices; revealing the effect of incidence on the magnitude and pattern of Görtler vortices formed

Calcineurin determines toxic versus beneficial responses to α-synuclein

Calcineurin (CN) is a highly conserved Ca[superscript 2+]–calmodulin (CaM)-dependent phosphatase that senses Ca[superscript 2+] concentrations and transduces that information into cellular responses. Ca[superscript 2+] homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca[superscript 2+], thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase’s activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.Jeffry M. and Barbara Picower FoundationJPB FoundationHoward Hughes Medical Institute (Collaborative Innovation Award)Eleanor Schwartz Charitable Foundatio

1,25‐dihydroxyvitamin D 3 influences cellular homocysteine levels in murine preosteoblastic MC3T3‐E1 cells by direct regulation of cystathionine β‐synthase

High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY owing to its negative impact on collagen maturation. HCY is cleared by cystathionine β‐synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3‐E1 murine preosteoblasts treated with 1,25‐dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH) 2 D 3 . Quantitative real‐time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH) 2 D 3 treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine, and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH) 2 D 3 ‐treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knockout osteoblasts, in which upregulation of cbs in response to 1,25(OH) 2 D 3 was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs . To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D 3 levels] and HCY levels. In conclusion, this study showed that cbs is a primary 1,25(OH) 2 D 3 target gene which renders HCY metabolism responsive to 1,25(OH) 2 D 3 . © 2011 American Society for Bone and Mineral ResearchPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88106/1/493_ftp.pd

iPSC-Derived Dopamine Neurons Reveal Differences between Monozygotic Twins Discordant for Parkinson’s Disease

SummaryParkinson’s disease (PD) has been attributed to a combination of genetic and nongenetic factors. We studied a set of monozygotic twins harboring the heterozygous glucocerebrosidase mutation (GBA N370S) but clinically discordant for PD. We applied induced pluripotent stem cell (iPSC) technology for PD disease modeling using the twins’ fibroblasts to evaluate and dissect the genetic and nongenetic contributions. Utilizing fluorescence-activated cell sorting, we obtained a homogenous population of “footprint-free” iPSC-derived midbrain dopaminergic (mDA) neurons. The mDA neurons from both twins had ∼50% GBA enzymatic activity, ∼3-fold elevated α-synuclein protein levels, and a reduced capacity to synthesize and release dopamine. Interestingly, the affected twin’s neurons showed an even lower dopamine level, increased monoamine oxidase B (MAO-B) expression, and impaired intrinsic network activity. Overexpression of wild-type GBA and treatment with MAO-B inhibitors normalized α-synuclein and dopamine levels, suggesting a combination therapy for the affected twin

Novel synthetic sulfoglycolipid IG20 facilitates exocytosis in chromaffin cells through the regulation of sodium channels

This is the peer reviewed version of the following article: Novel synthetic sulfoglycolipid IG20 facilitates exocytosis in chromaffin cells through the regulation of sodium channels: Journal of Neurochemistre, 135.5 (2015), which has been published in final form at http://dx.doi.org/10.1111/jnc.13357In searching for druggable synthetic lipids as potential modulators of synaptic transmission and plasticity, we synthesized sulfoglycolipid IG20 that stimulates neuritic outgrowth. Here we have explored its effects on ion channels and exocytosis in bovine chromaffin cells (BCCs). IG20 augmented the rate of basal catecholamine release. Such effect did not depend on Ca2+ mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca2+ entry through Lsubtype voltage-activated Ca2+ channels. Those channels were recruited by cell depolarization mediated by IG20 likely through its ability to enhance the recruitment of Na+ channels at more hyperpolarizing potentials. Confocal imaging with fluorescent derivative IG20-NBD revealed its rapid incorporation and confinement into the plasmalemma, supporting the idea that IG20 effects are exerted through a plasmalemmal-delimited mechanism. Thus, synthetic IG20 seems to mimic several physiological effects of endogenous lipids such as regulation of ion channels, Ca2+ signaling, and exocytosis. Therefore, sulfoglycolipid IG20 may become a pharmacological tool to investigate the role of the lipid environment on neuronal excitability, ion channels, neurotransmitter release, synaptic efficacy, and neuronal plasticity. It may also inspire the synthesis of druggable sulfoglycolipids aimed at increasing synaptic plasticity and efficacy in neurodegenerative diseases and traumatic brain – spinal cord injuryThis work was supported by grants from Ministerio de Economía y Competitividad, Spain (MINECO, SAF-2013-44108-P to AGG and LG; and FIS-PI11/01436 to AFM), Fundación Eugenio Rodríguez Pascual (to LG). We thank the continued support of Fundación Teófilo Hernando, Madrid, Spai

Dynamic physiological alpha-synuclein S129 phosphorylation is driven by neuronal activity

In Parkinson’s disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA−/− neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker

Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT’s role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects

Dopamine Induced Neurodegeneration in a PINK1 Model of Parkinson's Disease

Parkinson's disease is a common neurodegenerative disease characterised by progressive loss of dopaminergic neurons, leading to dopamine depletion in the striatum. Mutations in the PINK1 gene cause an autosomal recessive form of Parkinson's disease. Loss of PINK1 function causes mitochondrial dysfunction, increased reactive oxygen species production and calcium dysregulation, which increases susceptibility to neuronal death in Parkinson's disease. The basis of neuronal vulnerability to dopamine in Parkinson's disease is not well understood