Parkinson's disease gene, Synaptojanin1, dysregulates the surface maintenance of the dopamine transporter.

Missense mutations of PARK20/SYNJ1 (synaptojanin1/Synj1) have been linked to complex forms of familial parkinsonism, however, the molecular and cellular changes associated with dopaminergic dysfunction remains unknown. We now report fast depletion of evoked dopamine (DA) and altered maintenance of the axonal dopamine transporter (DAT) in the Synj1+/- neurons. While Synj1 has been traditionally known to facilitate the endocytosis of synaptic vesicles, we demonstrated that axons of cultured Synj1+/- neurons exhibit an increase of total DAT but a reduction of the surface DAT, which could be exacerbated by neuronal activity. We revealed that the loss of surface DAT is specifically associated with the impaired 5'-phosphatase activity of Synj1 and the hyperactive downstream PI(4,5)P2-PKCß pathway. Thus, our findings provided important mechanistic insight for Synj1-regulated DAT trafficking integral to dysfunctional DA signaling in early parkinsonism.

Cocaine-regulated trafficking of dopamine transporters in cultured neurons revealed by a pH sensitive reporter.

Cocaine acts by inhibiting plasma membrane dopamine transporter (DAT) function and altering its surface expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. In this study, we engineered and validated the use of DAT-pHluorin for studying DAT localization and its dynamic trafficking at neuronal processes of cultured mouse midbrain neurons. We demonstrate that unlike neuronal soma and dendrites, which contain a majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, whereas large varicosities contain abundant intracellular DAT within acidic intracellular structures. We also demonstrate that cocaine exposure leads to a Synaptojanin1-sensitive DAT internalization process followed by membrane reinsertion that lasts for days. Thus, our study reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.

Negative Feedback Role of Astrocytes in Shaping Excitation in Brain Cell Co-cultures.

Glial cells play an important role in maintaining neuronal homeostasis and may thus influence excitability in epileptogenesis. These cells in the brain have glutamate (Glu) transporters, which remove this neurotransmitter from the extracellular space. Lack of negative (-) feedback makes local neuronal circuits more excitable and potentially contributing to epileptogenic phenomena. In this study, the role of glial cells in providing (-) feedback is shown through different models of brain cells in culture imaged for intracellular calcium concentration [(Ca2+)i]. Moreover, here we study the individual cells by putting them in categories. Neuronal networks with high and low (-) feedback were established by using anti-mitotics to deplete glial cells. Separate stimuli with very low subthreshold concentrations of Glu (250-750 nM) were added to cultures to test if the order of stimulations matter in regard to calcium dynamics outcomes. Additionally, KCl and ATP were used to stimulate glial cells. We found that for cultures high in (-) feedback, order of the stimulus was not important in predicting cellular responses and because of the complexity of networks in low (-) feedback cultures the order of stimulus matters. As an additional method for analysis, comparison of high (-) feedback cultures, and pure astrocytes was also considered. Glial cells in pure astrocyte cultures tend to be larger in size than glial cells in high (-) feedback cultures. The potential effect of (-) feedback at the blood brain barrier (BBB) was also considered for the inflammatory responses of nitric oxide (NO) production and [Ca2+]i regulation using brain microvascular endothelial cells (BMVECs). The inflammatory and calcium signaling pathways both indicate the negative feedback role of astrocytes, poised between the BBB and structures deeper within the brain, where neuronal synapses are homeostatically maintained by glial uptake of neurotransmitters.

Morphological Changes in Astrocytes by Self-Oxidation of Dopamine to Polydopamine and Quantification of Dopamine through Multivariate Regression Analysis of Polydopamine Images.

Astrocytes, also known as astroglia, are important cells for the structural support of neurons as well as for biochemical balance in the central nervous system (CNS). In this study, the polymerization of dopamine (DA) to polydopamine (PDA) and its effect on astrocytes was investigated. The polymerization of DA, being directly proportional to the DA concentration, raises the prospect of detecting DA concentration from PDA optically using image-processing techniques. It was found here that DA, a naturally occurring neurotransmitter, significantly altered astrocyte cell number, morphology, and metabolism, compared to astrocytes in the absence of DA. Along with these effects on astrocytes, the polymerization of DA to PDA was tracked optically in the same cell culture wells. This polymerization process led to a unique methodology based on multivariate regression analysis that quantified the concentration of DA from optical images of astrocyte cell culture media. Therefore, this developed methodology, combined with conventional imaging equipment, could be used in place of high-end and expensive analytical chemistry instruments, such as spectrophotometry, mass spectrometry, and fluorescence techniques, for quantification of the concentration of DA after polymerization to PDA under in vitro and potentially in vivo conditions.

The Immunomodulatory Potential of Copper and Silver Based Self-Assembled Metal Organic Biohybrids Nanomaterials in Cancer Theranostics.

Copper high aspect ratio structures (CuHARS) and silver cystine nanoparticles (AgCysNPs) are two unique micro/nano particles under study here that show extensive anti-cancer effects on a glioma tumor cell line. These micro/nano particles have shown potent toxicity in the presence of inflammatory stimulus (combination of tumor necrosis factor, [TNF] and lipo-polysaccharide, LPS). CuHARS with a concentration of 20 µg/ml uniquely increased the catalytic generation of nitric oxide (NO), an important contributor in the immune system. This NO was generated in a cell culture tumor microenvironment (TME) in the presence of 25 µM S-nitrosothiol (cysteine-NO) and the inflammatory stimulus. CuHARS increased the NO production by 68.75% when compared to untreated glioma cells with CysNO and inflammatory stimulus. The production of NO was significantly higher under similar circumstances in the case of normal primary structural cells like brain microvascular endothelial cells (BMVECs). The production of NO by BMVECs went up by 181.25% compared to glioma cells. This significant increase in the NO concentration could have added up to tumorigenesis but the anti-cancer effect of CuHARS was prominent enough to lower down the viability of glioma cells by approximately 20% and increased the metabolism of structural cells, BMVECs by approximately 200%. The immunomodulatory effect of NO in the TME under these circumstances in the presence of the novel micro/nano material, CuHARS has risen up compared to the effect of inflammatory stimulus alone. The potency and specific nature of these materials toward tumor cells may make them suitable candidates for cancer treatment. Successive treatment of CuHARS to glioma cells also proved to be an effective approach considering the decrease in the total count of cells by 11.84 fold in case of three successive treatments compared to a single dose which only decreased the cell count by 2.45 fold showing the dose-dependent increasing toxicity toward glioma cells. AgCysNPs are another potent nanomaterial which also proved its significant toxic nature toward tumor cell lines as demonstrated here, but their immunomodulatory response is still unclear and needs to be explored further.

Self-Assembled Metal-Organic Biohybrids (MOBs) Using Copper and Silver for Cell Studies.

The novel synthesis of metal-containing biohybrids using self-assembly methods at physiological temperatures (37 °C) was compared for copper and silver using the amino acid dimer cystine. Once assembled, the copper containing biohybrid is a stable, high-aspect ratio structure, which we call CuHARS. Using the same synthesis conditions, but replacing copper with silver, we have synthesized cystine-capped silver nanoparticles (AgCysNPs), which are shown here to form stable colloid solutions in contrast to the CuHARS, which settle out from a 1 mg/mL solution in 90 min. Both the copper and silver biohybrids, as synthesized, demonstrate very low agglomeration which we have applied for the purpose of applications with cell culture methods, namely, for testing as anti-cancer compounds. AgCysNPs (1000 ng/mL) demonstrated significant toxicity (only 6.8% viability) to glioma and neuroblastoma cells in vitro, with concentrations as low as 20 ng/mL causing some toxicity. In contrast, CuHARS required at least 5 µg/mL. For comparative purposes, silver sulfate at 100 ng/mL decreased viability by 52% and copper sulfate at 100 ng/mL only by 19.5% on glioma cells. Using these methods, the novel materials were tested here as metal-organic biohybrids (MOBs), and it is anticipated that the functionalization and dynamics of MOBs may result in building a foundation of new materials for cellular applications, including cell engineering of both normal and diseased cells and tissue constructs.