The role of DJ-1 in enhancing mitochondrial quality control

Thesis (M.A.)--Boston UniversityDJ-1 is a cytosolic sensor for oxidative damage which acts on the Mitochondria. It works to curb the negative effects of high membrane potential in mitochondria, but the mechanism of action is still uncertain. This study measured DJ-1’s potential in enchancing mitochondrial quality control in the context of pancreatic B-cells treated with a palmitate and glucose media to promote glucolipotoxicity (GLT). DJ-1 was proven capable of reversing GLT induced changed in mitochondrial morphology in the arenas of Feret’s diameter, aspect ratio, and form factor. We also showed that the mitochondrial membrane potential did not vary with the presence or absence of DJ-1. In addition, DJ-1 was shown capable of limiting the upward boundary of GLT induced increase in mitochondrial membrane potential. Furthermore, an experiment using INS1 cells with GFP-LC3 showed that DJ-1 can decrease the average number of autophagosomes in the cell

Intrathecal Administration of the α1 Adrenergic Antagonist Phentolamine Upregulates Spinal GLT-1 and Improves Mirror Image Pain in SNI Model Rats

Mirror image pain (MIP) is a type of extraterritorial pain that results in contralateral pain or allodynia. Glutamate transporter-1 (GLT-1) is expressed in astrocytes and plays a role in maintaining low glutamate levels in the synaptic cleft. Previous studies have shown that GLT-1 dysfunction induces neuropathic pain. Our previous study revealed bilateral GLT-1 downregulation in the spinal cord of a spared nerve injury (SNI) rat. We hypothesized that spinal GLT-1 is involved in the mechanism of MIP. We also previously demonstrated noradrenergic GLT-1 regulation. Therefore, this study aimed to investigate the effect of an α1 adrenergic antagonist on the development of MIP. Rats were subjected to SNI. Changes in pain behavior and GLT-1 protein levels in the SNI rat spinal cords were then examined by intrathecal administration of the α1 adrenergic antagonist phentolamine, followed by von Frey test and western blotting. SNI resulted in the development of MIP and bilateral downregulation of GLT-1 protein in the rat spinal cord. Intrathecal phentolamine increased contralateral GLT-1 protein levels and partially ameliorated the 50% paw withdrawal threshold in the contralateral hind paw. Spinal GLT-1 upregulation by intrathecal phentolamine ameliorates MIP. GLT-1 plays a role in the development of MIPs

Neuronal activity mediated regulation of glutamate transporter GLT-1 surface diffusion in rat astrocytes in dissociated and slice cultures.

The astrocytic GLT-1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live-cell imaging to study the mechanisms regulating GLT-1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP-time lapse imaging, we show that GLT-1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity-dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT-1 is more stable than diffuse GLT-1 and that glutamate increases GLT-1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT-1 isoforms expressed in the brain, GLT-1a and GLT-1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT-1b more so. GLT-1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT-1 isoforms. Altogether, these data reveal that astrocytic GLT-1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission

Neuronal influences are necessary to produce mitochondrial co-localization with glutamate transporters in astrocytes.

yesAbstract Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial: GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments. Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity

Gene transfer of GLT-1, a glial glutamate transporter, into the spinal cord by recombinant adenovirus attenuates inflammatory and neuropathic pain in rats

<p>Abstract</p> <p>Background</p> <p>The glial glutamate transporter GLT-1 is abundantly expressed in astrocytes and is crucial for glutamate removal from the synaptic cleft. Decreases in glutamate uptake activity and expression of spinal glutamate transporters are reported in animal models of pathological pain. However, the lack of available specific inhibitors and/or activators for GLT-1 makes it difficult to determine the roles of spinal GLT-1 in inflammatory and neuropathic pain. In this study, we examined the effect of gene transfer of GLT-1 into the spinal cord with recombinant adenoviruses on the inflammatory and neuropathic pain in rats.</p> <p>Results</p> <p>Intraspinal infusion of adenoviral vectors expressing the GLT-1 gene increased GLT-1 expression in the spinal cord 2–21 days after the infusion. Transgene expression was primarily localized to astrocytes. The spinal GLT-1 gene transfer had no effect on acute mechanical and thermal nociceptive responses in naive rats, whereas it significantly reduced the inflammatory mechanical hyperalgesia induced by hindlimb intraplantar injection of carrageenan/kaolin. Spinal GLT-1 gene transfer 7 days before partial sciatic nerve ligation recovered the extent of the spinal GLT-1 expression in the membrane fraction that was decreased following the nerve ligation, and prevented the induction of tactile allodynia. However, the partial sciatic nerve ligation-induced allodynia was not reversed when the adenoviruses were infused 7 or 14 days after the nerve ligation.</p> <p>Conclusion</p> <p>These results suggest that overexpression of GLT-1 on astrocytes in the spinal cord by recombinant adenoviruses attenuates the induction, but not maintenance, of inflammatory and neuropathic pain, probably by preventing the induction of central sensitization, without affecting acute pain sensation. Upregulation or functional enhancement of spinal GLT-1 could be a novel strategy for the prevention of pathological pain.</p

The non-adrenergic imidazoline-1 receptor protein nischarin is a key regulator of astrocyte glutamate uptake

Astrocytic GLT-1 is the main glutamate transporter involved in glutamate buffering in the brain, pivotal for glutamate removal at excitatory synapses to terminate neurotransmission and for preventing excitotoxicity. We show here that the surface expression and function of GLT-1 can be rapidly modulated through the interaction of its N-terminus with the nonadrenergic imidazoline-1 receptor protein, Nischarin. The phox domain of Nischarin is critical for interaction and internalization of surface GLT-1. Using live super-resolution imaging, we found that glutamate accelerated Nischarin-GLT-1 internalization into endosomal structures. The surface GLT-1 level increased in Nischarin knockout astrocytes, and this correlated with a significant increase in transporter uptake current. In addition, Nischarin knockout in astrocytes is neuroprotective against glutamate excitotoxicity. These data provide new molecular insights into regulation of GLT-1 surface level and function and suggest new drug targets for the treatment of neurological disorders

Amoxicillin and amoxicillin/clavulanate reduce ethanol intake and increase GLT-1 expression as well as AKT phosphorylation in mesocorticolimbic regions.

Studies have shown that administration of the β-lactam antibiotic, ceftriaxone (CEF) attenuates ethanol consumption and cocaine seeking behavior as well as preventing ethanol-induced downregulation of glutamate transporter 1 (GLT-1) expression in central reward brain regions. However, it is not known if these effects are compound-specific. Therefore, the present study examined the effects of two other β-lactam antibiotics, amoxicillin (AMOX) and amoxicillin/clavulanate (Augmentin, AUG), on ethanol drinking, as well as GLT-1 and phosphorylated-AKT (pAKT) levels in the nucleus accumbens (Acb) and medial prefrontal cortex (mPFC) of alcohol-preferring (P) rats. P rats were exposed to free-choice of ethanol (15% and 30%) for five weeks and were given five consecutive daily i.p. injections of saline vehicle, 100 mg/kg AMOX or 100 mg/kg AUG. Both compounds significantly decreased ethanol intake and significantly increased GLT-1 expression in the Acb. AUG also increased GLT-1 expression in the mPFC. Results for changes in pAKT levels matched those for GLT-1, indicating that β-lactam antibiotic-induced reductions in ethanol intake are negatively associated with increases in GLT-1 and pAKT levels within two critical brains regions mediating drug reward and reinforcement. These findings add to a growing literature that pharmacological increases in GLT-1 expression are associated with decreases in ethanol intake and suggest that one mechanism mediating this effect may be increased phosphorylation of AKT. Thus, GLT-1 and pAKT may serve as molecular targets for the treatment of alcohol and drug abuse/dependence

Der Glutamattransporter GLT-1 des zentralen Nervensystems. Zellbiologische und elektrophysiologische Untersuchungen zur Transportfunktion

Der Glutamattransporter GLT-1 ist ein Transmembranprotein, das im ZNS überwiegend in Astrozyten lokalisiert ist. Seine Funktion als hochaffiner, Na+-abhängiger Transporter besteht in der Entfernung des Neurotansmitters Glutamat aus dem synaptischen Spalt. Dies ermöglicht eine getreue Signalweiterleitung und die Vermeidung von exzitatorischer Schädigung. Von den Glutamattransportern GLT-1 und den fluoreszierenden GLT-1 wie auch GLAST-1 und EAAC1, die am N-Terminus mit dem fluoreszierenden Polypeptid EGFP fusioniert sind, sind Konstrukte für die heterologe Expression in Xenopus laevis Oozyten und HEK293 Zellen (human embryonic kidney) erstellt worden. Die Untersuchungen dieser Arbeit belegen, daß GLT-1 als auch das Fusionsprotein während des Zell-targeting korrekt prozessiert und funktionell in die Plasmamembran eingebaut werden. Deshalb wurden die Konstrukte zur Charakterisierung von GLT-1 in Neurotransmitter Aufnahmestudien in GLT-1 transfizierten Xenopus laevis Oozyten und HEK293 Zellen mit radioaktiv markiertem L-[14C]-Glutamat, als auch in elektrophysiologischen whole-cell voltage- und patch-clamp Experimenten angewandt. Die Ionenspezifität für Na+-Ionen, KM-Werte von Glutamat und Na+-Ionen, die Kinetik der Glutamataufnahme und verschiedene Inhibitoren des GLT-1 Transporters wurden ermittelt. Die vorliegende Arbeit beschreibt zusätzlich den Einfluß von extrazellulären Ca2+-Ionen auf den Transport von Glutamat. Im Gegensatz zu Na+ wird Ca2+ nicht von GLT-1 transportiert, Ca2+-Ionen beeinflussen aber den Transport von L-Glutamat. Diese Beobachtungen erweitern unsere Kenntnisse über den Effekt von intrazellulärem Ca2+ hinaus auf die GluT Familie. Strukturanaloge Substrate des natürlichen Neurotransmitters Glutamat sind genauer klassifiziert worden. Zusätzlich zur endständigen geladenen Carboxylgruppe oder der SH-Gruppe in Liganden, konnte die essentielle Bedeutung der Aminogruppe in &#57527;-Position � wie in L-&#57527;-Aminoadipat � für die Bindung des Liganden bewiesen werden. Ungeladene Aminosäuren oder &#57528;-Aminosäuren, wie L-&#57528;-Aminoadipat, zeigen dagegen keinen Einfluß auf den Transport von L-Glutamat. Es wurde gezeigt, daß GLT-1, ähnlich wie GLAST-1, L-Aspartat transportieren kann

Kajian Pemilihan Struktur Fondasi Bangunan Empat Lantai

Fondasi adalah suatu konstruksi pada bagian dasar struktur bangunan yang berfungsi untuk meneruskan beban yang disebabkan struktur bangunan bagian atas. Dalam perencanaan suatu bangunan, diperlukan pemilihan fondasi yang efisien dalam hal biaya maupun waktu pengerjaan dan tidak melupakan kriteria desain yang ada. Penelitian&nbsp; ini&nbsp; dilakukan&nbsp; untuk menganalisis&nbsp; jenis fondasi yang efisien dan efektif digunakan pada&nbsp; Gedung Laboratorium Teknik 1 (GLT 1) &nbsp;dan Gedung Laboratorium Teknik 3 (GLT 3) Institut Teknologi Sumatera dengan membandingkan biaya dan waktu pekerjaan fondasi rakit pelat rata dan fondasi tiang bor. GLT 1 dan GLT 3 mempunyai kesamaan dalam jumlah lantai yaitu 4 lantai termasuk atap, namun berbeda dari jarak antar kolom. GLT 1 memiliki geometri gedung dengan panjang 56 m, lebar 24 m dan jarak antar kolom 8 m. Sedangkan GLT 3 memiliki geometri gedung dengan panjang 72 m, lebar 27 m dan jarak antar kolom 6 m. Berdasarkan analisis yang telah dilakukan, &nbsp;pekerjaan fondasi pada GLT 1 menyimpulkan bahwa dari aspek biaya pekerjaan fondasi tiang bor lebih hemat dibandingkan fondasi rakit. Sebaliknya, pekerjaan fondasi tiang bor pada GLT 3 lebih mahal sedikit dibandingkan pekerjaan fondasi rakit. Dari aspek waktu, pekerjaan fondasi rakit membutuhkan waktu lebih lama dari pekerjaan fondasi tiang bor