Dan Gincel - Academia.edu (original) (raw)

Papers by Dan Gincel

Journal of bioenergetics and biomembranes, 2000

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel... more The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel permeable to anions, cations, ATP, and other metabolites. VDAC was purified from sheep brain synaptosomes or rat liver mitochondria using a reactive red-agarose column, in addition to the hydroxyapatitate column. The red-agarose column allowed further purification (over 98%), concentration of the protein over ten-fold, decreasing Triton X-100 concentration, and/or replacing Triton X-100 with other detergents, such as Nonidet P-40 or octylglucoside. This purified VDAC reconstituted into planar-lipid bilayer, had a unitary maximal conductance of 3.7 +/- 0.1 nS in 1 M NaCl, at 10 mV and was permeable to both large cations and anions. In the maximal conducting state, the permeability ratios for Na(+), acetylcholine(+), dopamine,(+) and glutamate(-), relative to Cl(-), were estimated to be 0.73, 0.6, 0.44, and 0.4, respectively. In contrast, in the subconducting state, glutamate(-) was imper...

Investigative ophthalmology & visual science, 2002

To characterize and localize retinal voltage-dependent anion channel (VDAC) and to understand its... more To characterize and localize retinal voltage-dependent anion channel (VDAC) and to understand its possible contribution to mitochondrial function and dysfunction. VDAC was characterized by a method involving purification from isolated mitochondria and reconstitution into a planar lipid bilayer (PLB). The permeability transition pore (PTP) was monitored by Ca(2+) accumulation in isolated mitochondria and swelling of mitochondria. Localization was studied by immunocytochemistry and in situ hybridization. Retinal VDACs exhibited the electrophysiological fingerprint of the VDAC superfamily. It had a maximal chord conductance of 3.7 +/- 0.1 nanosiemens (nS) in 1 M NaCl, and a voltage-dependent conductance that was highest at transmembrane potential close to zero. It was modulated by glutamate, which decreased the channel's open probability, and by La(3+) and ruthenium amine binuclear complex (Ru360), which closed the channel. Energized and freshly prepared retinal mitochondria accumu...

Neuroscience Letters, 1997

Nature Genetics, 2006

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 ... more We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

Journal of Neurovirology, 2005

Changes in astrocyte shape and function are known to occur in association with human immunodefici... more Changes in astrocyte shape and function are known to occur in association with human immunodeficiency virus (HIV) dementia (HIVD). However, the causes and consequences of such changes are not completely understood. In vitro data suggest that changes in the expression of aquaporin 4 (AQP4), the aquaporin subtype expressed by astrocytes, can significantly influence cell shape and physiology. In the present study, the authors therefore investigated the possibility that AQP4 levels may be altered in HIVD. Using Western blot, the authors show that immunoreactivity for AQP4 is elevated in brain homogenates from the mid frontal gyrus of patients who died with HIVD (P < .005 HIV seronegative versus HIVD). Of interest, a significant increase was also observed in homogenates from HIV-infected individuals without dementia (P < .05 HIV seronegative versus neurologically normal HIV seropositive). In the present study the authors also examined the stimulated expression of AQP4 in cultured cells. Previous in vitro studies have shown that AQP4 expression may be increased by stimuli that induce cytoskeletal changes and/or the activation of p38 mitogen-activated protein (MAP) kinase. The authors therefore focused on tumor necrosis factor (TNF)-α, which has been linked to p38 MAP kinase activation, and thrombin, which may also induce changes in the actin cytoskeleton. Both may be elevated with HIVD. Again using Western blot, the authors show an increase in both AQP4 and phosphorylated p38 MAP kinase in homogenates from TNF-α-and thrombin-stimulated organotypic cerebellar and spinal cord cultures. Together, these studies suggest that AQP4 expression may be altered in HIVD and/or in response to exogenous proteinases. Additional studies may be warranted to determine whether altered AQP4 expression represents a protective and/or maladaptive response to central nervous system (CNS) inflammation. Journal of NeuroVirology (2005) 11, 535-543. AQP4 and HIV dementia C St Hillaire et al 537 * Cases used only for MFG analysis. * * Cases used only for DWM analysis.

Journal of Neurosurgery, 2014

The poor outcome of malignant gliomas is largely due to local invasiveness. Previous studies sugg... more The poor outcome of malignant gliomas is largely due to local invasiveness. Previous studies suggest that gliomas secrete excess glutamate and destroy surrounding normal peritumoral brain by means of excitotoxic mechanisms. In this study the authors assessed the effect on survival of 2 glutamate modulators (riluzole and memantine) in rodent glioma models. In an in vitro growth inhibition assay, F98 and 9L cells were exposed to riluzole and memantine. Mouse cerebellar organotypic cultures were implanted with F98 glioma cells and treated with radiation, radiation + riluzole, or vehicle and assessed for tumor growth. Safety and tolerability of intracranially implanted riluzole and memantine CPP:SA polymers were tested in F344 rats. The efficacy of these drugs was tested against the 9L model and riluzole was further tested with and without radiation therapy (RT). In vitro assays showed effective growth inhibition of both drugs on F98 and 9L cell lines. F98 organotypic cultures showed reduced growth of tumors treated with radiation and riluzole in comparison with untreated cultures or cultures treated with radiation or riluzole alone. Three separate efficacy experiments all showed that localized delivery of riluzole or memantine is efficacious against the 9L gliosarcoma tumor in vivo. Systemic riluzole monotherapy was ineffective; however, riluzole given with RT resulted in improved survival. Riluzole and memantine can be safely and effectively delivered intracranially via polymer in rat glioma models. Both drugs demonstrate efficacy against the 9L gliosarcoma and F98 glioma in vitro and in vivo. Although systemic riluzole proved ineffective in increasing survival, riluzole acted synergistically with radiation and increased survival compared with RT or riluzole alone.

Journal of Bioenergetics and Biomembranes, 2000

The amino acid glutamate, synthesized in the mitochondria, serves multiple functions, including a... more The amino acid glutamate, synthesized in the mitochondria, serves multiple functions, including acting as a neurotransmitter and participating in degradative and synthetic pathways. We have previously shown that glutamate modulates the channel activity of bilayer-reconstituted voltage-dependent anion channel (VDAC). In this study, we demonstrate that glutamate also modulates the opening of the mitochondrial permeability transition pore (PTP), of which VDAC is an essential component. Glutamate inhibited PTP opening, as monitored by transient Ca2+ accumulation, mitochondrial swelling and accompanying release of cytochrome c. Exposure to L-glutamate delayed the onset of PTP opening up to 3-times longer, with an IC50 of 0.5 mM. Inhibition of PTP opening by L-glutamate is highly specific, not being mimicked by D-glutamate, L-glutamine, L-aspartate, or L-asparagine. The interaction of L-glutamate with VDAC and its inhibition of VDAC&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s channel activity and PTP opening suggest that glutamate may also act as an intracellular messenger in the mitochondria-mediated apoptotic pathway.

Cell Biochemistry and Biophysics, 2003

Recently, it has been recognized that there is a metabolic coupling between the cytosol and mitoc... more Recently, it has been recognized that there is a metabolic coupling between the cytosol and mitochondria, where the outer mitochondrial membrane (OMM), the boundary between these compartments, has important functions. In this crosstalk, mitochondrial Ca2+ homeostasis and ATP production and supply play a major role. The primary transporter of ions and metabolites across the OMM is the voltage-dependent anion channel (VDAC). The interaction of VDAC with Ca2+, ATP glutamate, NADH, and different proteins was demonstrated, and these interactions may regulate OMM permeability. This review includes information on VDAC purification methods, characterization of its channel activity (selectivity, voltage-dependence, conductance), and the regulation of VDAC channel by ligands, such as Ca2+, glutamate and ATP and touches on many aspects of the physiological relevance of VDAC to Ca2+ homeostasis and mitochondria-mediated apoptosis.

Biophysical Journal, 2002

The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a rel... more The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 Ϯ 13 pS at ϩ60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K ϩ over Cl Ϫ (P K ϩ/P Cl Ϫ Ͼ 8) and preferred K ϩ over Li ϩ , Na ϩ , Rb ϩ , Cs ϩ , or choline ϩ . The synaptophysin channel is impermeable to Ca 2ϩ , which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission.

Biochemical Journal, 2001

Mitochondria play a central role in energy metabolism, Ca# + signalling, aging and cell death. To... more Mitochondria play a central role in energy metabolism, Ca# + signalling, aging and cell death. To control cytosolic or mitochondrial Ca# + concentration, mitochondria possess several Ca# +transport systems across the inner membrane. However, the pathway for Ca# + crossing the outer membrane has not been directly addressed. We report that purified voltage-dependent anion channel (VDAC) reconstituted into lipid bilayers or liposomes is highly permeable to Ca# + . VDAC contains Ca# +binding sites that bind Ruthenium Red (RuR), La$ + and that RuR completely closed VDACs in single or multichannel experiments. Energized, freshly prepared mitochondria accumulate Ca# + (500-700 nmol\mg of protein), and subsequently released it. The release of Ca# + is accompanied by cyclosporin A-inhibited swelling, suggesting activation of permeability transition pore (PTP). RuR and ruthenium amine binuclear complex, when added to mitochondria after Ca# + accumulation has reached a Key words : apoptosis, calcium transport, single channel.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2004

The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca 2 + and o... more The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca 2 + and other metabolites into and from mitochondria. Here, the intracellular localization and oligomeric organization of VDAC in brain mitochondria and ER are demonstrated. Immunohistochemical staining of VDAC in rat cerebellum showed high labeling of the Purkinje neurons. Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. Purified ER membranes contain VDAC, but not other mitochondrial proteins. Chemical cross-linking of isolated mitochondria, ER or purified VDAC demonstrated the existence of VDAC in oligomeric form. Based on the enrichment of VDAC in the junctional face of closely associated mitochondrial and ER membranes and the existence of VDAC oligomers, we propose an involvement of VDAC in specialized intermembrane communication between mitochondria or between ER and mitochondria, serving to complement the tight structural and functional coupling observed between these organelles. D

Experimental Neurology, 2007

The EAAT4 glutamate transporter helps regulate excitatory neurotransmission and prevents glutamat... more The EAAT4 glutamate transporter helps regulate excitatory neurotransmission and prevents glutamate-mediated excitotoxicity in the cerebellum. Immunohistochemistry and in situ hybridization have previously defined a cerebellar cell population expressing this protein. These methods, however, are not well suited for evaluating the dynamic regulation of the transporter and its gene-especially in living tissues. To better study EAAT4 expression and regulation, we generated bacterial artificial chromosome (BAC) promoter eGFP reporter transgenic mice. Histological analysis of the transgenic mice revealed that the EAAT4 promoter is active predominantly in Purkinje cells, but can also be modestly detected in other neurons early postnatally. EAAT4 promoter activity was not present in non-neuronal cells. Cerebellar organotypic slice cultures prepared from BAC transgenic mice provided a unique reagent to study transporter and Purkinje cell expression and regulation in living tissue. The correlation of promoter activity to protein expression makes the EAAT4 BAC promoter reporter a valuable tool to study regulation of EAAT4 expression.