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Stuart A Lipton

Publications and source records attributed to Stuart A Lipton.

At least 55 records · Page 3Linked to original sources
PubMed2003

GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2.

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Cell spreading controls endoplasmic and nuclear calcium: a physical gene regulation pathway from the cell surface to the nucleus.

Cells attaching to an extracellular matrix through integrins flatten out (spread) on the matrix, eliciting cellular signals needed for survival. We show that the shape of the nucleus changes and the nuclear calcium level increases in spreading cells. Moreover, cell spreading and osmotic stretching of isolated nuclei cause release of perinuclear Ca(2+), and patch clamping of nuclei reveals stretch-activated Ca(2+) permeable channels. Gene expression assays with myocyte enhancer factor 2, which is activated by calmodulin-dependent kinase IV, indicate that the elevation in nuclear Ca(2+) is functionally significant. We propose a mechano-transduction pathway in which spreading-induced nuclear stretching releases Ca(2+) from the perinuclear space, and the resulting Ca(2+) elevation in the nucleus provokes changes in gene expression.

3T3 Cells↗
PubMed2003

Mitochondrial fission in apoptosis, neurodegeneration and aging.

A decline in mitochondrial function is well recognized in neurodegenerative diseases and aging, and is thought to play a causal role in their biology. Unfortunately, the molecular basis underlying this detrimental loss in mitochondrial function remains mysterious. Interestingly, mitochondria undergo frequent fission and fusion. This process is regulated by molecular machinery that has been highly conserved during evolution, including dynamin-related GTPases that manifest opposing effects. A balance between mitochondrial fission and fusion events is required for normal mitochondrial and cellular function. Emerging evidence indicates that mitochondria undergo rapid and extensive fission at an early stage during apoptosis. A clue that these new findings are of significance for the pathogenesis of neurodegenerative disease is provided by the observation that OPA-1, a dynamin-related GTPase regulating mitochondrial fusion, is mutated in humans with dominant optic atrophy, which is characterized by degeneration of retinal ganglion cells and childhood blindness. Loss of function of OPA-1, analogous to deficiency of its yeast homologue, Mgm1p, is expected to lead to mitochondrial fission, loss of mitochondrial DNA, respiratory deficits and an increase in reactive oxygen species. Here we review the molecular mediators controlling mitochondrial fission and fusion, and how death effector molecules may hijack this ancient machinery. A shift in the rate of mitochondrial fission or fusion may provide a new mechanistic explanation for the mitochondrial dysfunction in neurodegenerative diseases and normal aging, and may offer a new target for therapeutic intervention.

Aging↗
PubMed2003

Possible role for memantine in protecting retinal ganglion cells from glaucomatous damage.

Glaucoma is a neurodegenerative disease typified by progressive loss of retinal ganglion cells (RGCs). Mild excitotoxicity has been implicated as one of the factors contributing to RGC death during the glaucomatous process. This type of excitotoxic cell death is due, at least in part, to somewhat excessive activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors. NMDA-receptor activity, however, is also essential for normal neuronal function. This means that potential neuroprotective agents that block virtually all NMDA-receptor activity will have unacceptable clinical side effects. Studies in our laboratory have shown that the adamantane derivative, memantine, blocks only excessive NMDA-receptor activity without disrupting normal activity. Past clinical use has demonstrated that memantine is safe, and it has recently been approved in Europe for the treatment of Alzheimer's disease and vascular dementia. Clinical studies of the safety and efficacy of memantine in glaucoma are currently underway. A series of second-generation memantine derivatives called nitro-memantines are currently in development and may prove to have even greater neuroprotective properties than does memantine.

Animals↗
PubMed2003

Translationally distinct populations of NMDA receptor subunit NR1 mRNA in the developing rat brain.

The translational activity of the NMDA subunit 1 (NR1) mRNA was examined in the developing rat brain by sucrose gradient fractionation. One translationally-active pool of NR1 mRNA was associated with large polyribosomes (polysomes) over the entire developmental period examined. A second NR1 mRNA pool, approximately half of the NR1 mRNA at post-natal day 4, sedimented only within the two to three ribosome range, indicating that it was translationally blocked during early brain development despite active translation of mRNAs coding for the NR2 subunits of the receptor. At post-natal day 4, both NR1 mRNA pools were distributed throughout the brain and contained similar profiles of NR1 mRNA splice variants, except that NR1-3 appeared to be present only in the translationally-blocked NR1 pool. After post-natal day 8, the translationally-blocked NR1 mRNA pool became progressively active within a background of globally-decreasing brain translational activity.

Age Factors↗
PubMed2003

BAG1 over-expression in brain protects against stroke.

The co-chaperone BAG1 binds and regulates 70 kDa heat shock proteins (Hsp70/Hsc70) and exhibits cytoprotective activity in cell culture models. Recently, we observed that BAG1 expression is induced during neuronal differentiation in the developing brain. However, the in vivo effects of BAG1 during development and after maturation of the central nervous system have never been examined. We generated transgenic mice over-expressing BAG1 in neurons. While brain development was essentially normal, cultured cortical neurons from transgenic animals exhibited resistance to glutamate-induced, apoptotic neuronal death. Moreover, in an in vivo stroke model involving transient middle cerebral artery occlusion, BAG1 transgenic mice demonstrated decreased mortality and substantially reduced infarct volumes compared to wild-type littermates. Interestingly, brain tissue from BAG1 transgenic mice contained higher levels of neuroprotective Hsp70/Hsc70 protein but not mRNA, suggesting a potential mechanism whereby BAG1 exerts its anti-apoptotic effects. In summary, BAG1 displays potent neuroprotective activity in vivo against stroke, and therefore represents an interesting target for developing new therapeutic strategies including gene therapy and small-molecule drugs for reducing brain injury during cerebral ischemia and neurodegenerative diseases.

Animals↗
PubMed2003

Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina.

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.

Aldehyde Oxidoreductases↗
PubMed2003

Divergent NMDA signals leading to proapoptotic and antiapoptotic pathways in the rat retina.

PURPOSE: To investigate the involvement of the p38 mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI-3) kinase-Akt signaling pathways after pathologic stimulation by N-methyl-D-aspartate (NMDA) receptors of retinal neurons in vivo. METHODS: NMDA (2-200 nmol), SB203580 (0.2-10 nmol, an inhibitor of p38 MAP kinase), LY294002 (6 nmol, an inhibitor of PI-3 kinase), or control solution was injected into the vitreous of Long-Evans rats. To assess retinal ganglion cell (RGC) death quantitatively, we labeled RGCs retrogradely by injecting aminostilbamidine (FluoroGold) into the superior colliculus and subsequently counting fluorescently labeled RGCs in retinal wholemounts. Phosphorylation of p38 and Akt was assessed by immunoblot of whole retinal lysates, and activity was measured with in vitro kinase assays. To localize phospho-p38 and phospho-Akt, immunohistochemistry was performed. TUNEL staining coupled with morphologic assessment was performed to assess apoptotic cell death. RESULTS: Intravitreous injection of more than 10 nmol NMDA induced RGC death. Before death, NMDA-stimulated retinas manifested increased phospho-p38 and phospho-Akt in the ganglion cell and inner nuclear layers. Subsequently, pyknotic, TUNEL-positive cells were also localized to these regions. SB203580 partially rescued RGCs, whereas LY294002 enhanced death of RGCs due to 10 nmol NMDA. SB203580 and LY294002 specifically inhibited the activity of p38 MAP kinase and Akt, respectively. CONCLUSIONS: The p38 MAP kinase and PI-3 kinase-Akt pathways are involved in signal transduction after excessive stimulation of NMDA receptors in the retina. These inhibitor studies suggest that the p38 MAP kinase pathway is proapoptotic, whereas the PI-3 kinase-Akt pathway is antiapoptotic in RGC death induced by NMDA.

Animals↗
PubMed2003

N-methyl-D-aspartate receptor subunit NR3A in the retina: developmental expression, cellular localization, and functional aspects.

PURPOSE: Recently, a novel N-methyl-D-aspartate receptor (NMDAR) subunit, NR3A, has been discovered in the brain and shown to decrease NMDAR activity by modulating the calcium permeability of the receptor channel. The insertion of NR3A within the NMDAR complex may thus alter NMDAR properties and play a crucial role during processes of neuronal development and degeneration. The present study is the first to investigate the expression and cellular localization of NR3A on the protein level in the retina and to elucidate its putative functional roles within the retinal circuitry. METHODS: The expression of NR3A in the retina was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. Functional aspects of NR3A in the retina were addressed by measuring the NMDA-induced increase in intracellular calcium, [Ca(2+)](i), in retinal cells prepared from wild-type (NR3A(+/+)) and NR3A knockout (NR3A(+/-), and NR3A(-/-)) mice. RESULTS: NR3A protein expression was initially observed in the first postnatal week and was predominantly localized to cell bodies in the ganglion cell layer. In older animals, two bands of NR3A immunoreactivity were additionally observed in the inner plexiform layer. NMDA-evoked [Ca(2+)](i) responses were found to be significantly greater in retinal cells in NR3A(-/-) mice than in wild-type retinas. CONCLUSIONS: The data indicate that NR3A is specifically expressed in the inner retina and may modulate NMDAR-mediated calcium influx and thus [Ca(2+)](i) levels in retinal ganglion cells and amacrine cells.

Animals↗
PubMed2002

Effect of the ubiquitous transcription factors, SP1 and MAZ, on NMDA receptor subunit type 1 (NR1) expression during neuronal differentiation.

The silencer factor NRSF/REST has been reported to restrict expression to neurons of a variety of genes, including that encoding NMDA receptor subunit type 1 (NR1), by suppressing transcription in nonneuronal cells. However, we recently reported that in addition to the absence of NRSF/REST-binding activity, another neuron-specific mechanism is necessary for high level expression of the NR1 gene in neurons. In this study, we explored the mechanism of induction of NR1 promoter activity during neuronal differentiation of the P19 cell line. We identified a 27 base pair GC-rich region in the promoter as an important element responsible for induction of the NR1 gene after neuronal differentiation. We found that the ubiquitous transcription factors SP1 and MAZ bind to this GC-rich region. Surprisingly, the binding activities of SP1 and MAZ are not remarkably changed after neuronal differentiation. Mutations in the SP1 and MAZ sites impair binding of SP1 and MAZ proteins and also decrease NR1 promoter activity. These findings suggest that SP1 and MAZ mediate enhancement of NR1 promoter activity during neuronal differentiation despite the fact that their binding activity does not change.

Animals↗
PubMed2002

Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain.

NR3A is a developmentally regulated N-methyl-D-aspartate receptor (NMDAR) subunit that was previously known as NMDAR-L or chi-1. Unlike other NMDAR subunits, NR3A inhibits the NMDAR-associated ion channel in a novel manner, and a role in synaptogenesis has been suggested for this subunit. Here, we report a comprehensive study to delineate the temporal and anatomic expression of NR3A protein in the mammalian brain by using a monoclonal anti-NR3A antibody. NR3A protein was found to peak at postnatal day (P) 8, and to decrease gradually from P12 to adulthood in the rat central nervous system. Moreover, NR3A protein was heavily expressed in all areas of the isocortex, portions of the amygdaloid nuclei, and selective cell layers and nuclei of the hippocampus, thalamus, hypothalamus, brainstem, and spinal cord. NR3A protein was also expressed in the cerebellar cortex, whereas only weak signal was detected in the previous in situ studies by using riboprobes. At an ultrastructural level, NR3A was associated specifically with asymmetrical synapses and localized to postsynaptic membranes. This information will facilitate future research on NMDARs by providing clues to possible inclusion of the NR3A subunit in NMDARs in many brain regions.

Animals↗
PubMed2002

S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.

Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of neurodegenerative diseases and stroke. However, the mechanism of MMP activation remains unclear. We report that MMP activation involves S-nitrosylation. During cerebral ischemia in vivo, MMP-9 colocalized with neuronal nitric oxide synthase. S-Nitrosylation activated MMP-9 in vitro and induced neuronal apoptosis. Mass spectrometry identified the active derivative of MMP-9, both in vitro and in vivo, as a stable sulfinic or sulfonic acid, whose formation was triggered by S-nitrosylation. These findings suggest a potential extracellular proteolysis pathway to neuronal cell death in which S-nitrosylation activates MMPs, and further oxidation results in a stable posttranslational modification with pathological activity.

Animals↗
PubMed2002

Caspase cascades in human immunodeficiency virus-associated neurodegeneration.

Many patients infected with human immunodeficiency virus-1 (HIV-1) develop a syndrome of neurologic deterioration known as HIV-associated dementia (HAD). Neurons are not productively infected by HIV-1; thus, the mechanism of HIV-induced neuronal injury remains incompletely understood. Several investigators have observed evidence of neuronal injury, including dendritic degeneration, and apoptosis in CNS tissue from patients with HAD. Caspase enzymes, proteases associated with the process of apoptosis, are synthesized as inactive proenzymes and are activated in a proteolytic cascade after exposure to apoptotic signals. Here we demonstrate that HAD is associated with active caspase-3-like immunoreactivity that is localized to the soma and dendrites of neurons in affected regions of the human brain. Additionally, the cascade of caspase activation was studied using an in vitro model of HIV-induced neuronal apoptosis. Increased caspase-3 proteolytic activity and mitochondrial release of cytochrome c were observed in cerebrocortical cultures exposed to the HIV coat protein gp120. Specific inhibitors of both the Fas/tumor necrosis factor-alpha/death receptor pathway and the mitochondrial caspase pathway prevented gp120-induced neuronal apoptosis. Caspase inhibition also prevented the dendrite degeneration observed in vivo in transgenic mice with CNS expression of HIV/gp120. These findings suggest that pharmacologic interventions aimed at the caspase enzyme pathways may be beneficial for the prevention or treatment of HAD.

AIDS Dementia Complex↗
PubMed2002

Dominant-interfering forms of MEF2 generated by caspase cleavage contribute to NMDA-induced neuronal apoptosis.

Myocyte enhancer factor-2 (MEF2) transcription factors are activated by p38 mitogen-activated protein kinase during neuronal and myogenic differentiation. Recent work has shown that stimulation of this pathway is antiapoptotic during development but proapoptotic in mature neurons exposed to excitotoxic or other stress. We now report that excitotoxic (N-methyl-D-aspartate) insults to mature cerebrocortical neurons activate caspase-3, -7, in turn cleaving MEF2A, C, and D isoforms. MEF2 cleavage fragments containing a truncated transactivation domain but preserved DNA-binding domain block MEF2 transcriptional activity via dominant interference. Transfection of constitutively active MEF2 (MEF2C-CA) rescues MEF2 transcriptional activity after N-methyl-D-aspartate insult and prevents neuronal apoptosis. Conversely, dominant-interfering MEF2 abrogates neuroprotection by MEF2C-CA. These results define a pathway to excitotoxic neuronal stress/apoptosis via caspase-catalyzed cleavage of MEF2. Additionally, we show that similar MEF2 cleavage fragments are generated in vivo during focal stroke damage. Hence, this pathway appears to have pathophysiological relevance in vivo.

Animals↗
PubMed2002

Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits.

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.

Animals↗
PubMed2002

Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the same thiol group on a single cysteine residue, forming a molecular switch composed of a latticework of possible redox, NO or Zn(2+) modifications to control protein function. Thiol-mediated regulation of protein function can also involve reactions of cysteine residues that affect ligand binding allosterically. This article reviews the basis for these molecular cysteine switches, drawing on the NMDA receptor as an exemplary protein, and proposes a molecular model for the action of S-nitrosylation based on recently derived crystal structures.

Animals↗