Dysregulation of Rac or Rho elicits death of motor neurons and activation of these GTPases is altered in the G93A mutant hSOD1 mouse model of amyotrophic lateral sclerosis.

Rho GTPases play a central role in neuronal survival; however, the antagonistic relationship between Rac and Rho in the regulation of motor neuron survival remains poorly defined. In the current study, we demonstrate that treatment with NSC23766, a selective inhibitor of the Rac-specific guanine nucleotide exchange factors, Tiam1 and Trio, is sufficient to induce the death of embryonic stem cell (ESC)-derived motor neurons. The mode of cell death is primarily apoptotic and is characterized by caspase-3 activation, de-phosphorylation of ERK5 and AKT, and nuclear translocation of the BH3-only protein Bad.

The Bcl-2 Homology-3 Domain (BH3)-Only Proteins, Bid, DP5/Hrk, and BNip3L, Are Upregulated in Reactive Astrocytes of End-Stage Mutant SOD1 Mouse Spinal Cord.

The molecular mechanisms leading to motor neuron death in amyotrophic lateral sclerosis (ALS) are unknown; however, several studies have provided evidence of a central role for intrinsic apoptosis. Bcl-2 homology-3 domain (BH3)-only proteins are pro-apoptotic members of the Bcl-2 family whose enhanced expression acts as a trigger for the intrinsic apoptotic cascade. Here, we compared the relative expression of BH3-only proteins in the spinal cord of end-stage G93A mutant SOD1 mice to age-matched wild-type (WT) mice.

Varicella zoster virus infection of human fetal lung cells alters mitochondrial morphology.

Varicella zoster virus (VZV) is a ubiquitous alphaherpesvirus that establishes latency in ganglionic neurons throughout the neuraxis after primary infection. Here, we show that VZV infection induces a time-dependent significant change in mitochondrial morphology, an important indicator of cellular health, since mitochondria are involved in essential cellular functions. VZV immediate-early protein 63 (IE63) was detected in mitochondria-rich cellular fractions extracted from infected human fetal lung fibroblasts (HFL) by Western blotting.

Differential Mitochondrial Adaptation in Primary Vascular Smooth Muscle Cells from a Diabetic Rat Model.

Diabetes affects more than 330 million people worldwide and causes elevated cardiovascular disease risk. Mitochondria are critical for vascular function, generate cellular reactive oxygen species (ROS), and are perturbed by diabetes, representing a novel target for therapeutics. We hypothesized that adaptive mitochondrial plasticity in response to nutrient stress would be impaired in diabetes cellular physiology via a nitric oxide synthase- (NOS-) mediated decrease in mitochondrial function.

Raw and processed microscope images of fixed cells at baseline and following various experimental perturbations.

The data included in this article comprise raw and processed images of fixed cells at baseline and subjected to various experimental perturbations. This dataset includes images of HUVEC cells fixed and subsequently incubated at either 37 °C or room temperature, primary rat vascular smooth muscle cells exposed to 25 mM glucose, and SH-SY5Y neurons exposed to hydrogen peroxide. Raw images appear exactly as they were captured on the microscope, while processed images show the binarization provided by software used for measurements of mitochondrial morphology.

Fully automated software for quantitative measurements of mitochondrial morphology.

Mitochondria undergo dynamic changes in morphology in order to adapt to changes in nutrient and oxygen availability, communicate with the nucleus, and modulate intracellular calcium dynamics. Many recent papers have been published assessing mitochondrial morphology endpoints. Although these studies have yielded valuable insights, contemporary assessment of mitochondrial morphology is typically subjective and qualitative, precluding direct comparison of outcomes between different studies and likely missing many subtle effects.

Regulation of cyclic AMP response element-binding protein during neuroglial interactions.

Communications between neurons and glial cells play an important role in regulating homeostasis in the central nervous system. cAMP response element-binding protein (CREB), a transcription factor, is down-regulated by neurotoxins, which are known to be released by activated glial cells. To determine the role of CREB signaling in neuroglial interactions, we used three neuroglial coculture models consisting of human neuroprogenitor cell (NPC)-derived neurons and human microglia.

Dipeptidyl peptidase-4 inhibition in diabetic rats leads to activation of the transcription factor CREB in β-cells.

Incretin therapies are effective in controlling blood glucose levels in type 2 diabetic patients by improving the survival and function of β-cells. They include dipeptidyl peptidase-4 (DPP-4) inhibitors and long-acting glucagon-like peptide-1 (GLP-1) analogs. We have previously reported that GLP-1 enhances the survival of cultured human islets by activation of the transcription factor CREB.

Neuronal apoptosis induced by selective inhibition of Rac GTPase versus global suppression of Rho family GTPases is mediated by alterations in distinct mitogen-activated protein kinase signaling cascades.

Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766).

Laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture.

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures.

Induction of an inflammatory loop by interleukin-1β and tumor necrosis factor-α involves NF-kB and STAT-1 in differentiated human neuroprogenitor cells.

Proinflammatory cytokines secreted from microglia are known to induce a secondary immune response in astrocytes leading to an inflammatory loop. Cytokines also interfere with neurogenesis during aging and in neurodegenerative diseases. The present study examined the mechanism of induction of inflammatory mediators at the transcriptional level in human differentiated neuroprogenitor cells (NPCs).

C-terminal binding proteins are essential pro-survival factors that undergo caspase-dependent downregulation during neuronal apoptosis.

C-terminal binding proteins (CtBPs) are transcriptional co-repressors that are subject to proteasome-dependent downregulation during apoptosis. Alternative mechanisms that regulate CtBP expression are currently under investigation and the role of CtBPs in neuronal survival is largely unexplored. Here, we show that CtBPs are downregulated in cerebellar granule neurons (CGNs) induced to undergo apoptosis by a variety of stressors.

N-terminal cleavage of the mitochondrial fusion GTPase OPA1 occurs via a caspase-independent mechanism in cerebellar granule neurons exposed to oxidative or nitrosative stress.

Neuronal cell death via apoptosis or necrosis underlies several devastating neurodegenerative diseases associated with aging. Mitochondrial dysfunction resulting from oxidative or nitrosative stress often acts as an initiating stimulus for intrinsic apoptosis or necrosis. These events frequently occur in conjunction with imbalances in the mitochondrial fission and fusion equilibrium, although the cause and effect relationships remain elusive.

Neuroprotective actions of glucagon-like peptide-1 in differentiated human neuroprogenitor cells.

Glucagon-like peptide-1 (GLP-1)-based therapies are currently available for the treatment of type 2 diabetes, based on their actions on pancreatic β cells. GLP-1 is also known to exert neuroprotective actions. To determine its mechanism of action, we developed a neuron-rich cell culture system by differentiating human neuroprogenitor cells in the presence of a combination of neurotrophins and retinoic acid.

Signal transducer and activator of transcription-5 mediates neuronal apoptosis induced by inhibition of Rac GTPase activity.

In several neuronal cell types, the small GTPase Rac is essential for survival. We have shown previously that the Rho family GTPase inhibitor Clostridium difficile toxin B (ToxB) induces apoptosis in primary rat cerebellar granule neurons (CGNs) principally via inhibition of Rac GTPase function. In the present study, incubation with ToxB activated a proapoptotic Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and a pan-JAK inhibitor protected CGNs from Rac inhibition.

Calpain plays a central role in 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity in cerebellar granule neurons.

1-Methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity has previously been attributed to either caspase-dependent apoptosis or caspase-independent cell death. In the current study, we found that MPP(+) induces a unique, non-apoptotic nuclear morphology coupled with a caspase-independent but calpain-dependent mechanism of cell death in primary cultures of rat cerebellar granule neurons (CGNs). Using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay in CGNs exposed to MPP(+), we observed that these neurons are essentially devoid of caspase-dependent DNA fragments indicative of apoptosis.

CREB downregulation in vascular disease: a common response to cardiovascular risk.

Objective: To examine the impact of low-density lipoprotein (LDL), an established mediator of atherosclerosis, on the transcription factor cAMP-response element-binding protein (CREB), which is a regulator of vascular smooth muscle cell (VSMC) quiescence.

Methods And Results: VSMC CREB content is diminished in rodent models of diabetes and pulmonary hypertension. We examined aortic CREB content in rodent models of aging, hypertension, and insulin resistance, and we determined nuclear CREB protein in the medial VSMC of high-fat-fed LDL receptor-null mice.

Caspases indirectly regulate cleavage of the mitochondrial fusion GTPase OPA1 in neurons undergoing apoptosis.

The critical processes of mitochondrial fission and fusion are regulated by members of the dynamin family of GTPases. Imbalances in mitochondrial fission and fusion contribute to neuronal cell death. For example, increased fission mediated by the dynamin-related GTPase, Drp1, or decreased fusion resulting from inactivating mutations in the OPA1 GTPase, causes neuronal apoptosis and/or neurodegeneration.

Green tea epigallocatechin 3-gallate accumulates in mitochondria and displays a selective antiapoptotic effect against inducers of mitochondrial oxidative stress in neurons.

Epigallocatechin-3-gallate (EGCG) is a major flavonoid component of green tea that displays antiapoptotic effects in numerous models of neurotoxicity. Although the intrinsic free radical scavenging activity of EGCG likely contributes to its antiapoptotic effect, other modes of action have also been suggested. We systematically analyzed the antiapoptotic action of EGCG in primary cultures of rat cerebellar granule neurons (CGNs).

Glutathione binding to the Bcl-2 homology-3 domain groove: a molecular basis for Bcl-2 antioxidant function at mitochondria.

Bcl-2 protects cells against mitochondrial oxidative stress and subsequent apoptosis. However, the mechanism underlying the antioxidant function of Bcl-2 is currently unknown. Recently, Bax and several Bcl-2 homology-3 domain (BH3)-only proteins (Bid, Puma, and Noxa) have been shown to induce a pro-oxidant state at mitochondria (1-4).

KSR v. Teleflex. Part 1: Impact of U.S Supreme Court Patent Law on Canadian intellectual property and regulatory rights landscape.

In KSR, SCOTUS retooled the standard for obviousness to bring it back in line with the court's previous decisions in Hotchkiss and Graham. A comparative review of the law of obviousness in the United States and Canada, and its relation to innovation and competition, was undertaken in Sections II and III. The focal point of observed differences is the inherent creativity and inventiveness of the PHOSITA, which in turn informs several binary and highly rigid aspects of Canadian patent law relevant to a statutory determination of obviousness.