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S Strickland

Publications and source records attributed to S Strickland.

At least 19 recordsLinked to original sources
PubMed2000

Tissue plasminogen activator-mediated fibrinolysis protects against axonal degeneration and demyelination after sciatic nerve injury.

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.

Animals↗
PubMed2000

Completion of meiosis in Drosophila oocytes requires transcriptional control by grauzone, a new zinc finger protein.

Mutations in grauzone or cortex cause abnormal arrest in Drosophila female meiosis. We cloned grauzone and identified it as a C2H2-type zinc finger transcription factor. The grauzone transcript is present in ovaries and at later developmental stages. A Grauzone-GFP fusion protein is functional and localizes to nuclei of both nurse cells and follicle cells during oogenesis. Three lines of evidence indicate that grauzone and cortex interact: reducing cortex function enhanced the grauzone mutant phenotype; cortex transcript abundance is reduced in the absence of grauzone function and Grauzone protein binds to the cortex promoter. These results demonstrate that activation of cortex transcription by grauzone is necessary for the completion of meiosis in Drosophila oocytes, and establish a new pathway that specifically regulates the female meiotic cell cycle.

Alleles↗
PubMed1999

Neuronal death and blood-brain barrier breakdown after excitotoxic injury are independent processes.

Neuronal damage in the CNS after excitotoxic injury is correlated with blood-brain barrier (BBB) breakdown. We have used a glutamate analog injection model and genetically altered mice to investigate the relationship between these two processes in the hippocampus. Our results show that BBB dysfunction occurs too late to initiate neurodegeneration. In addition, plasma infused directly into the hippocampus is not toxic and does not affect excitotoxin-induced neuronal death. To test plasma protein recruitment in neuronal degeneration, we used plasminogen-deficient (plg(-/-)) mice, which are resistant to excitotoxin-induced degeneration. Plasminogen is produced in the hippocampus and is also present at high levels in plasma, allowing us to determine the contribution of each source to cell death. Intrahippocampal delivery of plasminogen to plg(-/-) mice restored degeneration to wild-type levels, but intravenous delivery of plasminogen did not. Finally, although the neurons in plg(-/-) mice do not die after excitotoxin injection, BBB breakdown occurs to a similar extent as in wild-type mice, indicating that neuronal death is not necessary for BBB breakdown. These results indicate that excitotoxin-induced neuronal death and BBB breakdown are separable events in the hippocampus.

Animals↗
PubMed1999

Dissociation of mRNA cytoplasmic polyadenylation from translational activation by structural modification of the 5'-UTR.

During early metazoan development, certain maternal mRNAs are translationally activated by elongation of their poly(A) tails. Bicoid ( bcd ) mRNA is a Drosophila maternal mRNA that is translationally activated by cytoplasmic polyadenylation during the first hour after egg deposition. The sequences necessary and sufficient to promote its poly(A) elongation, and hence translation, are contained within its 3'-untranslated region (UTR). The mechanism by which poly(A) elongation at the 3'-end affects translational initiation at the 5'-end remains unknown. To investigate this question, we have analyzed a bicoid mRNA whose 5'-UTR contains a short antisense sequence directed against a portion of the coding region. This mutated RNA is efficiently translated in vitro. After injection into Drosophila embryos, this RNA is stable and polyadenylated, but inefficiently translated. These experiments show that structural modification of the 5'-end of an mRNA can perturb the translational activation normally conferred by polyadenylation in vivo.

3' Untranslated Regions↗
PubMed1999

Assaying the polyadenylation state of mRNAs.

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.

Blotting, Northern↗
PubMed1999

Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system.

Tissue plasminogen activator mediates excitotoxin-induced neurodegeneration and microglial activation in the mouse hippocampus. Here we show that tissue plasminogen activator (tPA) acts in a protease-independent manner to modulate the activation of microglia, the cells of the central nervous system with macrophage properties. Cultured microglia from tPA-deficient mice can phagocytose as efficiently as wild-type microglia. However, tPA-deficient microglia in mixed cortical cultures exhibit attenuated activation in response to lipopolysaccharide, as judged by morphological changes, increased expression of the activation marker F4/80 and the release of the pro-inflammatory cytokine tumor necrosis factor-(&agr;). When tPA is added to tPA deficient cortical cultures prior to endotoxin stimulation, microglial activation is restored to levels comparable to that observed in wild-type cells. Proteolytically-inactive tPA can also restore activation of tPA-deficient microglia in culture and in vivo. However, this inactive enzyme does not restore susceptibility of tPA-deficient hippocampal neurons to excitotoxin-mediated cell death. These results dissociate two different functions of tPA: inactive enzyme can mediate microglial activation, whereas proteolytically-competent protein also promotes neuronal degeneration. Thus tPA is identified as a new cytokine in the central nervous system.

Animals↗
PubMed1998

Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice.

Intravenous tissue plasminogen activator (tPA) is used to treat acute stroke because of its thrombolytic activity and its ability to restore circulation to the brain. However, this protease also promotes neurodegeneration after intracerebral injection of excitotoxins such as glutamate, and neuronal damage after a cerebral infarct is thought to be mediated by excitotoxins. To investigate the effects of tPA on cerebral viability during ischemia/reperfusion, we occluded the middle cerebral artery in wild-type and tPA-deficient mice with an intravascular filament. This procedure allowed us to examine the role of tPA in ischemia, independent of its effect as a thrombolytic agent. tPA-deficient mice exhibited approximately 50% smaller cerebral infarcts than wild-type mice. Intravenous injection of tPA into tPA-/- or wild-type mice produced larger infarcts, indicating that tPA can increase stroke-induced injury. Since tPA promotes desirable (thrombolytic) as well as undesirable (neurotoxic) outcomes during stroke, future therapies should be aimed at countering the excitotoxic damage of tPA to afford even better neuroprotection after an acute cerebral infarct.

Animals↗
PubMed1998

Mapping of Drosophila mutations using site-specific male recombination.

Although recombination does not usually occur in the male Drosophila germline, site-specific recombination can be induced at the ends of P elements. This finding suggested that male recombination could be used to map Drosophila mutations. In this article, we describe the general method and its application to the mapping of two EMS-induced female-sterile mutations, grauzone and cortex. Within two months, the grauzone gene was mapped relative to seven different P-element insertion sites, and cortex was mapped relative to 23 different P-elements. The results allowed us to map grauzone to a region of about 50 kb, and cortex distal to the chromosomal region 33E. These experiments demonstrate that P-element-induced site-specific male recombination is an efficient and general method to map Drosophila autosomal mutations.

Animals↗
PubMed1998

Cytoplasmic polyadenylation of Toll mRNA is required for dorsal-ventral patterning in Drosophila embryogenesis.

Toll encodes a receptor that is critical for dorsal-ventral patterning in the early Drosophila embryo. Previous data have suggested that the accumulation of Toll protein in the embryo temporally correlates with elongation of the poly (A) tail of the message. Here, we demonstrate that Toll mRNA is translationally activated by regulated cytoplasmic polyadenylation. We also identify a 192 nucleotide regulatory element in the Toll 3' UTR that is necessary for robust translational activation of Toll mRNA and also regulates polyadenylation. UV crosslinking analyses suggest that two proteins bind specifically to the 192 nucleotide element. One or both of these proteins may be factors that are required for translational regulation or cytoplasmic polyadenylation. These studies demonstrate that regulated polyadenylation plays a critical role in the Drosophila dorsal-ventral patterning system.

Animals↗
PubMed1997

Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin.

Excess excitatory amino acids can provoke neuronal death in the hippocampus, and the extracellular proteases tissue plasminogen activator (tPA) and plasmin (ogen) have been implicated in this death. To investigate substrates for plasmin that might influence neuronal degeneration, extracellular matrix (ECM) protein expression was examined. Laminin is expressed in the hippocampus and disappears after excitotoxin injection. Laminin disappearance precedes neuronal death, is spatially coincident with regions that exhibit neuronal loss, and is blocked by either tPA-deficiency or infusion of a plasmin inhibitor, both of which also block neuronal degeneration. Preventing neuron-laminin interaction by infusion of anti-laminin antibodies into tPA-deficient mice restores excitotoxic sensitivity to their hippocampal neurons. These results indicate that disruption of neuron-ECM interaction via tPA/plasmin catalyzed degradation of laminin sensitizes hippocampal neurons to cell death.

Animals↗
PubMed1997

Removal of tissue plasminogen activator does not protect against neuronal degeneration in the cerebellum of the weaver mouse.

Tissue plasminogen activator (tPA) is a serine protease that has been shown to be involved in neuronal degeneration. Recently, elevated cerebellar tPA has been reported in a naturally occurring mutant mouse, weaver. Weaver mice suffer extensive degeneration of cerebellar granular neurons during development, leading to severe malformation of the cerebellum as well as abnormal behavior (ataxia). The observations that the developing weaver cerebellum displays a 10-fold increase in tPA activity over wild-type and that a serine protease inhibitor was able to rescue weaver granule cells from premature death in culture suggested that tPA might mediate the death of these mutant neurons. We tested this possibility by introducing the weaver mutation into tPA-deficient mice and comparing the weaver phenotype in the presence or absence of tPA. Analysis at 28 days after birth indicates that tPA-deficient weaver mice are indistinguishable from tPA-containing weaver mice in behavior, cerebellar anatomy, histology, and laminin expression (also reported to be increased in weaver). These results suggest that removal of tPA activity from weaver mice does not protect against neuronal degeneration in the cerebellum and, thus, tPA does not appear to mediate this form of cell death.

Animals↗
PubMed1997

Neuronal death in the central nervous system demonstrates a non-fibrin substrate for plasmin.

Mice deficient for plasminogen exhibit a variety of pathologies, all of which examined to date are reversed when the animals are also made fibrin(ogen) deficient. These results suggested that the predominant, and perhaps exclusive, physiological role of plasminogen is clearance of fibrin. Plasminogen-deficient mice also display resistance to excitotoxin-induced neurodegeneration, in contrast with wild-type mice, which are sensitive. Based on the genetic interaction between plasminogen and fibrinogen, we investigated whether resistance to neuronal cell death in the plasminogen-deficient mice is dependent on fibrin(ogen). Unexpectedly, mice lacking both plasminogen and fibrinogen are resistant to neurodegeneration to levels comparable to plasminogen-deficient mice. Therefore, plasmin acts on substrates other than fibrin during experimental neuronal degeneration, and may function similarly in other pathological settings in the central nervous system.

Animals↗
PubMed1997

An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus.

Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.

Animals↗
PubMed1997

Regulated localization confers multiple functions on the protease urokinase plasminogen activator.

We have investigated the role of the plasminogen activation cascade in skeletal muscle differentiation. Migrating, undifferentiated myoblasts express urokinase plasminogen activator (uPA) and its cell surface receptor (uPAR). Consequently, uPA is localized predominantly to the cell surface. Preventing uPA from associating with its receptor with a noncatalytic form of uPA (NC-uPA) hinders migration of myoblasts and inhibits differentiation. When myoblasts reach confluence, cease migrating, and start to differentiate, uPAR gets downregulated, and uPA becomes redistributed from the cell surface to the extracellular space. The function of uPA at this stage was tested using the protease inhibitors aprotinin, alpha2-antiplasmin, or plasminogen activator inhibitor-1 (PAI-1). Contrary to the role of cell-associated uPA, inhibition of soluble uPA/plasmin stimulates differentiation of myoblasts. Aprotinin can inhibit activation of latent TGFbeta and stimulates differentiation, suggesting PAI-1 and alpha2-antiplasmin also may stimulate differentiation via this mechanism. These data suggest that regulation of uPA localization allows a dual function for this protease in regulating cell migration and controlling cell differentiation.

Animals↗
PubMed1997

Oocyte selection of mutations affecting cytoplasmic polyadenylation of maternal mRNAs.

Translational activation by cytoplasmic polyadenylation is a conserved mechanism in metazoan early development. In Xenopus and mouse, the regulatory sequences that control this process during oocyte meiotic maturation have been identified in the 3' untranslated region (3'-UTR) of a class of maternal messenger RNAs (mRNAs). In this report, we have investigated sequences controlling cytoplasmic polyadenylation of a mouse maternal mRNA. Pools of RNAs, transcribed from DNA randomly mutated by a PCR-based method, were micro-injected into the cytoplasm of mouse primary oocytes to allow in vivo selection of inefficiently polyadenylated transcripts. After oocyte maturation, the nonelongated RNAs were gel-isolated, and single base substitutions that alter poly(A) addition were identified. Analysis of these mutant RNAs identified single nucleotides that influence efficiency of cytoplasmic polyadenylation during mouse oocyte maturation. In addition, this strategy should facilitate identification of yet unknown sequence elements responsible for basic biological mechanisms during and after early development.

Animals↗
PubMed1997

Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA.

Nanos protein promotes abdominal structures in Drosophila embryos by repressing the translation of maternal hunchback mRNA in the posterior. To study the mechanism of nanos-mediated translational repression, we first examined the mechanism by which maternal hunchback mRNA is translationally activated. In the absence of nanos activity, the poly(A) tail of hunchback mRNA is elongated concomitant with its translation, suggesting that cytoplasmic polyadenylation directs activation. However, in the presence of nanos the length of the hunchback mRNA poly(A) tail is reduced. To determine if nanos activity represses translation by altering the polyadenylation state of hunchback mRNA, we injected various in vitro transcribed RNAs into Drosophila embryos and determined changes in polyadenylation. Nanos activity reduced the polyadenylation status of injected hunchback RNAs by accelerating their deadenylation. Pumilio activity, which is necessary to repress the translation of hunchback, is also needed to alter polyadenylation. An examination of translation indicates a strong correlation between poly(A) shortening and suppression of translation. These data indicate that nanos and pumilio determine posterior morphology by promoting the deadenylation of maternal hunchback mRNA, thereby repressing its translation.

Animals↗