Biomedical subjects

A J Windebank

Publications and source records attributed to A J Windebank.

At least 37 records · Page 2Linked to original sources

PubMed1999

Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the adult rat spinal cord: regulation by the glutamate receptor agonist kainic acid.

Previous in vitro studies indicate that select members of the neurotrophin gene family, namely brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), contribute to survival and differentiation of spinal cord motoneurons. To investigate the potential roles of these factors in the adult spinal cord, we examined their cellular localization and regulation after systemic exposure to an excitotoxic stimulus, kainic acid (KA). Of the neurotrophins examined, NT-4/5 mRNA was most robustly expressed in the lumbosacral spinal cord of the normal adult rat, including expression by neurons throughout the gray matter, and in a subpopulation of white and gray matter glia. Both BDNF and NT-3 mRNAs were also densely expressed by alpha motoneurons of lamina IX, but were detected at lower levels elsewhere in the gray matter. NT-3 mRNA was additionally expressed by spinal cord glia, but was less widespread compared to NT-4/5. In response to systemic administration of KA, NT-4/5 and BDNF mRNAs were dramatically upregulated in a spatially and temporally restricted fashion, whereas levels of NT-3 mRNA were unchanged. These results provide strong in vivo evidence to support the idea that BDNF, NT-3, and in particular, NT-4/5, play a role in the normal function of the adult spinal cord. Furthermore, our results indicate that the actions of BDNF and NT-4/5 participate in the response of the cord to excitotoxic stimuli, and that those of NT-4/5 and NT-3 include both neurons and glia.

Animals↗

PubMed1999

Role of the blink reflex in the evaluation of sensory neuronopathy.

Because of an incidental observation that the blink reflex was normal in paraneoplastic sensory neuronopathy (SN) and frequently abnormal in nonparaneoplastic SN, the authors reviewed the electromyographic records of patients with SN in whom blink reflex studies were performed. The blink reflex was normal in all 17 patients with paraneoplastic SN and abnormal in 20 of 43 patients with nonparaneoplastic SN. Although it does not exclude paraneoplastic SN, an abnormal blink reflex favors a nonparaneoplastic etiology.

Action Potentials↗

PubMed1999

Exclusion of the ninjurin gene as a candidate for hereditary sensory neuropathies type I and type II.

Ninjurin is a protein that is up-regulated in Schwann cells and neurons after peripheral nerve injury. Its role in promoting nerve regeneration and its expression in sensory neurons of dorsal root ganglia, as well as the chromosomal localization of the ninjurin gene, makes this gene a candidate for hereditary sensory neuropathies (HSN). In the present report, the human ninjurin gene was analyzed in 17 unrelated patients with HSN type I, two patients with HSN type II, and 10 normal controls, by single strand conformation polymorphism and by direct sequencing. All three exons and splice junctions of the gene were investigated and no mutations were found in our sample of patients. Our results rule out a mutation in the translated region of the ninjurin gene as a cause of HSN type I and type II.

Cell Adhesion Molecules, Neuronal↗

PubMed1999

Congenital hypomyelination due to myelin protein zero Q215X mutation.

Congenital hypomyelination (CH) is a hereditary demyelinating peripheral neuropathy characterized by early infancy onset, distal muscle weakness, hypotonia, areflexia, and severe slowing of nerve conduction velocities. In the present report, the clinical, morphological, and immunohistochemical features of a CH case and the identification of a mutation in the gene (MPZ) for protein zero (P0) associated with this phenotype are described. This "de novo" mutation in a patient presenting with clinical features quite distinct from those of the more frequent Charcot-Marie-Tooth type 1B disease (CMT1B) or Dejerine-Sottas syndrome (DSS) confirms that CH is allelic with other disorders characterized by a less severe phenotype and a different clinical and neuropathological profile.

Demyelinating Diseases↗

PubMed1999

Neurons undergo apoptosis in animal and cell culture models of diabetes.

Recent clinical trials indicate that the severity of diabetic neuropathy is correlated with the level of patient glycemic control. In the current study, hyperglycemia induces apoptotic changes in dorsal root ganglion neurons and Schwann cells in vivo both in streptozotocin-treated diabetic rats and in rats made acutely hyperglycemic with infused glucose. Typical apoptotic nuclear and cytoplasmic changes are observed. In addition mitochondrial changes recently reported to occur as part of the apoptotic cascade, such as ballooning of mitochondria and disruption of the internal cristae, are seen in diabetic dorsal root ganglion neurons and Schwann cells. Similar changes have been reported in neurons in the presence of oxidative stress. In order to study the neurotoxic effects of high glucose we developed an in vitro model using rat dorsal root ganglion neurons. In dorsal root ganglion cultured in defined medium, addition of moderate glucose levels results in neurite degeneration and apoptosis. These changes are coupled with activation of caspase-3, dependent on the concentration of glucose. The apoptotic changes observed in vitro are similar to those observed in vivo. In contrast, addition of IGF-I, even at physiological concentrations, prevents activation of caspase-3 and neuronal apoptosis in vitro. We suggest that oxidative stress may promote the mitochondrial changes in diabetic animals and lead to activation of programmed cell death caspase pathways. These results imply a new pathogenetic mechanism for diabetic sensory neuropathy.

Animals↗

PubMed1999

Chemotherapeutic neuropathy.

Peripheral neurotoxicity is a dose-limiting side-effect for a number of effective chemotherapeutic agents, including platinum compounds, taxanes, and vinca alkaloids. New experimental chemotherapy drugs that cause neuropathy include suramin and Dolostatin-10. A better understanding of cellular mechanisms will lead to novel treatment strategies that will protect neurons without decreasing therapeutic efficacy.

Animals↗

PubMed1999

Phase I trial of dolastatin-10 (NSC 376128) in patients with advanced solid tumors.

Dolastatin-10 (dola-10) is a potent antimitotic peptide, isolated from the marine mollusk Dolabela auricularia, that inhibits tubulin polymerization. Preclinical studies of dola-10 have demonstrated activity against a variety of murine and human tumors in cell cultures and mice models. The purpose of this Phase I clinical trial was to characterize the maximum tolerated dose, pharmacokinetics, and biological effects of dola-10 in patients with advanced solid tumors. Escalating doses of dola-10 were administered as an i.v. bolus every 21 days, using a modified Fibonacci dose escalation schema. Pharmacokinetic studies were performed with the first treatment cycle. Neurological testing was performed on each patient prior to treatment with dola-10, at 6 weeks and at study termination. Thirty eligible patients received a total of 94 cycles (median, 2 cycles; maximum, 14 cycles) of dola-10 at doses ranging from 65 to 455 microg/m2. Dose-limiting toxicity of granulocytopenia was seen at 455 microg/m2 for minimally pretreated patients (two or fewer prior chemotherapy regimens) and 325 microg/m2 for heavily pretreated patients (more than two prior chemotherapy regimens). Nonhematological toxicity was generally mild. Local irritation at the drug injection site was mild and not dose dependent. Nine patients developed new or increased symptoms of mild peripheral sensory neuropathy that was not dose limiting. This toxicity was more frequent in patients with preexisting peripheral neuropathies. Pharmacokinetic studies demonstrated a rapid drug distribution with a prolonged plasma elimination phase (t 1/2z = 320 min). The area under the concentration-time curve increased in proportion to administered dose, whereas the clearance remained constant over the doses studied. Correlation analysis demonstrated a strong relationship between dola-10 area under the concentration-time curve values and decrease from baseline for leukocyte counts. In conclusion, dola-10 administered every 3 weeks as a peripheral i.v. bolus is well tolerated with dose-limiting toxicity of granulocytopenia. The maximum tolerated dose (and recommended Phase II starting dose) is 400 microg/m2 for patients with minimal prior treatment (two or fewer prior chemotherapy regimens) and 325 microg/m2 for patients who are heavily pretreated (more than two prior chemotherapy regimens).

Adult↗

PubMed1998

Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal.

Insulin-like growth factor-I (IGF-I) is emerging as an important growth factor able to modulate the programmed cell death (PCD) pathway mediated by the cysteine-dependent aspartate proteases (caspases); however, little is known about the effect of IGF-I after nerve growth factor (NGF) withdrawal in neurons. To begin to understand the neuronal death-sparing effect of IGF-I under NGF-free conditions, we tested whether embryonic sensory dorsal root ganglion neurons (DRG) were able to survive in defined serum-free medium in the presence of IGF-I. We further studied the role of IGF-I signaling and caspase inhibition after NGF withdrawal. NGF withdrawal produced histological changes of apoptosis including chromatin condensation, shrinkage of the perikaryon and nucleus, retention of the plasma membrane, and deletion of single cells. Both IGF-I and Boc-aspartyl (OMe)-fluoromethylketone (BAF), a caspase inhibitor, equally reduced apoptosis after NGF withdrawal. The antiapoptotic effect of IGF-I was completely blocked by LY294002, an inhibitor of PI 3-kinase signaling, but not by the mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) activated protein kinase inhibitor PD98059. Functional IGF-I receptors were extensively expressed both in rat and human DRG neurons, although they were most abundant in the neuronal growth cone. Collectively, these findings indicate that IGF-I, signaling though the PI-3 kinase pathway, is important in modulating PCD in cultured DRG neurons after NGF withdrawal, and IGF-I may be important in DRG embryogenesis.

Animals↗

PubMed1998

Paracrine production of nerve growth factor during rat dorsal root ganglion development.

Nerve growth factor is a target derived growth factor. In the peripheral nervous system, it is produced by tissues innervated by the sympathetic nervous system and small sensory neurons. In the present study, we tested the hypothesis that an alternate source of nerve growth factor must be available to support dorsal root ganglion neurons before they make connection with the target. Using reverse transcriptase polymerase chain reaction (RT-PCR), we detected nerve growth factor mRNA at embryonic day 12 to 17, but not in adult dorsal root ganglia. In situ hybridization studies revealed positive staining in satellite/supportive cells juxtaposed to dorsal root ganglion neurons. Our study suggests that nerve growth factor from supporting cells may have a paracrine function during development of primary sensory neurons.

Animals↗

PubMed1998

Thrombin induced inhibition of neurite outgrowth from dorsal root ganglion neurons.

Thrombin is a multifunctional protease. Recent studies on cultured neuronal cells have suggested a function for thrombin in the development and maintenance of the nervous system. Thrombin has been found to induce neurite retraction and reverse stellation in neuroblastoma cell lines and rat astrocytes, respectively. The major focus of our study was to investigate the potential role of thrombin in peripheral nervous system development using the rat embryonic dorsal root ganglion model. We found a dose dependent inhibition of neurite outgrowth from explant dorsal root ganglion cultures upon exposure to 2 to 200 nM thrombin. This effect was reversed by the specific thrombin inhibitor, hirudin. A synthetic peptide that imitates the fully active receptor, thrombin receptor activating peptide, was also found to inhibit neurite outgrowth from dorsal root ganglia. bis-Benzimide stained neuronal cultures did not show any evidence of cell death after exposure to thrombin or thrombin receptor activating peptides. Immunohistochemical studies revealed specific staining of the thrombin receptor on neurons, with intense labeling along neurites. Enriched neuronal cultures exposed to thrombin and thrombin receptor activating peptides revealed rapid activation of phospholipase Cgamma-1, a second messenger associated with the thrombin receptor. These findings are the first to describe the localization of the thrombin receptor to dorsal root ganglion neurons. We propose that receptor activation is associated with thrombin induced inhibition of neurite outgrowth.

Animals↗

PubMed1998

Cisplatin-induced apoptosis in rat dorsal root ganglion neurons is associated with attempted entry into the cell cycle.

Platinum compounds induce apoptosis in malignant cells and are used extensively in the treatment of cancer. Total dose is limited by development of a sensory neuropathy. We now demonstrate that when rats are administered cisplatin (2 mg/kg i.p. for 5 d), primary sensory neurons in the dorsal root ganglion die by apoptosis. This was reproduced by exposure of dorsal root ganglion neurons and PC12 cells to cisplatin (3 microg/ml) in vitro. Apoptosis was confirmed by electron microscopy, DNA laddering, and inhibition by the caspase inhibitor z-VAD.fmk (100 microM). Cell death in vitro was preceded by upregulation of cyclin D1, cdk4, and increased phosphorylation of retinoblastoma protein; all are indicators of cell cycle advancement. The level of p16(INK4a), an endogenous inhibitor of the cyclin D1/cdk4 complex decreased. Exposure of PC12 cells and dorsal root ganglion neurons to increased levels of nerve growth factor (100 ng/ ml) prevented both apoptosis and upregulation of the cell cycle markers. Cancer cells without nerve growth factor receptors (gp140TrkA) were not protected by the neurotrophin. This indicated that cisplatin may kill cancer cells and neurons by a similar mechanism. In postmitotic neurons, this involves an attempt to re-enter the cell cycle resulting in apoptosis which is specifically prevented by nerve growth factor.

Animals↗

PubMed1998

Autocrine regulation of neurite outgrowth from PC12 cells by nerve growth factor.

The PC12 cell line may be used as a model of NGF-induced neuronal differentiation. Exposure to NGF is accompanied by extension of neurites, cessation of growth and differentiation into cells resembling sympathetic neurons. In this study neurite outgrowth from PC12 cells was induced in serum-free, NGF-free medium conditions. Neurite outgrowth in serum-free conditions was abolished by exposure to anti-NGF antisera. Reverse transcription combined with polymerase chain reaction (RT-PCR) and in situ hybridization of PC12 cells in serum-free medium conditions revealed NGF transcripts. Western blot analysis of these cells revealed tyrosine phosphorylation of the high affinity NGF receptor (TrkA/gp140) and activation of a downstream signal cascade element, ERK-1/MAP kinase. NGF was also detected by a specific enzyme-linked immunoabsorbant assay (ELISA) revealing picogram levels of protein in conditioned medium and cell lysates. Survival of embryonic rat dorsal root ganglion neurons was maintained in cultures grown in this serum-free conditioned medium. This demonstrated that NGF may act as an autocrine or paracrine growth factor for PC12 cell differentiation.

Animals↗

PubMed1998

Myelination by Schwann cells in the absence of extracellular matrix assembly.

Assembly of extracellular collagen fibrils and Schwann cell basal lamina has previously been identified as a prerequisite for compact myelin formation in the peripheral nervous system. Synthesis of this extracellular matrix (ECM) in vitro required the presence of serum and ascorbic acid. Using rat embryonic dorsal root ganglion neurons and Schwann cells, we have developed a fully defined medium in which myelination occurs. In the absence of ascorbic acid, normal myelin was formed without ECM assembly. This demonstrates that although myelination and ECM assembly are usually closely linked, ECM formation is not a prerequisite for myelination in vitro.

Animals↗

PubMed1998

Direct activation of the high-affinity nerve growth factor receptor by a non-peptide symmetrical polyanion.

The high-affinity nerve growth factor receptor (gp140TrkA) is a tyrosine kinase receptor. The dimeric ligand, nerve growth factor, activates the receptor by stabilizing homodimer formation, which initiates transautophosphorylation. Suramin is a symmetrical planar polyanionic molecule which is being used as a novel experimental anti-neoplastic agent. Proposed mechanisms of the drug's anti-proliferative activity include blocking mitogenic stimulatory growth factors or inhibition of tumor-specific cellular enzymes. In PC12 cells and in dorsal root ganglion neurons, suramin has been shown to act as a partial agonist for gp140TrkA. We now demonstrate direct activation of gp140TrkA by suramin using in vitro protein kinase assays and receptor dimerization studies. Additionally, activation of phosphatidylinositol-3-kinase by suramin and nerve growth factor was observed with 10-min exposure. The addition of anti-nerve growth factor antibodies along with suramin did not reduce the level of gp140TrkA phosphorylation, excluding induction of an autocrine loop of nerve growth factor release and activation. This demonstrates that a small polyanion can directly activate gp140TrkA via receptor dimerization. Our study reveals a suramin-induced homodimerization of gp140TrkA. This finding correlated with significant neurite outgrowth in naive PC12 cells exposed to the drug. Studies will be initiated to design structural analogs of suramin which possess neurotrophic properties with no associated neurotoxicity.

Animals↗

PubMed1998

Activation of the high affinity nerve growth factor receptor by two polyanionic chemotherapeutic agents: role in drug induced neurotoxicity.

Suramin is a polyanionic chemotherapeutic agent which causes severe peripheral neuropathy. The mechanisms of antineoplastic and neurotoxic activities are still poorly understood. Interference with growth factor receptor function has been suggested for suramin's chemotherapeutic function. Previous studies from our laboratory have demonstrated that suramin interfered with the function of nerve growth factor (NGF) and induced lysosomal storage defects within dorsal root ganglion neurons. Pentosan polysulfate (PPS) was used as another polyanionic agent, to compare these two cellular functions; NGF receptor interaction and disruption in glycolipid metabolism. Like suramin and NGF, PPS induced neurite outgrowth from the PC12 cell line which correlated with tyrosine phosphorylation of the high affinity NGF receptor (TrkA/gpl40) and ERK-1/MAP kinase. Ultrastructural studies of dorsal root ganglion exposed to PPS for various time periods were normal. This contrasted with suramin exposed cultures which consistently developed lamellar inclusion bodies (LIB) within 6 h. LIB formation with suramin treatment was associated with neuronal cell death, while PPS treatment did not cause any neurotoxic effects. These results indicated that PPS mimicked the effect of suramin on NGF receptors but did not cause similar accumulation of LIB. This suggested that the effect of polyanionic compounds on TrkA was not involved in LIB accumulation and subsequent induction of neurotoxicity.

Animals↗

PubMed1998

Suramin induced ceramide accumulation leads to apoptotic cell death in dorsal root ganglion neurons.

Suramin is an experimental antineoplastic agent that is currently being tested in clinical trials for a number of human cancers. In previous clinical trials, it has been noted that a significant percentage of patients treated with suramin develop a peripheral neuropathy. Both the cytotoxic (chemotherapeutic) and neurotoxic mechanisms of action of this compound are unknown. Evidence presented in this study suggests that both effects may be due to extensive disruption in glycolipid transport and/or metabolism. Suramin treated dorsal root ganglion cultures revealed an accumulation of the GM1 ganglioside and ceramide. Exposure of cultures to suramin, a cell permeable ceramide analog, or sphingomyelinase lead to apoptotic cell death demonstrated by electron microscopy, bis-benzimide staining and DNA laddering on gel electrophoresis. Furthermore, a significant increase in intracellular ceramide preceded cell death in suramin treated neurons. We propose that suramin induced ceramide accumulation within neurons leads to apoptotic cell death.

Animals↗

PubMed1997

Role of ceramide in suramin-induced cancer cell death.

Suramin is an experimental antineoplastic agent which is currently being tested in clinical trials for its utility in treating breast and prostate cancer. Recent in vitro studies from our laboratory report a disruption in glycolipid metabolism and cell death in suramin-treated neurons. Evidence presented in this study proposes to consolidate the neurotoxic and cytotoxic effects of suramin. Electron microscopic studies, bis-benzimide staining and DNA gel electrophoresis of suramin and C2-ceramide treatment revealed apoptotic cells in human breast, prostate and rat neuron like cell lines. Apoptotic cell death was preceded by an elevation in intracellular ceramide.

Animals↗

PubMed1997

1,25-dihydroxyvitamin D3 regulates the expression of N-myc, c-myc, protein kinase C, and transforming growth factor-beta2 in neuroblastoma cells.

1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3) alters the proliferation of neuroblastoma cells in culture in part via a nerve growth factor (NGF)-mediated pathway. This suggests that factors other than NGF also play a role in the growth arrest induced by 1,25(OH)2D3. To more fully characterize the effect of 1,25(OH)2D3 on neuroblastoma cells, we treated the cells with 10(-8) M 1,25(OH)2D3 and examined the cells for changes in the expression of N-myc, c-myc, transforming growth factor-beta2 (TGF-beta2), and protein kinase C (PKC) activity. Our results show that 1,25(OH)2D3 causes a decrease in the expression of N-myc and c-myc, as well as a two-fold increase in total PKC activity and a dose-dependent increase in TGF-beta2 expression. These results show that 1,25(OH)2D3 regulates the expression of growth-regulatory factors other than NGF in neuroblastoma cells and that 1,25(OH)2D3 influences the growth of neural cells via multiple growth regulatory pathways.

Animals↗