DFP initiated early alterations of PKA/p-CREB pathway and differential persistence of beta-tubulin subtypes in the CNS of hens contributes to OPIDN.

Organophosphorus ester-induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken, which results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 mL/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were sacrificed at different time points such as 2, 4, and 8 h, as well as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum brainstem and spinal cord were quickly dissected and frozen for protein (western) and mRNA (northern) studies. Subcellular fractionation, SDS-PAGE and immunoblotting of the nuclear and supernatant fractions using standard protocols from spinal cord and cerebrum showed differential expression of protein levels of PKA, CREB and phosphorylated CREB (p-CREB). There was an increase in PKA level in spinal cord nuclear fraction after 4 h (130+/-5%) and 8 h (133+/-6 %), while cerebrum nuclear fraction showed decrease (77+/-5%) at 4 h and remained at the same level at 8 h. No change was seen in either spinal cord or cerebrum soluble fraction at any time points. There was an increase in CREB level in the spinal cord supernatant (133+/-3%) after 5 days, while nuclear and supernatant fraction of the cerebrum did not show any alterations at any time point. p-CREB was induced in the spinal cord nuclear fraction at 1 day (150+/-3%) and 5 days (173+/-7%) of treatment, in contrast to the decreased levels p-CREB (72+/-4%) at 10 days in cerebrum nuclear fraction. Supernatant fraction of spinal cord and cerebrum did not show any changes in pCREB at time points studied. Similarly another set of animals were treated with DFP and perfused using standard protocols and immunohistochemistry for p-CREB in the brain and spinal cord confirmed the overall protein expression pattern identified by western analysis. Expression of beta-tubulin subtypes (1, 2, 3, and 4), studied by Northern blotting showed complex and differential pattern, while immunohistochemistry of the anti-beta-tubulin for the entire period of OPIDN developmental stages showed early induction and persistence even in the disintegrating axonal and non-neuronal structures of the CNS. These data thus strongly suggest that early cytoskeletal damage at molecular level mediated by PKA/p-CREB pathways leads to the culmination of gross (microscopically observable) level cytoskeletal changes in various components of central nervous system (CNS), consistent with our earlier findings. Thus, the differential protein expression of PKA, CREB, p-CREB and beta-tubulin subtypes appear to contribute to the initiation, progression and development of OPIDN, probably by recruiting other molecular pathways specific to various components of nervous system.

Therapeutic targets in focal and segmental glomerulosclerosis.

PURPOSE OF REVIEW: Focal and segmental glomerulosclerosis occurs due to a defect in the glomerular filtration barrier. This review highlights contributions from the past year that have enhanced our understanding of the pathophysiology of focal and segmental glomerulosclerosis with emphasis on discoveries which may lead to the identification of therapeutic targets. RECENT FINDINGS: Slit diaphragm proteins have become increasingly important in signal transduction and in mediating downstream events. Actin polymerization occurs after the podocin-nephrin-Neph-1 complex is phosphorylated by Src kinase and Fyn. Recent studies of angiotensin receptor antagonists, corticosteroids and erythropoietin unravel new mechanisms that ameliorate proteinuria by targeting the cell cycle within the podocyte. The discovery that an N-acetylmannosamine kinase (MNK) mutant mouse has glomerulopathy is suggestive that human sialylation pathways may represent therapeutic targets. Proteinuria before podocyte effacement demonstrated in laminin-beta2 null mice highlights the importance of the glomerular basement membrane. Interferon-beta reduced proteinuria in three models of kidney injury, showing greatest effect on glomerular endothelial cells in vitro. SUMMARY: Basic research has illuminated mechanisms by which classic therapies have antiproteinuric effects directly on the podocyte. As knowledge expands with improved molecular techniques, understanding signaling pathways in health and proteinuric states should lead to potential therapeutic targets in focal and segmental glomerulosclerosis.

TRPC6 and FSGS: the latest TRP channelopathy.

Focal and segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome in children and adults throughout the world. In the past 50 years, significant advances have been made in the identification and characterization of familial forms of nephrotic syndrome and FSGS. Resultant to these pursuits, several podocyte structural proteins such as nephrin, podocin, alpha-actinin 4 (ACTN4), and CD2-associated protein (CD2AP) have emerged to provide critical insight into the pathogenesis of hereditary nephrotic syndromes. The latest advance in familial FSGS has been the discovery of a mutant form of canonical transient receptor potential cation channel 6 (TRPC6), which causes an increase in calcium transients and essentially a gain of function in this cation channel located on the podocyte cell membrane. The TRP ion channel family is a diverse group of cation channels united by a common primary structure which contains six membrane-spanning domains, with both carboxy and amino termini located intracellularly. TRP channels are unique in their ability to activate independently of membrane depolarization. TRPC6 channels have been shown to be activated via phospholipase C stimulation. The mechanisms by which mutant TRPC6 causes an increase in intracellular calcium and leads to glomerulosclerosis are unknown. Mutant TRPC6 may affect critical interactions with the aforementioned podocyte structural proteins, leading to abnormalities in the slit diaphragm or podocyte foot processes. Mutant TRPC6 may also amplify injurious signals mediated by Ang II, a common final pathway of podocyte apoptosis in various mammalian species. Current evidence also suggests that blocking TRPC6 channels may be of therapeutic benefit in idiopathic FSGS, a disease with a generally poor prognosis. Preliminary experiments reveal the commonly used immunosuppressive agent FK-506 can inhibit TRPC6 activity in vivo. This creates the exciting possibility that blocking TRPC6 channels within the podocyte may translate into long-lasting clinical benefits in patients with FSGS.

Toxicogenomic studies of the rat brain at an early time point following acute sarin exposure.

We have studied sarin-induced global gene expression patterns at an early time point (2 h: 0.5 x LD50) using Affymetrix Rat Neurobiology U34 chips and male Sprague-Dawley rats. A total of 46 genes showed statistically significant alterations from control levels. Three gene categories contained more of the altered genes than any other groups: ion channel (8 genes) and calcium channel and binding proteins (6 genes). Alterations were also found in the following gene groups: ATPases and ATP-based transporters (4), growth factors (4), G-protein-coupled receptor pathway-related molecules (3), neurotransmission and neurotransmitter transporters (3), cytoskeletal and cell adhesion molecules (2), hormones (2), mitochondria-associated proteins (2), myelin proteins (2), stress-activated molecules (2), cytokine (1), caspase (1), GABAnergic (1), glutamergic (1), immediate early gene (1), prostaglandin (1), transcription factor (1), and tyrosine phosphorylation molecule (1). Persistent alteration of the following genes also were noted: Arrb1, CaMKIIa, CaMKIId, Clcn5, IL-10, c-Kit, and Plp1, suggesting altered GPCR, kinase, channel, and cytokine pathways. Selected genes from the microarray data were further validated using relative RT-PCR. Some of those genes (GFAP, NF-H, CaMKIIa, Calm, and MBP) have been shown by other laboratories and ours, to be involved in the pathogenesis of sarin-induced pathology and organophosphate-induced delayed neurotoxicity (OPIDN). Induction of both proapoptotic (Bcl2l11, Casp6) and antiapoptotic (Bcl-X) genes, besides suppression of p21, suggest complex cell death/protection-related mechanisms operating early on. Principal component analysis (PCA) of the expression data confirmed that the changes in gene expression are a function of sarin exposure, since the control and treatment groups separated clearly. Our model (based on current and previous studies) indicates that both degenerative and regenerative pathways are activated early and contribute to the level of neurodegeneration at a later time, leading to neuro-pathological alterations.

Gene expression profiles of the rat brain both immediately and 3 months following acute sarin exposure.

We have studied sarin-induced global gene expression patterns at an early time point (15 min; 0.5xLD50) and a later time point (3 months; 1xLD50) using Affymetrix: Rat Neurobiology U34 chips in male, Sprague-Dawley rats and have identified a total of 65 (early) and 38 (late) genes showing statistically significant alterations from control levels at 15 min and 3 months, respectively. At the early time point, those that are classified as ion channel, cytoskeletal and cell adhesion molecules, in addition to neuropeptides and their receptors predominated over all other groups. The other groups included: cholinergic signaling, calcium channel and binding proteins, transporters, chemokines, GABAnergic, glutamatergic, aspartate, catecholaminergic, nitric oxide synthase, purinergic, and serotonergic signaling molecules. At the late time point, genes that are classified as calcium channel and binding proteins, cytoskeletal and cell adhesion molecules and GABAnergic signaling molecules were most prominent. Seven molecules (Ania-9, Arrb-1, CX-3C, Gabab-1d, Nos-2a, Nrxn-1b, PDE2) were identified that showed altered persistent expression in both time points. Selected genes from each of these time points were further validated using semi quantitative RT-PCR approaches. Some of the genes that were identified in the present study have been shown to be involved in organophosphate-induced neurotoxicity by both other groups as well as ours. Principal component analysis (PCA) of the expression data from both time points was used for comparative analysis of the gene expression, which indicated that the changes in gene expression were a function of dose and time of euthanasia after the treatment. Our model also predicts that besides dose and duration of post-treatment period, age and possibly other factors may be playing important roles in the regulation of pathways, leading to the neurotoxicity.

Sarin (nerve agent GB)-induced differential expression of mRNA coding for the acetylcholinesterase gene in the rat central nervous system.

We carried out a time-course study on the effects of a single intramuscular (i.m.) dose (0.5x LD(50)) of sarin (O-isopropyl methylphosphonofluoridate), also known as nerve agent GB, on the mRNA expression of acetylcholinesterase (AChE) in the brain of male Sprague-Dawley rats. Sarin inactivates the enzyme AChE which is responsible for the breakdown of the neurotransmitter acetylcholine (ACh), leading to its accumulation at ACh receptors and overstimulation of the cholinergic system. Rats were treated with 50 microg/kg of sarin (0.5x LD(50)) in 1 mL saline/kg and terminated at the following time points: 1 and 2 hr and 1, 3, and 7 days post-treatment. Control rats were treated with normal saline. Total RNA was extracted, and northern blots were hybridized with cDNA probes for AChE and 28S RNA (control). Poly-A RNA from both treated and control cortex was used for reverse transcription-polymerase chain reaction (RT-PCR)-based verification of the data from the northern blots. The results obtained indicate that a single (i.m.) dose of sarin (0.5x LD(50)) produced differential induction and persistence of AChE mRNA levels in different regions of the brain. Immediate induction of AChE transcripts was noted in the brainstem (126+/-6%), cortex (149+/-4%), midbrain (153+/-5%), and cerebellum (234+/-2%) at 1 hr. The AChE expression level, however, increased over time and remained elevated after a decline at 1 day in the previously shown more susceptible brainstem. The transcript levels remained elevated at a later time point (3 days) in the midbrain, after a dramatic decline at day 1 (110+/-2%). In the cortex, transcript levels came down to control values by day 1. The cerebellum also showed a decline of the elevated levels observed at 2 hr (275+/-2%) to control values by day 1. RT-PCR analysis of the AChE transcript at 30 min in the cortex showed an induction to 213+/-3% of the control level, confirming the expression pattern obtained by the northern blot data. The immediate induction followed by the complex pattern of the AChE mRNA time-course in the CNS may indicate that the activation of both cholinergic-related and unrelated functions of the gene plays an important role in the pathological manifestations of sarin-induced neurotoxicity.

Sarin causes altered time course of mRNA expression of alpha tubulin in the central nervous system of rats.

Sarin induced neurotoxicity is suspected to be one of the key factors responsible for Gulf-war syndrome. We studied the effect of a single (50 microg/kg/i.m) dose of sarin (0.5 x LD50) on the mRNA expression of alpha tubulin in the central nervous system (CNS) of rats which were sacrificed at different time points i.e. 1 and 2 hrs, as well as, 1, 3 and 7 days post-treatment. Northern data collected from CNS regions indicate differential, spatial, and temporal regulation of alpha tubulin mRNA levels. Immediate induction and persistence of alpha tubulin transcripts in sarin-treated CNS suggest that sarin-induced neurotoxicity is in part mediated by the altered expression of cytoskeletal genes which may be regulated at multiple levels.

Early differential elevation and persistence of phosphorylated cAMP-response element binding protein (p-CREB) in the central nervous system of hens treated with diisopropyl phosphorofluoridate, an OPIDN-causing compound.

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester-induced delayed neurotoxicity in sensitive species. We studied the effect of single dose of DFP on the expression of phosphorylated cAMP-response element binding protein (p-CREB), which is a well known transcription factor involved in several pathways mediating different types of external stimuli. The hens were perfused with neutral buffered formalin at different time points, i.e., 0.5, 1.0, and 2.0 hrs, as well as 1, 2, 5, and 20 days after dosing. The central nervous system regions of the whole brain were dissected and 7-micron sections were stained for either p-CREB immunopositivity or with hematoxylin and eosin. Results indicated an early differential increase of p-CREB immunopositivity in susceptible regions such as cerebellum, brainstem, and midbrain within 2 hrs. These induced levels persisted upto 5 days in these tissues, although the time course of p-CREB immunopositivity was distinctly different for each region. In the cerebellum induction of p-CREB was seen in the granular layer where both the granulocytes and the glial cells showed induction. Increased immunopositivity for p-CREB in the Purkinje cells and in some basket cells of the molecular layer was noticed over time, but the induction was not as great as in the granular layer. Of all the tissues cerebellum showed the strongest intensity of immunopositivity of the cells as well as the highest (absolute) number of pCREB-positive cells. The brainstem showed a similar fluctuating pattern like the cerebellum with the highest percentage increase of the immunoreactive cells at 5 days preceded by the lowest dip in immunopositivity at 2 days. In the midbrain, there was a time-dependent increase in the immunopositivity from 0.5 hr onwards until reaching control levels at 20 days. Immunopositivity was also noted in portions of the spina medularis and spina oblongata. The cerebrum (non-susceptible tissue) of DFP-treated hens did not show much deviation from the controls. The endothelial cells of the susceptible regions showed induction at early time points, in contrast to the absence of induction in cerebrum. Spatial and temporal differences in the immunopositivity pattern indicate probable involvement of CREB-independent pathways also. Overall, the complex induction pattern of p-CREB, along with our earlier observations of the early induction of c-fos, c-jun and Protein Kinase A (PKA) as well as the induction of Calcium2+/Calmodulin dependent Protein Kinase II (CaM kinase II) at later periods, strongly suggest an activator role of CREB mediated pathways that may lead to the clinical development of delayed neurotoxicity.

Differential alteration of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the central nervous system of hens treated with diisopropylphosphorofluoridate (DFP).

A single dose (1.7 mg/kg, s.c.) of diisopropylphosphorofluoridate (DFP) causes organophosphorus ester-induced delayed neurotoxicity (OPIDN) in susceptible species. We studied the effects of DFP administration on the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic protein at different time points (1, 2, 5, 10 and 20 days) post-treatment. Total RNA was extracted from cerebrum, cerebellum, brainstem, midbrain, and spinal cord of the control and DFP-treated hens, and northern blots were prepared using standard protocols and hybridized with GAPDH, as well as beta-actin and 28S RNA cDNA (control) probes. There was a distinct spatial/temporal mRNA expression pattern for the different tissues studied. Non-susceptible tissue, cerebrum showed a dramatic increase in GAPDH mRNA at day 1, post-treatment and levels remained high at all time points, suggestive of protective mechanisms from the beginning. In contrast, highly susceptible tissues like brainstem, spinal cord and midbrain showed either no elevation or slight down-regulation at day 1, suggesting trauma and cell injury/cell death. Overall, there was moderate level of induction during the subsequent time points in these tissues, indicative of pathways of either recovery or degeneration. Cerebellum being the less susceptible tissue showed moderate increase initially, followed by higher induction, suggestive of rapid recovery. Our current data on GAPDH provides an important link in this complex network of molecular changes involving pathways identified by our group and others, such as nitric oxide (NO), CaM kinase-II (CaMK-II), protein kinase-A (PKA), c-fos, and phosphorylated-CREB (p-CREB) in DFP-induced OPIDN.