Tirupapuliyur V. Damodaran

Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia

The deficiency of glucose-6-phosphatase (G6Pase) underlies life-threatening hypoglycemia and growth retardation in glycogen storage disease type Ia (GSD-Ia). An adeno-associated virus (AAV) vector encoding G6Pase was pseudotyped as AAV8 and administered to 2-week-old GSD-Ia mice (n=9). Median survival was prolonged to 7 months following vector administration, in contrast to untreated GSD-Ia mice that survived for only 2 weeks. Although GSD-Ia mice were initially growth-retarded, treated mice increased fourfold in weight to normal size. Blood glucose was partially corrected by 2 weeks following treatment, whereas blood cholesterol normalized. Glucose-6-phosphatase activity was partially corrected to 25% of the normal level at 7 months of age in treated mice, and blood glucose during fasting remained lower in treated, affected mice than in normal mice. Glycogen storage was partially corrected in the liver by 2 weeks following treatment, but reaccumulated to pre-treatment levels by 7 months old (m.o.). Vector genome DNA decreased between 3 days and 3 weeks in the liver following vector administration, mainly through the loss of single-stranded genomes; however, double-stranded vector genomes were more stable. Although CD8+ lymphocytic infiltrates were present in the liver, partial biochemical correction was sustained at 7 m.o. The development of efficacious AAV vector-mediated gene therapy could significantly reduce the impact of long-term complications in GSD-Ia, including hypoglycemia, hyperlipidemia and growth failure.

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.

Alterations in Levels of mRNAs Coding for Glial Fibrillary Acidic Protein (GFAP) and Vimentin Genes in the Central Nervous System of Hens Treated with Diisopropyl Phosphorofluoridate (DFP)

Diisopropyl phophorofluoridate (DFP) produces organophosphorus-ester induced delayed neurotoxicity (OPIDN) in the hen, human and other sensitive species. We studied the effect of DFP admimistration (1.7 mg/kg/s.c.) on the expression of Intermediate Filament (IF) proteins: Glial Fibrillary Acidic Protein (GFAP) and vimentin which are known indicators of neurotoxicity and astroglial pathology. The hens were sacrificed at different time points i.e. 1,2,5,10 and 20 days. Total RNA was extracted from the following brain regions: cerebrum, cerebellum, and brainstem as well as spinal cord. Northern blots prepared using standard protocols were hybridized with GFAP and vimentin as well as β-actin and 18S RNA cDNA (controls) probes. The results indicate a differential/spatial/temporal regulation of GFAP and vimentin levels which may be due to the result of disruption of glial-neuronal network. The GFAP transcript levels reached near control levels (88% and 95%) at 20 days post DFP treatment after an initial down-regulation (60% and 73%) in highly susceptible tissues like spinal cord and brainstem respectively. However vimentin transcript levels remained down-regulated (61% and 53%) at 20 days after an early reduced levels(47% and 55%) for spinal cord and brainstem respectively. This may be due to the astroglial pathology resulting in neuronal alterations or vice-versa. In cerebellum (less susceptile tissue) GFAP levels were moderately down-regulated at 1,2 and 5 days and reached near control values at 10 and 20 days. Vimentin was rapidly reinduced (128%) in cerebellum at 5 days and remained at the same level at 10 days and then returned to control values at 20 days after an initial down-regulation at 1 and 2 days. Thus these alterations were less drastic in cerebellum as indicated by initial susceptibility followed by rapid recovery. On the other hand both GFAP and vimentin levels were upregulated from 2 days onwards in the non-susceptible tissue cerebrum, implying protective mechanisms from the beginning. Hence the DFP induced astroglial pathology as indicated by the complex expression profile of GFAP and vimentin mRNA levels may be playing an important role in the delayed degeneration of axons or is the result of progressive degeneration of axons in OPIDN.

C-fos mRNA induction in the central and peripheral nervous systems of diisopropyl phosphorofluoridate (DFP)-treated hens

A single dose of diisopropyl phosphorofluoridate (DFP), an organophosphorus ester, produces delayed neurotoxicity (OPIDN) in hen. DFP produces mild ataxia in hens in 7–14 days, which develops into severe ataxia or paralysis as the disease progresses. Since, OPIDN is associated with alteration in the expression of several proteins (e.g., Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) α-subunit, tau, tubulin, neurofilament (NF) protein, vimentin, GFAP) as well as their mRNAs (e.g., NF, CaM kinase II α-subunit), we determined the effect of a single dose of DFP on the expression of one of the best known immediate-early gene (IEG), c-fos. C-fos expression was measured by Northern hybridization in cerebrum, cerebellum, brainstem, midbrain, spinal cord, and the sciatic nerves of hens at 0.5 hr, 1 hr, 2 hr, 1 day, 5 days, 10 days, and 20 days after a single 1.7 mg/kg, sc. injection of DFP. All the tissues (cerebrum, 52%; cerebellum, 55%; brainstem, 49%; midbrain, 23%; spinal cord, 80%; sciatic nerve, 157%;) showed significant increase in c-fos expression in 30 min and this elevated level persisted at least up to 2 hr. Expressions of β-actin mRNA and 18S RNA were used as internal controls. The significant increase in c-fos expression in DFP-treated hens suggests that c-fos may be one of the IEGs involved in the development of OPIDN.

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 μg/kg/i.m) dose of sarin (0.5 × 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.

Altered time course of mRNA expression of alpha tubulin in the central nervous system of hens treated with diisopropyl phosphorofluoridate (DFP).

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester induced delayed neurotoxicity (OPIDN) in the hen, human and other sensitive species. We studied the effect of single dose of DFP (1.7 mg/kg/s.c.) on the expression of alpha tubulin which is one of the major sub-unit of tubulin polymers that constitute an important constituent of cellular architecture. The hens were sacrificed at different time points i.e. 1, 2, 5, 10, and 20 days. Total RNA was extracted from the following brain regions: cerebrum, cerebellum, and brainstem as well as spinal cord. Northern blots prepared using standard protocols were hybridized with alpha tubulin as well as with β-actin and 28S RNA cDNA (controls) probes. The results indicate a differential /spatial /temporal regulation of alpha tubulin levels which may be the result of perturbed microtubule dynamics not only in the axons but also in perikarya of neurons in the CNS of DFP treated hens. In the highly susceptible tissues like brainstem and spinal cord the initial down-regulation of mRNA levels could be attributed to DFP induced stress response resulting in inhibited cell metabolism and or cell injury / cell death. Increase in levels of mRNA at 5 days and thereafter coincided with increased tubulin transport which may be due to increased phosphorylation of tubulins in both axons and perikarya and other intraaxonal changes resulting in impaired axonal transport. DFP induced decreased rate of tubulin polymerization resulting in increased levels of free tubulin monomers may be involved in the altered alpha tubulin mRNA expression at different time points by autoregulatory circuits. Cerebellum being the less susceptible tissue showed only a moderate decline at day 2, while the alpha tubulin remained at near control levels at day 1. Delayed down-regulation may be due to the co-ordinated up or down- regulation of different sub-types of alpha and beta tubulins as well as the differential response of specialised cell types in cerebellum. Continuous overexpression of alpha tubulin in cerebrum from the beginning may be its effective protective strategy to safeguard itself from neurotoxicity. Differential expression pattern observed could be due to the differential susceptibility and variability in the rate of axonal transport of different regions besides the tubulin heterogenity of CNS. Hence our results indicte differential expression of alpha tubulin is either one of the reasons for the development of OPIDN or the result of progressive changes taking place during OPIDN.

Therapeutic targets in focal and segmental glomerulosclerosis.

Purpose of reviewFocal 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 findingsSlit 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-β2 null mice highlights the importance of the glomerular basement membrane. Interferon-β reduced proteinuria in three models of kidney injury, showing greatest effect on glomerular endothelial cells in vitro. SummaryBasic 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.

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.

Early Differential Induction of C-jun in the Central Nervous System of Hens Treated with Diisopropylphosphorofluoridate (DFP)

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester induced delayed neurotoxicity (OPIDN) in the hen, human and other sensitive species. We studied the effect of a single dose of DFP (1.7 mg/kg/sc) on the expression of c-jun, which is one of the heterodimerizing ITFs (Inducible Transcriptional Factors) of the AP-1 family. The hens were sacrificed at different time points ie 0.25,0.50, 1 and 2 hrs. Total RNA was extracted from the following brain regions: cerebrum, cerebellum, brainstem, midbrain and as well as spinal cord. Northern blots prepared using standard protocols were hybridized with c-jun as well as b-actin and 18S RNA cDNA (control) probes. The results indicate differential regulation of c-jun levels which may be due to the activation of both cholinergic and non-cholinergic pathways of CNS, besides changing roles of c-jun (as mediator of degeneration or regeneration) depending on heterodimerization with other ITFs. In the highly susceptible tissues like brainstem and spinal cord c-jun transcript levels increased at 15 minutes and continued to increase gradually till it reached the maximum at 2 hrs. Overall spinal cord showed the maximum levels of c-jun induction (207%) at 2 hrs time point of all the CNS tissues. The enhancement of cholinergic transmisson by the inhibition of cholinestrase may be responsible for the gradual increase mediated by neural and vascular factors. In contrast, less susceptible tissue, cerebellum showed almost immediate induction to high level of (179%) at 15 minutes and the levels stayed more or less the same until it peaked to 185% at 2 hrs. Relatively low abundance of cholinergic neurons and high number of sensitized specialized cell types like Bergman glia and Purkinje cells may be responsible for the immediate higher induction. Non-susceptible tissue cerebrum did not show any changes in the c-jun levels. In midbrain the induction pattern was very similar to that of brainstem. This differential induction pattern of c-jun encomposing the differences in the quantity and time course was directly proportionate to the degree of susceptibility and cellular heterogeneity of different regions of CNS. The significant increase in c-jun levels along with our earlier observation on the increased c-fos levels indicate that AP-1 family of genes may be one of the IEGs involved in the long term changes which eventually lead to OPIDN.

DFP initiated early alterations of PKA/p-CREB pathway and differential persistence of β-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.