Jahan Ara

Bone Morphogenetic Proteins 4, 6, and 7 Are Up-Regulated in Mouse Spinal Cord during Experimental Autoimmune Encephalomyelitis

Although spontaneous remyelination occurs in multiple sclerosis (MS), the extent of myelin repair is often inadequate to restore normal function. Oligodendrocyte precursors remaining in nonremyelinating MS plaques may be restricted by an inhibitory signal. Bone morphogenetic proteins (BMPs) have been implicated as repressors of oligodendrocyte development and inducers of astrogliogenesis. We hypothesized that BMPs are up‐regulated in MS lesions and play a role in demyelination and astrogliosis. We examined expression of BMPs in an animal model of MS, chronic experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide in C57BL/6 mice. By 14 days postimmunization, compared to those of control mice, the lumbar spinal cords of MOG‐peptide EAE mice demonstrated prominent astrogliosis, infiltration of inflammatory cells, and disrupted expression of myelin proteins. Quantitative RT‐PCR showed that expression of BMP4, BMP6, and BMP7 mRNA increased 2‐ to 4‐fold in the lumbar spinal cords of animals with symptomatic EAE versus in vehicle‐treated and untreated controls on days 14, 21, and 42 postimmunization. BMP2 mRNA expression was not altered. BMP4 mRNA was much more abundant in the spinal cords of all animals than was mRNA encoding BMP2, BMP6, and BMP7. Immunoblot analysis confirmed the increased expression of BMP4 in the EAE animals. Immunohistochemistry revealed increased BMP4 immunoreactivity in areas of inflammation in MOG‐peptide EAE animals. BMP4 labeling was mostly limited to macrophages but was sometimes associated with astrocytes and oligodendrocytes. These results indicate that members of the BMP family are differentially expressed in adult spinal cord and are up‐regulated during EAE.

Induction of neuropilins-1 and -2 and their ligands, Sema3A, Sema3F, and VEGF, during Wallerian degeneration in the peripheral nervous system

The neuropilins, NP-1 and NP-2, are coreceptors for Sema3A and Sema3F, respectively, both of which are repulsive axonal guidance molecules. NP-1 and NP-2 are also coreceptors for vascular endothelial growth factor (VEGF). The neuropilins and their ligands are known to play prominent roles in axonal pathfinding, fasciculation, and blood vessel formation during peripheral nervous system (PNS) development. We confirmed a prior report (Exp. Neurol. 172 (2001) 398) that VEGF mRNA levels rise during Wallerian degeneration in the PNS and herein demonstrate that NP-1, NP-2, Sema3A, and Sema3F mRNA levels increase in peripheral nerves distal to a transection or crush injury. In a sciatic nerve crush model, in which axonal regeneration is robust, the highest levels of Sema3F mRNA below the injury site are in the epi- and perineurium. Our results suggest the possibility that the neuropilins and their semaphorin ligands serve to guide, rather than to impede, regenerating axons in the adult PNS.

Modulation of sciatic nerve expression of class 3 semaphorins by nerve injury.

Beginning with the unexpected finding by cDNA array analysis that neuropilin-2 is induced in sciatic nerve distal to a transection, we document, for the first time, up-regulation in the axotomized adult peripheral nervous system of class 3 semaphorins and their receptors, which are known to play prominent roles in axonal guidance during neural development. Previously, we described the use of cDNA arrays to screen for novel peripheral nervous system axotomy-induced candidate neurotrophic proteins. A novel finding of that prior study was substantial induction of neuropilin 2 (NP2) mRNA in the axotomized nerve segments. Following up on that initial observation, we have now used real-time quantitative reverse transcription-polymerase chain reaction to demonstrate induction of genes encoding neuropilin 1 (NP1), which, like NP2, serves as a coreceptor for members of the class 3 semaphorin family of axonal guidance molecules and of five of the six known class 3 semaphorins (Sema3A, Sema3B, Sema3C, Sema3E, and Sema3F, but not Sema3D) in crushed or transected sciatic nerves.

Hypoxic-preconditioning induces neuroprotection against hypoxia-ischemia in newborn piglet brain.

Preconditioning-induced ischemic tolerance has been documented in the newborn brain, however, the signaling mechanisms of this preconditioning require further elucidation. The aims of this study were to develop a hypoxic-preconditioning (PC) model of ischemic tolerance in the newborn piglet, which emulates important clinical similarities to human situation of birth asphyxia, and to characterize some of the molecular mechanisms shown to be implicated in PC-induced neuroprotection in rodent models. One day old piglets were subjected to PC (8% O2/92% N2) for 3 h and 24 h later were exposed to hypoxia-ischemia (HI) produced by a combination of hypoxia (5% FiO2) for a period of 30 min and ischemia induced by a period of hypotension (10 min of reduced mean arterial blood pressure; ≤70% of baseline). Neuropathologic analysis and unbiased stereology, conducted at 24 h, 3 and 7 days of recovery following HI, indicated a substantial reduction in the severity of brain damage in PC piglets compared to non-PC piglets (P<0.05). PC significantly increased the mRNA expression of hypoxia-inducible factor-1α (HIF-1α) and its target gene, vascular endothelial growth factor (VEGF) at 0 h, 6h, 24 h, 3 and 7 days of recovery. Immunoblot analysis demonstrated that PC resulted in HIF-1α protein stabilization and accumulation in nuclear extracts of cerebral cortex of newborn piglet brain compared to normoxic controls. Protein levels of VEGF increased in a time-dependent manner in both cortex and hippocampus following PC. Double-immunolabeling indicated that VEGF is mainly expressed in neurons, endothelial cells and astroglia. Our study demonstrates for the first time the protective efficacy of PC against hypoxic-ischemic injury in newborn piglet model, which recapitulates many pathophysiological features of asphyxiated human neonates. Furthermore, as has been shown in rodent models of preconditioning, our results suggest that PC-induced protection in neonatal piglets may involve upregulation of VEGF.

Reactive oxygen species modified DNA fragments of varying size are the preferred antigen for human anti-DNA autoantibodies.

In vitro studies were carried out to determine if reactive oxygen species modified DNA molecules are the preferred antigen for anti-DNA antibodies found in SLE sera. Reactive oxygen species were generated by 254 nm irradiation of hydrogen peroxide. Single stranded breaks, decrease in Tm and modification of adenine (21.7%) and thymine (48%) were the major effects observed on native DNA fragments of 300 bp in length. The ROS-modified DNA showed increased binding with naturally occurring anti-DNA autoantibodies as compared to unmodified DNA fragments. These results were substantiated by competition ELISA. Measurement of binding with DNA fragments of varying size revealed considerably increased binding as the fragment size increased from 50 bp to 800 bp. The relative affinity of anti-DNA IgG for ROS-modified and native DNA fragments of 300 bp were in the order of 6.26 x 10(-8) M and 4.07 x 10(-8) M, respectively.

Polynucleotide specificity of anti-reactive oxygen species (ROS) DNA antibodies.

Hydrogen peroxide in the presence of short wavelength UV light was able to induce alterations in native DNA fragments of 300 bp (ROS‐DNA). thereby rendering it immunogenic in experimental animals. The specificity of induced antibodies was investigated by direct binding and competition ELISA. Inhibition studies revealed nearly 89% inhibition in the antibody binding by the immunogen and recognition of native B‐, A‐ and allied conformations presented by various synthetic polynucleotides. Gel retardation assay reiterated the formation of immune complexes between induced antibodies and native and ROS‐DNA fragments. It was observed that naturally occurring anti‐DNA autoantibodies from systemic lupus erythemalosus(SLE) sera recognize ROS‐DNA. The comparison of the specificities of anti‐DNA auloantibodies from 10 SLE patients showed a 20 50‐fold preference for ROS‐DNA over native DNA. These results demonstrate that anti‐DNA antibodies can be induced by ROS‐DNA, and that some of the autoimmune DNA binding antibodies found in SLE may result from response to reactive oxygen species.

Peripheral nerve regeneration is delayed in neuropilin 2–deficient mice

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin‐1 and neuropilin‐2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin‐dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin‐2 facilitates peripheral‐nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin‐2‐deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin‐2‐deficient mice than in the control mice. Thus, neuropilin‐2 facilitates peripheral‐nerve axonal regeneration.

Schwann cell-autonomous role of neuropilin-2

Neuropilins and group A plexins are components of receptor complexes for class 3 semaphorins, gradients of which help to guide migration of neural progenitor cells and axonal growth cones during development. We demonstrated previously that neuropilins and class 3 semaphorins are induced in sciatic nerve by crush or transection. We now report that in cultured rat Schwann cells, expression of mRNA encoding neuropilin‐2 (NRP2) and plexin‐A3 (PlexA3), proteins involved in semaphorin‐3F (Sema3F) signal transduction, is diminished markedly by forskolin, an adenylate cyclase activator that, like axonal contact, induces Schwann cell synthesis of myelin lipids and proteins. Interestingly, Schwann cell expression of mRNA encoding NRP1, which participates in Sema3A signaling, is not downregulated by forskolin. Antibodies that recognize ectodomains of NRP2 but not control antibodies prevented cultured Schwann cells from aligning in parallel and forming columns. These results are consistent with the view that in nerves undergoing Wallerian degeneration, Schwann cell NRP2 facilitates assembly of Schwann cells into the tubular aggregates (bands of Büngner) that guide regenerating axons.

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multipotent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.

Hypoxic-preconditioning enhances the regenerative capacity of neural stem/progenitors in subventricular zone of newborn piglet brain

Perinatal hypoxia-ischemia (HI) results in brain injury, whereas mild hypoxic episodes result in preconditioning, which can significantly reduce the vulnerability of the brain to subsequent severe hypoxia-ischemia. Hypoxic-preconditioning (PC) has been shown to enhance cell survival and differentiation of progenitor cells in the central nervous system (CNS). The purpose of this study was to determine whether pretreatment with PC prior to HI stimulates subventricular zone (SVZ) proliferation and neurogenesis in newborn piglets. One-day-old piglets were subjected to PC (8% O2/92% N2) for 3h and 24h later were exposed to HI produced by combination of hypoxia (5% FiO2) for a pre-defined period of 30min and ischemia induced by a period of 10min of hypotension. Here we demonstrate that SVZ derived neural stem/progenitor cells (NSPs) from PC, HI and PC+HI piglets proliferated as neurospheres, expressed neural progenitor and neurodevelopmental markers, and that greater proportion of the spheres generated are multipotential. Neurosphere assay revealed that preconditioning pretreatment increased the number of NSP-derived neurospheres in SVZ following HI compared to normoxic and HI controls. NSPs from preconditioned SVZ generated twice as many neurons and astrocytes in vitro. Injections with 5-Bromo-2-deoxyuridine (BrdU) after PC revealed a robust proliferative response within the SVZ that continued for one week. PC also increased neurogenesis in vivo, doublecortin positive cells with migratory profiles were observed streaming from the SVZ to striatum and neocortex. These findings show that the induction of proliferation and neurogenesis by PC might be a positive adaptation for an efficient repair and plasticity in the event of a hypoxic-ischemic insult.