MAB1580 Sigma-AldrichAnti-Oligodendrocytes Antibody, clone NS-1

Oligodendrocytes are responsible for producing and maintaining myelin throughout the CNS. One of the pathological features observed following traumatic brain injury (TBI) is the progressive demyelination and degeneration of axons within white matter tracts. While the effect of TBI on axonal health has been well documented, there is limited information regarding the response of oligodendrocytes within these areas. The aim of this study was to characterize the response of both mature oligodendrocytes and immature proliferative oligodendrocyte lineage cells across a 3 month timecourse following TBI. A computer-controlled cortical impact model was used to produce a focal lesion in the left motor cortex of adult mice. Immunohistochemical analyses were performed at 48 hours, 7 days, 2 weeks, 5 weeks and 3 months following injury to assess the prevalence of mature CC-1+ oligodendrocyte cell death, immature Olig2+ cell proliferation and longer term survival in the corpus callosum and external capsule. Decreased CC-1 immunoreactivity was observed in white matter adjacent to the site of injury from 2 days to 2 weeks post TBI, with ongoing mature oligodendrocyte apoptosis after this time. Conversely, proliferation of Olig2+ cells was observed as early as 48 hours post TBI and significant numbers of these cells and their progeny survived and remained in the external capsule within the injured hemisphere until at least 3 months post injury. These findings demonstrate that immature oligodendrocyte lineage cells respond to TBI by replacing oligodendrocytes lost due to damage and that this process occurs for months after injury.

Primary coenzyme Q10 (CoQ10) deficiency is due to mutations in genes involved in CoQ biosynthesis. The disease has been associated with five major phenotypes, but a genotype-phenotype correlation is unclear. Here, we compare two mouse models with a genetic modification in Coq9 gene (Coq9(Q95X) and Coq9(R239X)), and their responses to 2,4-dihydroxybenzoic acid (2,4-diHB). Coq9(R239X) mice manifest severe widespread CoQ deficiency associated with fatal encephalomyopathy and respond to 2,4-diHB increasing CoQ levels. In contrast, Coq9(Q95X) mice exhibit mild CoQ deficiency manifesting with reduction in CI+III activity and mitochondrial respiration in skeletal muscle, and late-onset mild mitochondrial myopathy, which does not respond to 2,4-diHB. We show that these differences are due to the levels of COQ biosynthetic proteins, suggesting that the presence of a truncated version of COQ9 protein in Coq9(R239X) mice destabilizes the CoQ multiprotein complex. Our study points out the importance of the multiprotein complex for CoQ biosynthesis in mammals, which may provide new insights to understand the genotype-phenotype heterogeneity associated with human CoQ deficiency and may have a potential impact on the treatment of this mitochondrial disorder.

We developed a combined conditional cytotoxic, i.e., herpes simplex type 1-thymidine kinase (TK), plus immune-stimulatory, i.e., fms-like tyrosine kinase ligand-3-mediated gene therapy for glioblastoma multiforme (GBM). Therapeutic transgenes were encoded within high-capacity adenoviral vectors (HC-Ad); TK was expressed constitutively, while Flt3L was under the control of the TetOn regulatable promoter. We previously assessed efficacy and safety in intracranial GBM rodent models. But, since this approach involves expression of a cytokine within the brain, we chose the nonhuman primate, i.e., Callithrix jaccus (marmoset) as it has been established that its immune response shares similarities with man. We characterized the safety, cell-type specific expression, and doxycycline (DOX)-inducibility of HC-Ad-TetOn-Flt3L delivered within the striatum. We used allometrically scaled DOX doses delivered orally, twice daily for one month, mimicking the route and duration of DOX administration planned for the GBM trial. Flt3L was effectively expressed within astrocytes, microglia, oligodendrocytes, and neurons. No evidence of brain or systemic toxicities due to the treatment was encountered. Our data indicate that DOX doses equivalent to those used in humans to treat infections can be safely used "off-label" to turn "on" therapeutic gene expression from HC-Ad-TetOn-Flt3L; providing evidence for the safety of this approach in the clinic.

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in regulating CNS myelination. BDNF mutant mice exhibit a hypomyelinating phenotype, and BDNF exerts distinct effects upon oligodendroglial proliferation, differentiation, and myelination in vitro. To investigate the precise influence that BDNF exerts in regulating CNS myelination in vivo, we have generated conditional knock-out mice in which TrkB has been deleted specifically in oligodendrocytes. Deletion of TrkB disrupted normal oligodendrocyte myelination, resulting in a significant reduction in myelin protein expression and myelination of CNS white matter tracts during development. Importantly, conditional knock-out mice exhibited normal numbers of mature oligodendrocytes and normal numbers of myelinated axons; however, myelin thickness was significantly reduced during development. These data indicate that while TrkB expression in oligodendrocytes plays no role in the initial contact with axons, it exerts an important influence in subsequent stages to promote myelin ensheathment. The conditional knock-out mice also exhibited an increased density of oligodendrocyte progenitor cells (OPCs) in CNS white matter tracts. Concordant with these results, in vitro analyses using OPCs subjected to TrkB knockdown also revealed increased OPC proliferation. Our data suggested this effect was dependent upon TrkC and p75 expression. Thus, our data demonstrate that TrkB expression in oligodendroglia exerts a direct effect on oligodendrocytes to promote myelination and an indirect effect upon the OPC population, modifying their proliferative potential.

The beneficial effect of interventions with chondroitinase ABC enzyme to reduce axon growth-inhibitory chondroitin sulphate side chains after central nervous system injuries has been mainly attributed to enhanced axonal sprouting. After traumatic brain injury (TBI), it is unknown whether newly sprouting axons that occur as a result of interventional strategies are able to functionally contribute to existing circuitry, and it is uncertain whether maladaptive sprouting occurs to increase the well-known risk for seizure activity after TBI. Here, we show that after a controlled cortical impact injury in rats, chondroitinase infusion into injured cortex at 30 min and 3 days reduced c-Fos⁺ cell staining resulting from the injury alone at 1 week postinjury, indicating that at baseline, abnormal spontaneous activity is likely to be reduced, not increased, with this type of intervention. c-Fos⁺ cell staining elicited by neural activity from stimulation of the affected forelimb 1 week after injury was significantly enhanced by chondroitinase, indicating a widespread effect on cortical map plasticity. Underlying this map plasticity was a larger contribution of neuronal, rather than glial cells and an absence of c-Fos⁺ cells surrounded by perineuronal nets that were normally present in stimulated naïve rats. After injury, chondroitin sulfate proteoglycan digestion produced the expected increase in growth-associated protein 43-positive axons and perikarya, of which a significantly greater number were double labeled for c-Fos after intervention with chondroitinase, compared to vehicle. These data indicate that chondroitinase produces significant gains in cortical map plasticity after TBI, and that either axonal sprouting and/or changes in perineuronal nets may underlie this effect. Chondroitinase dampens, rather than increases nonspecific c-Fos activity after brain injury, and induction of axonal sprouting is not maladaptive because greater numbers are functionally active and provide a significant contribution to forelimb circuitry after brain injury.

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.

Coenzyme Q10 (CoQ(10)) or ubiquinone is a well-known component of the mitochondrial respiratory chain. In humans, CoQ(10) deficiency causes a mitochondrial syndrome with an unexplained variability in the clinical presentations. To try to understand this heterogeneity in the clinical phenotypes, we have generated a Coq9 Knockin (R239X) mouse model. The lack of a functional Coq9 protein in homozygous Coq9 mutant (Coq9(X/X)) mice causes a severe reduction in the Coq7 protein and, as consequence, a widespread CoQ deficiency and accumulation of demethoxyubiquinone. The deficit in CoQ induces a brain-specific impairment of mitochondrial bioenergetics performance, a reduction in respiratory control ratio, ATP levels and ATP/ADP ratio and specific loss of respiratory complex I. These effects lead to neuronal death and demyelinization with severe vacuolization and astrogliosis in the brain of Coq9(X/X) mice that consequently die between 3 and 6 months of age. These results suggest that the instability of mitochondrial complex I in the brain, as a primary event, triggers the development of mitochondrial encephalomyopathy associated with CoQ deficiency.

Introduction: Cervical compressive myelopathy is the most serious complication of cervical spondylosis or ossification of the posterior longitudinal ligament (OPLL) and the most frequent cause of spinal cord dysfunction. There is little information on the exact pathophysiological mechanism responsible for the progressive loss of neural tissue in the spinal cord of such patients. In this study, we used the spinal hyperostotic mouse (twy/twy) as a suitable model of human spondylosis, and OPLL to investigate the cellular and molecular changes in the spinal cord. Mutant twy/twy mouse developed ossification of the ligamentum flavum at C2-C3 and exhibited progressive paralysis. <br />Materials and methods: The mutant twy/twy mice, aged 16 and 24 weeks, were used in the present study. The cervical spinal cord was analyzed histologically and immunohistochemically. <br />Results: We observed that a significant correlation between the proportion of apoptotic oligodendrocytes in the compressed area of the spinal cord and the magnitude of cord compression. Immunohistochemical analysis indicated overexpression of TNFR1, CD95, and p75NTR in the twy/twy mice, which was localized by the immunofluorescence in the neurons and oligodendrocytes. <br />Conclusion: The expression of such factors seems to play at least some role in the apoptotic process, which probably contributes to axonal degeneration and demyelination in the twy/twy mice spinal cords with severe compression.

Adenoviral vectors (Ads) have been evaluated in clinical trials for glioma. However, systemic immunity against the vectors can hamper therapeutic efficacy. We demonstrated that combined immunostimulation and cytotoxic gene therapy provides long-term survival in preclinical glioma models. Because helper-dependent high-capacity Ads (HC-Ads) elicit sustained transgene expression, in the presence of antiadenoviral immunity, we engineered HC-Ads encoding conditional cytotoxic herpes simplex type 1 thymidine kinase and immunostimulatory cytokine Fms-like tyrosine kinase ligand-3 under the control of the TetOn system. Escalating doses of combined HC-Ads (1×10(8), 1×10(9), and 1×10(10) viral particles [VP]) were delivered into the rat brain. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points after vector delivery. Histopathological analysis did not reveal any evidence of toxicity or long-term inflammation at the lower doses tested. Vector genomes were restricted to the injection site. Serum chemistry did not uncover adverse systemic side effects at any of the doses tested. Taken together, our data indicate that doses of up to 1×10(9) VP of each HC-Ad can be safely administered into the normal brain. This comprehensive toxicity and biodistribution study will lay the foundations for implementation of a phase 1 clinical trial for GBM using HC-Ads.

Adenovirus-mediated delivery of the immune-stimulatory cytokine Flt3L and the conditionally cytotoxic thymidine kinase (TK) induces tumor regression and long-term survival in preclinical glioma (glioblastoma multiforme [GBM]) models. Flt3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. Although pDCs can present antigen and produce powerful inflammatory cytokines, that is, interferon α (IFN-α), their role in tumor immunology remains debated. Thus, we studied the role of pDCs and IFN-α in Ad.TK/GCV+ Ad.Flt3L-mediated anti-GBM therapeutic efficacy. Our data indicate that the combined gene therapy induced recruitment of plasmacytoid DCs (pDCs) into the tumor mass; which were capable of in vivo phagocytosis, IFN-α release, and T-cell priming. Thus, we next used either pDCs or an Ad vector encoding IFN-α delivered within the tumor microenvironment. When rats were treated with Ad.TK/GCV in combination with pDCs or Ad-IFN-α, they exhibited 35% and 50% survival, respectively. However, whereas intracranial administration of Ad.TK/GCV + Ad.Flt3L exhibited a high safety profile, Ad-IFN-α led to severe local inflammation, with neurologic and systemic adverse effects. To elucidate whether the efficacy of the immunotherapy was dependent on IFN-α-secreting pDCs, we administered an Ad vector encoding B18R, an IFN-α antagonist, which abrogated the antitumoral effect of Ad.TK/GCV + Ad.Flt3L. Our data suggest that IFN-α release by activated pDCs plays a critical role in the antitumor effect mediated by Ad.TK/GCV + Ad.Flt3L. In summary, taken together, our results demonstrate that pDCs mediate anti-GBM therapeutic efficacy through the production of IFN-α, thus manipulation of pDCs constitutes an attractive new therapeutic target for the treatment of GBM.