• Epigenetics of Fetal Brain Development in the Valproic Acid Model of Autism

      Konopko, Melissa A.; Krueger, Bruce K.; 0000-0001-9515-398X (2017)
      Use of the drug, valproic acid (VPA) by pregnant women increases the risk of autism in their children. This led to the development of the VPA mouse model of autism in which a pregnant dam is given a single dose of the drug at E12.5 which results in her offspring expressing autistic phenotypes. Our lab has found that using the in utero VPA model of autism results in a transient increase in Bdnf mRNA and protein in the mouse fetal brain. The goal of my research is to elucidate the epigenetic underpinnings of VPA and the related increased expression of the neurotrophin gene, Bdnf. VPA is a histone deacetylase inhibitor. Changes in histone acetylation engage in molecular crosstalk with other epigenetic marks and can result in alterations in histone and DNA methylation. Several histone modifications were analyzed using chromatin immunoprecipitation at three exons of interest, Bdnf exons 1, 4, and 6. A significant increase was found in each excitatory mark examined including acetylation of various histones as well as H3K4me3. Sex differences were found at some sites in histone acetylation and at most sites for H3K4me3. The greater increase in these activating marks was found in females. Two inhibitory marks were also analyzed, H3K27me3 and DNA CpG methylation. No effect of VPA was found on H3K27me3 and a small, but statistically significant effect was found on DNA methylation across four CpGs upstream of Bdnf exon 4. Bdnf transcript levels were measured in both sexes and found that there is more stimulation of exons 1 and 4 in females. Furthermore, we found that most stimulation of Bdnf transcript expression occurred in the caudal regions of the fetal brain and not the telencephalon. Together, these experiments help to elucidate the epigenetic contributions to VPA's ability to increase Bdnf expression in the brains of fetuses exposed to the drug in utero.
    • Fetal valproate syndrome and sensory processing by the superior colliculus

      Dendrinos, Georgia; Keller, Asaf (2010)
      Sensory processing disorder (SPD) is characterized by an inability to filter sensory information, particularly simultaneously arriving multimodal inputs. We examined the effects of prenatal exposure to valproic acid (VPA), an SPD-linked teratogen, on the behavior of juvenile and adult rats, and on physiology and the prenatal and postnatal of the superior colliculus, a critical multisensory integration center in the brain. VPA exposed rats showed dose-dependent deficits in colliculus-dependent behaviors including startle response, prepulse inhibition and nociceptive thresholds. Some deficits reversed with age. In extracellular recordings from anesthetized rats, collicular neurons of control and VPA-treated rats showed no difference in spontaneous firing rates or evoked responses to presentations of unimodal stimuli. However, neurons from VPA-treated rats responded with significantly higher magnitudes to concurrent stimulus presentations of different modalities. Individual neurons from control and VPA-treated rats responded less to a multimodal stimulus than a unimodal stimulus. This response depression was exhibited differentially for control and VPA-treated rats depending on stimulus intensity. At low intensity combinations of multimodal stimuli, more neurons of VPA-treated rats showed a significant change in response to multimodal stimuli than unimodal stimuli. At high intensity combinations, neurons responded significantly differently to multimodal stimuli than unimodal stimuli, regardless of treatment. Stereological analyses revealed that the embryonic brain of animals exposed to VPA in utero were significantly smaller with volumetric decreases in several structures including the cortex, thalamus, and superior colliculus. Postnatally, colliculi of VPA-treated rats had significantly fewer parvalbumin-positive neurons, a subset of GABAergic cells. These results suggest that prenatal VPA treatment affects the development of the superior colliculus and leads to persistent anatomical and physiological changes which are evidenced by aberrant behavior in tasks that require sensory processing.
    • Oncolytic Mechanisms of the HSV-2 Mutant deltaPK and Enhancement by Valproic Acid

      Bollino, Dominique; Aurelian, Laure (2015)
      Carcinogenesis is the process by which normal cells become malignant. It typically occurs through the accumulation of mutations that dysregulate intracellular signaling pathways and lead to unchecked growth and proliferation. Oncolytic viruses (OV) are replication conditional virus mutants that take advantage of these overactivated growth pathways to selectively replicate in and lyse tumor cells. In addition to the direct infection and lysis of tumor cells, OVs also kill tumor cells through disruption of tumor vasculature and the induction of potent anti-tumor immune responses. Clinical efficacy of OVs remains relatively poor, attributed to therapeutic barriers such as poor tumor penetration, premature viral clearance, and the presence of highly resistant cancer stem cell (CSC) subpopulations. Efforts to enhance OV efficacy include the addition of transgenes to enhance anti-tumor immunity, as well as combination therapy with cytotoxic and/or immunosuppressive drugs to increase tumor cell death and reduce innate antiviral responses. The growth compromised HSV-2 mutant ΔPK, has robust oncolytic activity in both melanoma cultures and xenografts associated with the induction of multiple pathways of programmed cell death. However, the impact of ΔPK on putative CSC populations, as well as its ability to harness immune responses that contribute to tumor cell death is still poorly understood. This thesis work sought to answer these questions as well as investigate the potential benefits of combining ΔPK with valproic acid (VPA), a histone deacetylase inhibitor with demonstrated cytotoxic and immunosuppressive properties. We report that: (i) ΔPK prevents anchorage-independent growth and lyses 3D cultures through calpain-dependent clearance of the autophagy protein p62/SQSTM1, (ii) ΔPK oncolysis includes several features of immunogenic cell death, such as the inhibition of Th2-based immunosuppressive conditions, promotion of a Th1-biased microenvironment, and the induction of anti-tumor immune surveillance mechanisms, (iii) VPA induces a novel calpain-dependent necroptotic form of cell death in neuronal cells, and (iv) the combination of VPA and ΔPK treatment in melanoma increases ΔPK -induced cell death through enhanced caspase activation. These findings suggest that ΔPK is a multi-mechanistic OV with particularly promising cancer therapeutic potential, and warrant further in vivo investigation into the oncolytic potential of the ΔPK and VPA combination.