Ellen M. Carpenter

Enhanced sensitivity to N-methyl-D-aspartate receptor activation in transgenic and knockin mouse models of Huntington's disease

We used two mouse models of Huntingtons disease (HD) to examine changes in glutamate receptor sensitivity and striatal electrophysiology. One model, a transgenic, consisted of mice expressing exon 1 of the human HD gene and carrying 141–157 CAG repeat sequences (R6/2 line). The second model, a CAG repeat “knockin,” consisted of mice with different lengths of CAG repeats (CAG71 and CAG94 repeats). The effects of glutamate receptor activation were examined by visualizing neurons in brain slices with infrared videomicroscopy and differential interference contrast optics to determine changes in somatic area (cell swelling). Striatal and cortical neurons in both models (R6/2 and CAG94) displayed more rapid and increased swelling to N‐methyl‐D‐aspartate (NMDA) than those in controls. This effect was specific as there were no consistent group differences after exposure to α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) or kainate (KA). Intracellular recordings revealed that resting membrane potentials (RMPs) in the R6/2 transgenics were significantly more depolarized than those in their respective controls. RMPs in CAG94 mice also were more depolarized than those in CAG71 mice or their controls in a subset of striatal neurons. Confirming previous results, R6/2 mice expressed behavioral abnormalities and nuclear inclusions. However, CAG71 and CAG94 knockins did not, suggesting that increased sensitivity to NMDA may occur early in the disease process. These findings imply that NMDA antagonists or compounds that alter sensitivity of NMDA receptors may be useful in the treatment of HD. J. Neurosci. Res. 58:515–532, 1999.

Hox Genes and Spinal Cord Development

The spinal cord is differentiated along the rostrocaudal axis into large domains with regional distinctions reflected in the position and projection of specific cell types. Spinal cord patterning is likely to be mediated by the local expression and activity of transcription factors. This review will examine the expression of one class of transcription factors, encoded by the Hox genes, that are active in spinal cord patterning. Hox genes encode homeodomain-containing proteins with overlapping rostrocaudal domains of expression in the developing spinal cord. Rostrally expressed Ant-p/Ubx/Abd-D-related Hox genes may function in patterning the cervical spinal cord, while Abd-D-related, caudally expressed Hox genes may pattern the lumbar spinal cord. Changes in spinal cord patterning are apparent following Hox gene inactivation, supporting a role for these genes in defining or establishing this pattern.

miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis

The regulation of epithelial proliferation during organ morphogenesis is crucial for normal development, as dysregulation is associated with tumor formation. Non-coding microRNAs (miRNAs), such as miR-200c, are post-transcriptional regulators of genes involved in cancer. However, the role of miR-200c during normal development is unknown. We screened miRNAs expressed in the mouse developing submandibular gland (SMG) and found that miR-200c accumulates in the epithelial end buds. Using both loss- and gain-of-function, we demonstrated that miR-200c reduces epithelial proliferation during SMG morphogenesis. To identify the mechanism, we predicted miR-200c target genes and confirmed their expression during SMG development. We discovered that miR-200c targets the very low density lipoprotein receptor (Vldlr) and its ligand reelin, which unexpectedly regulate FGFR-dependent epithelial proliferation. Thus, we demonstrate that miR-200c influences FGFR-mediated epithelial proliferation during branching morphogenesis via a Vldlr-dependent mechanism. miR-200c and Vldlr may be novel targets for controlling epithelial morphogenesis during glandular repair or regeneration.

Early Effects of Lipopolysaccharide-Induced Inflammation on Foetal Brain Development in Rat

Studies in humans and animal models link maternal infection and imbalanced levels of inflammatory mediators in the foetal brain to the aetiology of neuropsychiatric disorders. In a number of animal models, it was shown that exposure to viral or bacterial agents during a period that corresponds to the second trimester in human gestation triggers brain and behavioural abnormalities in the offspring. However, little is known about the early cellular and molecular events elicited by inflammation in the foetal brain shortly after maternal infection has occurred. In this study, maternal infection was mimicked by two consecutive intraperitoneal injections of 200 μg of LPS (lipopolysaccharide)/kg to timed-pregnant rats at GD15 (gestational day 15) and GD16. Increased thickness of the CP (cortical plate) and hippocampus together with abnormal distribution of immature neuronal markers and decreased expression of markers for neural progenitors were observed in the LPS-exposed foetal forebrains at GD18. Such effects were accompanied by decreased levels of reelin and the radial glial marker GLAST (glial glutamate transporter), and elevated levels of pro-inflammatory cytokines in maternal serum and foetal forebrains. Foetal inflammation elicited by maternal injections of LPS has discrete detrimental effects on brain development. The early biochemical and morphological changes described in this work begin to explain the sequelae of early events that underlie the neurobehavioural deficits reported in humans and animals exposed to prenatal insults.

Identification of a Hoxd10-regulated transcriptional network and combinatorial interactions with Hoxa10 during spinal cord development.

Hoxd10 is expressed in the posterior spinal cord and hindlimbs of the mouse. Hoxd10, along with other Hox transcription factors, is thought to regulate the activity of genes involved in nervous system patterning and motor neuron development, but little is known about the downstream targets regulated by this gene. cDNA microarrays were used to investigate the transcriptional network regulated by Hoxd10 in homozygous knockout animals. Sixty‐nine genes were identified with altered expression levels in mutant spinal cords. Among these were genes involved in such diverse cellular events as cellular communication, cell cycle control, development and differentiation, and neuronal survival. The expression of some of these genes was investigated using reverse transcriptase‐polymerase chain reaction (RT‐PCR) and in situ hybridization. Nine genes showed changes in expression of the same sign and similar magnitude using RT‐PCR in Hoxd10 single mutant animals, with additional changes in expression seen in Hoxa10/Hoxd10 double mutant animals. In situ hybridization studies also demonstrated changes in expression consistent with microarray results. Analysis of putative promoter regions for Hox protein binding sites suggested that some genes may be direct Hoxd10 targets, whereas others likely are regulated through intermediate steps. Using cDNA microarrays to study a single gene knockout during critical developmental stages has identified a large number of genes regulated by Hoxd10, many of which would not have been approached as candidates for Hox gene regulation based on function or expression.

L1 CAM expression is increased surrounding the lesion site in rats with complete spinal cord transection as neonates.

L1 is a cell adhesion molecule associated with axonal outgrowth, fasciculation, and guidance during development and injury. In this study, we examined the long-term effects of spinal cord injury with and without exercise on the re-expression of L1 throughout the rat spinal cord. Spinal cords from control rats were compared to those from rats receiving complete mid-thoracic spinal cord transections at postnatal day 5, daily treadmill step training for up to 8 weeks, or both transection and step training. Three months after spinal cord transection, we observed substantially higher levels of L1 expression by both Western blot analysis and immunocytochemistry in rats with and without step training. Higher expression levels of L1 were seen in the dorsal gray matter and in the dorsal lateral funiculus both above and below the lesion site. In addition, L1 was re-expressed on the descending fibers of the corticospinal tract above the lesion. L1-labeled axons also expressed GAP-43, a protein associated with axon outgrowth and regeneration. Treadmill step training had no effect on L1 expression in either control or transected rats despite the fact that spinal transected rats displayed improved stepping patterns indicative of spinal learning. Thus, spinal cord transection at an early age induced substantial L1 expression on axons near the lesion site, but was not additionally augmented by exercise.

Differential Pax6 promoter activity and transcript expression during forebrain development

Three different Pax6 promoters -- P0, P1, and P alpha -- show differential activity in the developing eye and spinal cord. To examine promoter usage during forebrain development, we performed in situ hybridization and reverse transcription-polymerase chain reaction to detect transcripts initiated from each promoter. Promoter-specific transcripts are expressed within subdomains of total Pax6 expression, but differ from one another in their spatial localization and expression over time. Additionally, we identified a novel P0-initiated transcript and detected a developmentally regulated antisense transcript.

Developmental expression of REGA-1, a regionally expressed glial antigen in the central nervous system of grasshopper embryos

Glial cells are a large component of the developing nervous system, appearing before the onset of axon outgrowth in a variety of developing systems. Their time of appearance and their location in conjunction with developing axon pathways may allow them to define the position of axon pathways. Specific glial cells may be utilized as guideposts by growing axons, allowing them to recognize the appropriate pathway, or conversely, glial cells may inhibit axons from growing along an inappropriate pathway. The 7F7 monoclonal antibody labels a subset of glial cells in grasshopper embryos that may play a role in defining the location of selected axonal pathways. This antibody recognizes the REGA- 1 molecule, a cell-surface antigen with a molecular weight of 60 kDa, which is regionally expressed on developing glial cells. REGA-1 is expressed around the edges of clusters of glial cells and on lamellae extending from glial cells to line the edges of some axonal pathways. REGA-1 expression is first seen in the neuroblast sheet, surrounding neuroblast 4–1. Slightly later in development, 2 glial cells extend processes that express REGA-1 and demarcate the caudal edge of the anterior commissure. As the animal matures, cell processes expressing REGA-1 line the edges of the longitudinal connective, then expand to surround the central neuropil of the segmental ganglia. REGA-1 expression is also seen in conjunction with axons leaving the segmental ganglia via the segmental nerves and the intersegmental connectives. REGA-1 expression is limited to a subset of glial cells; some known glial cells such as the segment boundary cell do not express REGA-1. Glial cell processes expressing REGA-1 are seen only in association with axons, which suggests that these processes may act as borders or guard rails confining axons to the appropriate regions of the developing CNS. Axons navigating a path through the CNS may be prohibited from growing into inappropriate regions based on their inability to cross the boundaries established by glial cells expressing REGA-1.

Particulate Bone Allograft Incorporation in Regeneration of Osseous Defects; Importance of Particle Sizes

Packing of bone defect with particulate allografts is a commonly performed clinical procedure. However, the ideal size of bone particles used to fill bone defects is ill-defined. For this reason the study of biology of bone allografts with different particle sizes has been performed. Standard size bone defects in the femur and the tibia of experimental animals were filled with freeze-dried cortical bone allografts with particle sizes of 1-2mm, 800-500μm, 500-300μm, 300-90μm, 250-125μm, 125-106μm, 106 to 75μm and 75-25μm. Unfilled defects and those filled with autologous bone served as controls. Cortical bone was chosen because it produced better clinical results than did cancellous bone. Likewise freeze-dried particulate bone effected more rapid healing than did frozen bone. Numerical scores were assigned to each defect based on the gross, radiographic and histomorphometric studies. Particles in the range of 300 to 90 microns produced rapid healing by direct ossification. Particles below 100µm had a significantly reduced osteogenic potential. Particles in the range of 75-25μm failed to heal the defects all together. Healing of defects packed with particles larger than 300μm was slower than with 300 – 90 μm grafts. Rapid healing of bone defects packed with particulate bone allografts in the range of 300 to 90μm indicates such allografts can be used effectively in the filling of bone defects. This is of clinical relevance.

Decreased reelin expression and organophosphate pesticide exposure alters mouse behaviour and brain morphology

Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders, including ASDs (autism spectrum disorders). In this study, we examined the combinatorial effect of two factors thought to be involved in autism – reduction in the expression of the extracellular matrix protein reelin and prenatal exposure to an organophosphate pesticide, CPO (chlorpyrifos oxon). Mice with reduced reelin expression or prenatal exposure to CPO exhibited subtle changes in ultrasound vocalization, open field behaviour, social interaction and repetitive behaviour. Paradoxically, mice exposed to both variables often exhibited a mitigation of abnormal behaviours, rather than increased behavioural abnormalities as expected. We identified specific differences in males and females in response to both of these variables. In addition to behavioural abnormalities, we identified anatomical alterations in the olfactory bulb, piriform cortex, hippocampus and cerebellum. As with our behavioural studies, anatomical alterations appeared to be ameliorated in the presence of both variables. While these observations support an interaction between loss of reelin expression and CPO exposure, our results suggest a complexity to this interaction beyond an additive effect of individual phenotypes.