Identification of a conserved 125 base-pair Hb9 enhancer that specifies gene expression to spinal motor neurons

The homeobox gene Hb9 is expressed selectively by motor neurons (MNs) in the developing CNS. Previous studies have identified a 9-kb 5' fragment of the mouse Hb9 gene that is sufficient to direct gene expression to spinal MNs in vivo. Here, we sought to identify more discrete MN-specifying elements, using homology searches between genomic sequences of evolutionarily distant species. Based on homology screening of the mouse and human Hb9 promoters, we identified a 3.6-kb Hb9 enhancer that proved sufficient to drive MN-specific lacZ expression. We then compared mouse, human, and pufferfish (Fugu rubripes) genomic sequences, and identified a conserved 438-bp sequence, consisting of noncontiguous 313-bp and 125-bp fragments, residing within the 3.6-kb Hb9 enhancer. The zebrafish (Danio rerio) Hb9 genomic region was then found to have two identical copies of the 125-bp sequence, but no counterpart for the 313-bp sequence. Transgenic analysis showed that the 125-bp alone was both necessary and sufficient to direct spinal MN-specific lacZ expression, whereas the 313-bp sequence had no such enhancer activity. Moreover, the 125-bp Hb9 enhancer was found to harbor two Hox/Pbx consensus-binding sequences, mutations of which completely disrupted thoracolumbar Hb9 expression. These data suggest that Hox/Pbx plays a critical role in the segmental specification of spinal MNs. Together, these results indicate that the molecular pathways regulating Hb9 expression are evolutionarily conserved, and that MN-specific gene expression may be directed and achieved using a small 125-bp 5' enhancer.     

Retinoid receptors in chronic degeneration of the spinal cord: observations in a rat model of amyotrophic lateral sclerosis

Changes in distribution and expression of retinoid receptors may be part of a spinal cord protective response to acute injury and to chronic degeneration. In this study, we have combined RNA and protein expression analysis to characterize the expression profile of retinoid receptors in the lumbar spinal cord of the superoxide dismutase 1 G93A mutant rat model of amyotrophic lateral sclerosis, a fatal neurodegenerative disorder causing extensive motor neuron loss. We also report a nonsignificant change in RNA expression of binding proteins and metabolizing enzymes for retinol and retinoic acid in the mutant rat spinal cord at end-stage disease. Only retinoid X receptor beta (RXRbeta), and to a lesser extent retinoic acid receptor beta and alpha (RARbeta/alpha) were reliably detected in lumbar spinal cord at an early pre-symptomatic phase and throughout the disease progression. The expression of RXRbeta in lamina II neurons in the dorsal horn of transgenic and wild type (WT) animals was associated with extensive astrocyte staining in end-stage lumbar spinal cord from transgenic rats. RARbeta and RARalpha diffuse staining of large motor neurons in the pre-symptomatic transgenic and in the WT lumbar cord appear to decline in end-stage disease, when a selective and strong gamma motor neuron RARalpha staining becomes evident. As gliosis and motor neuron loss are key pathogenic features in amyotrophic lateral sclerosis, the selective expression of retinoid receptors in astrocytes and motor neurons may provide further clues to the role of retinoid signalling in neurodegeneration and suggest new treatment strategies based on retinoid-modulating agents.     

Neural expression of alpha-internexin promoter in vitro and in vivo

alpha-Internexin is a 66 kDa neuronal intermediate filament protein found most abundantly in the neurons of the nervous systems during early development. To characterize the function of mouse alpha-internexin promoter, we designed two different expression constructs driven by 0.7 kb or 1.3 kb of mouse alpha-internexin 5'-flanking sequences; one was the enhanced green fluorescent protein (EGFP) reporter for monitoring specific expression in vitro, and the other was the cre for studying the functional DNA recombinase in transgenic mice. After introducing DNA constructs into non-neuronal 3T3 fibroblasts and a neuronal Neuro2A cell line by lipofectamine transfection, we observed that the expression of EGFP with 1.3 kb mouse alpha-internexin promoter was in a neuron-dominant manner. To establish a tissue-specific pattern in the nervous system, we generated a transgenic mouse line expressing Cre DNA recombinase under the control of 1.3 kb alpha-Internexin promoter. The activity of the Cre recombinase at postnatal day 1 was examined by mating the cre transgenic mice to ROSA26 reporter (R26R) mice with knock-in Cre-mediated recombination. Analyses of postnatal day 1 (P1) newborns showed that beta-galactosidase activity was detected in the peripheral nervous system (PNS), such as cranial nerves innervating the tongue and the skin as well as spinal nerves to the body trunk. Furthermore, X-gal-labeled dorsal root ganglionic (DRG) neurons showed positive for alpha-Internexin in cell bodies but negative in their spinal nerves. The motor neurons in the spinal cord did not exhibit any beta-galactosidase activity. Therefore, the cre transgene driven by mouse alpha-internexin promoter, described here, provides a useful animal model to specifically manipulate genes in the developing nervous system.     

Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors

Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg(SOD1*G93A)dl1 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1 ^G93A. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1 ^G93A mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1 ^G93A mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2–3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + AGF can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.     

In vivo imaging of single axons in the mouse spinal cord

We provide a protocol that describes imaging of single fluorescently labeled axons in the spinal cord of living mice. This method takes advantage of transgenic mouse lines in which the thy1-promoter drives the expression of variants of the green fluorescent protein in a small percentage (less than 1%) of sensory neurons. As a consequence, single axons can be resolved in the surgically exposed dorsal column using wide-field epifluorescence microscopy. This approach allows direct observation of axonal degeneration and regeneration in mouse models of spinal cord pathology for several hours or repetitively over the course of several days.     

Hoxa5 gene regulation: A gradient of binding activity to a brachial spinal cord element

The Hox genes cooperate in providing positional information needed for spatial and temporal patterning of the vertebrate body axis. However, the biological mechanisms behind spatial Hox expression are largely unknown. In transgenic mice, gene fusions between Hoxa5 (previously called Hox-1.3) 5' flanking regions and the lacZ reporter gene show tissue- and time-specific expression in the brachial spinal cord in day 11-13 embryos. A 604-bp regulatory region with enhancer properties directs this spatially specific expression. Fine-detail mapping of the enhancer has identified several elements involved in region-specific expression, including an element required for expression in the brachial spinal cord. Factors in embryonic day 12.5 nuclear extracts bind this element in electrophoretic mobility shift assays (EMSA) and protect three regions from DNase digestion. All three sites contain an AAATAA sequence and mutations at these sites reduce or abolish binding. Furthermore, this element binds specific individual embryonic proteins on a protein blot. The binding activity appears as a gradient along the anterior-posterior axis with two- to threefold higher levels observed in extracts from anterior regions than from posterior regions. In parallel with the EMSA, the proteins on the protein blot also show reduced binding to probes with mutations at the AAATAA sites. Most importantly, transgenic mice carrying Hoxa5/lacZ fusions with the three AAATAA sites mutated either do not express the transgene or have altered transgene expression. The brachial spinal cord element and its binding proteins are likely to be involved in spatial expression of Hoxa5 during development.     

Inducible Nitric Oxide Synthase Up-Regulation in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age-matched transgenic mice expressing normal SOD1 and of wild-type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end-stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS-immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region unaffected by mSOD1. Similarly, no up-regulation of iNOS is detected in the spinal cord of age-matched transgenic mice expressing normal SOD1 or of wild-type mice. The time course of the spinal cord gliosis and iNOS up-regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age-matched transgenic mice expressing normal SOD1 and wild-type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up-regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.     

Decreased GLT-1 and increased SOD1 and HO-1 expression in astrocytes contribute to lumbar spinal cord vulnerability of SOD1-G93A transgenic mice

The SOD1-G93A transgenic mouse is a widely used ALS model, but the death of lower motor neurons is the hallmark. Here, we show that the SOD1-G93A transgene and HO-1 are preferentially over-expressed in the lumbar spinal cord, particularly in the activated astrocytes of the transgenic mice. We also show down-regulation of GLT-1 in spite of the proliferating astrocytes. However, GLT-1, SOD1-G93A transgene and HO-1 expression were not obviously changed in the motor cortex. Our data link spinal cord vulnerability to relatively decreased expression of GLT-1, and high expression of the transgene and HO-1 in astrocytes in SOD1-G93A transgenic mice.     

The prostate apoptosis response-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. Here, we report higher Par-4 levels in lumbar spinal cord samples from patients with amyotrophic lateral sclerosis (ALS) than in lumbar spinal cord samples from neurologically normal patients. We also compared the levels of Par-4 in lumbar spinal cord samples from wild-type and transgenic mice expressing the human Cu/Zn-superoxide dismutase gene with a familial ALS mutation. Relative to control samples, higher Par-4 levels were observed in lumbar spinal cord samples prepared from the transgenic mice at a time when they had hind-limb paralysis. Immunohistochemical analyses of human and mouse lumbar spinal cord sections revealed that Par-4 is localized to motor neurons in the ventral horn region. In culture studies, exposure of primary mouse spinal cord motor neurons or NSC-19 motor neuron cells to oxidative insults resulted in a rapid and large increase in Par-4 levels that preceded apoptosis. Pretreatment of the motor neuron cells with a Par-4 antisense oligonucleotide prevented oxidative stress-induced apoptosis and reversed oxidative stress-induced mitochondrial dysfunction that preceded apoptosis. Collectively, these data suggest a role for Par-4 in models of motor neuron injury relevant to ALS.     

A role for A/T-rich sequences and Pit-1/GHF-1 in a distal enhancer located in the human growth hormone locus control region with preferential pituitary activity in culture and transgenic mice.

A region located remotely upstream of the human pituitary GH (GH-N) gene and required for efficient GH-N gene expression in the pituitary of transgenic mice was cloned as a 1.6-kb Bg/II (1.6G) fragment. The 1.6G fragment in the forward or reverse orientation increased -496GH-N promoter activity significantly in pituitary GC and GH3 cells after gene transfer. The 1.6G fragment was also able to stimulate activity from a minimal thymidine kinase (TK) promoter which, unlike -496GH-N, lacked any Pit-1/GHF-1 element. Enhancer activity was localized by deletion analysis to a 203-bp region in the 3'-end of the 1.6G fragment and was characterized by the presence of a diffuse 136-bp nuclease-protected site, observed with pituitary (GC) but not nonpituitary (HeLa) cell nuclear protein. A major low-mobility complex was observed by electrophoretic mobility shift assay (EMSA) with GC cell nuclear protein, and the pattern was distinct from that seen with a HeLa cell extract. The nuclease-protected region contains three A/T-rich Pit-1/ GHF-1-like elements, and their disruption, in the context of the 203-bp region fused to the TK promoter, reduced enhancer activity significantly in pituitary cells in culture. A mutation in this region was also shown to decrease enhancer activity in transgenic mice and correlated with a decrease in the 203-bp enhancer region complex observed by EMSA. The participation of Pit-1/GHF-1 in this complex is indicated by competition studies with Pit-1/GHF-1 elements and antibodies, and direct binding of Pit-1/GHF-1 to the A/T-rich sequences was shown by EMSA using recombinant protein. These studies link the A/T-rich sequences to the distal enhancer activity associated with the GH locus control region in vitro and in vivo.