Williams Rodriguez

Expression of arginase isozymes in mouse brain

The two forms of arginase (AI and AII) in man, identical in enzymatic function, are encoded in separate genes and are expressed differentially in various tissues. AI is expressed predominantly in the liver cytosol and is thought to function primarily to detoxify ammonia as part of the urea cycle. AII, in contrast, is predominantly mitochondrial, is more widely expressed, and is thought to function primarily to produce ornithine. Ornithine is a precursor in the synthesis of proline, glutamate, and polyamines. This study was undertaken to explore the cellular and regional distribution of AI and AII expression in brain using in situ hybridization and immunohistochemistry. AI and AII were detected only in neurons and not in glial cells. AI presented stronger expression than AII, but AII was generally coexpressed with AI in most cells studied. Expression was particularly high in the cerebral cortex, cerebellum, pons, medulla, and spinal cord neurons. Glutamic acid decarboxylase 65 and glutamic acid decarboxylase 67, postulated to be related to the risk of glutamate excitotoxic and/or γ‐aminobutyric acid inhibitoxic injury, were similarly ubiquitous in their expression and generally paralleled arginase expression patterns, especially in cerebral cortex, hippocampus, cerebellum, pons, medulla, and spinal cord. This study showed that AI is expressed in the mouse brain, and more strongly than AII, and sheds light on the anatomic basis for the arginine→ornithine→glutamate→GABA pathway. J. Neurosci. Res. 66:406–422, 2001.

Axotomy‐induced changes in pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP receptor gene expression in the adult rat facial motor nucleus

It has been demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) promotes the survival of neurons in culture and can inhibit neuronal cell death after experimental injury. Furthermore, peripheral axotomy results in increased PACAP gene expression in sensory and sympathetic neurons, suggesting that PACAP might be a mediator in the injury response in certain parts of the nervous system. However, changes in PACAP expression have not been reported in injured motor neurons, despite the significant problem of motor neuron degeneration in injury and in several neurological diseases. We examined here changes in gene expression of PACAP and two high‐affinity PACAP receptors, PAC1 and VPAC2, in adult rat motor neurons after facial nerve axotomy by in situ hybridization. PACAP gene expression was very low in facial motor neurons of normal rats. However, a robust time‐dependent increase in PACAP mRNA was observed in the facial motor nucleus in most or all axotomized motor neurons. This induction was detectable 6 hr after axotomy, and peaked at 48 hr, when expression on the injured side averaged more than 20‐fold higher than that on the contralateral side. Thereafter, PACAP mRNA levels decreased slightly, but remained more than 10‐fold elevated for as long as 30 days after axotomy. In contrast to PACAP, gene expression for both the PAC1 and VPAC2 receptor was high in facial motor neurons of normal rats. No significant change was observed for VPAC2 receptor gene expression in facial motor neurons after axotomy, whereas gene expression for the PAC1 receptor became significantly decreased. The results indicate that the PACAP ligand receptor system is tightly regulated in the facial motor nucleus after axotomy, providing evidence that PACAP may be involved in motor injury responses. J. Neurosci. Res. 57:953–961, 1999.

Lymphocyte regulation of neuropeptide gene expression after neuronal injury.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase‐activating peptide (PACAP) are induced strongly in neurons after several types of injury, and exhibit neuroprotective actions in vitro and in vivo. It is thought that changes in expression of neuropeptides and other molecules in injured neurons are mediated by new factors produced in Schwann and immune cells at the injury site, a loss of target‐derived factors, or a combination of mediators. To begin to determine the role of the inflammatory mediators, we investigated axotomy‐induced changes in VIP and PACAP gene expression in the facial motor nucleus in severe combined immunodeficient (SCID) mice, and in mice with targeted mutations in specific cytokine genes. In normal mice, VIP and PACAP mRNA was induced strongly in facial motor neurons 4 days after axotomy. The increase in PACAP mRNA was blocked selectively in SCID mice, indicating that mechanisms responsible for VIP and PACAP gene induction are not identical. The loss of PACAP gene expression in SCID mice after axotomy was fully reversed by an infusion of normal splenocytes, suggesting that PACAP mRNA induction requires inflammatory mediators. PACAP and VIP mRNA inductions, however, were maintained in mice lacking leukemia inhibitory factor (LIF) and interleukin‐6 (IL‐6), and in mice lacking both receptors for tumor necrosis factor α (TNFα). The data suggest that an inflammatory response, most likely involving T lymphocytes, is necessary for the axotomy‐induced increase in PACAP but not in VIP. LIF, IL‐6, and TNFα, however, are not required for this response to injury.

Induction of neuropeptide gene expression and blockade of retrograde transport in facial motor neurons following local peripheral nerve inflammation in severe combined immunodeficiency and BALB/C mice

Peripheral nerve inflammation is a common clinical problem that accompanies nerve injury and several diseases including Guillain-Barre syndrome and acute and chronic inflammatory demyelinating polyneuropathy. To determine if neuropeptides are induced in motor neurons after inflammation and to study the mechanisms involved, a nerve cuff soaked in complete Freunds adjuvant (CFA) was applied locally to the facial nerve of Balb/C mice. This procedure resulted in an influx of lymphocytes and macrophages to the affected area and a blockade of retrograde axonal transport distal, but not proximal, to the site of application. The same treatment resulted in a strong ipsilateral induction of pituitary adenylyl cyclase activating peptide (PACAP) gene expression in motor neurons in the facial motor nucleus. Because the changes could have occurred due to the loss of target-derived factors or to the production of new factors by immune cells, we studied the effect of the inflammatory stimulus on PACAP mRNA in mice with severe combined immunodeficiency (SCID). As expected, SCID mice showed a severely reduced influx of T-lymphocytes but not macrophages to the peripheral nerve. Moreover, although retrograde transport distal to the inflammation site was blocked similarly in control and SCID mice, the number of motor neurons expressing PACAP mRNA after CFA application was significantly reduced in SCID mice. The data indicate that the induction of PACAP mRNA during nerve inflammation requires the involvement of lymphocytes. However, because the induction of PACAP gene expression was only partially blocked in SCID mice, macrophages, loss of target-derived factors, or other mechanisms may also contribute to the upregulation of PACAP gene expression in motor neurons after nerve inflammation.

Restoration of axotomy-induced PACAP gene induction in SCID mice with CD4+ T-lymphocytes.

PACAP is a neuropeptide with putative neuroprotective, regenerative, and immunomodulatory actions. PACAP mRNA is up-regulated in motor neurons following facial nerve axotomy in wild type, but not immunodeficient SCID mice. Because CD4+ lymphocytes appear to be neuroprotective in facial nerve and other injury models, we studied PACAP gene expression in SCID mice preinfused with CD4+ enriched splenocytes. Whereas the mean number of PACAP hybridizing neurons after axotomy was reduced by 75% in uninfused SCID mice, infusion of CD4+ enriched splenocytes restored the number to a value not significantly different than controls. The CD4+ cell-dependent induction of PACAP in motor neurons may thus be a factor in the cascade of events triggered by immune cells that ultimately lead to nerve regeneration.

Embryonic expression of pituitary adenylyl cyclase-activating polypeptide and its selective type I receptor gene in the frog Xenopus laevis neural tube.

The genes encoding pituitary adenylyl cyclase‐activating peptide (PACAP) and its selective type I receptor (PAC1) are expressed in the embryonic mouse neural tube, where they may be involved in neurogenesis and neural tube development. We examined here the early expression and potential actions of PACAP and PAC1 in the vertebrate developmental model Xenopus laevis. PACAP and PAC1 mRNAs were first detected by RT‐PCR in stage 16–18 embryos (18 hours after fertilization). Two distinct PACAP precursor mRNAs were identified. One encoded both growth hormone‐releasing hormone and PACAP, whereas the other encoded only full‐length PACAP. Unlike that in the adult, the latter represented the predominant embryonic PACAP mRNA species. In situ hybridization revealed that PACAP and PAC1 mRNAs were restricted to neural cells. PAC1 gene expression was observed mainly in the ventricular zone in the ventral parts of the prosencephalon, mensencephalon, rhombencephalon, and anterior spinal cord. In contrast, PACAP mRNA was localized exclusively in postmitotic cells in the dorsolateral parts of the rhombencephalon and entire spinal cord. Most PACAP mRNA‐containing cells were characterized as Rohon‐Beard neurons. Exposure of early embryos to UV irradiation, which ventralizes embryos and inhibits neural induction, reduced the expression of PACAP and PAC1 genes. These results suggest that PACAP may be involved in the early development of the embryonic Xenopus neural tube. J. Comp. Neurol. 441:266–275, 2001.

Targeting of Embryonic and Postnatal Autonomic and Enteric Neurons with a Vasoactive Intestinal Peptide Transgene

Abstract : The neuropeptide vasoactive intestinal peptide (VIP) is expressed in several distinct sites in the CNS, in cholinergic and enteric ganglia, and in a small subpopulation of neurons within sympathetic ganglia. Previous studies on the human VIP gene indicate that transcription in neural crest‐derived neuroblastoma and pheochromocytoma cell lines is controlled in part by multiple regulatory elements located along 4.5 kb of upstream 5′ flanking sequence. In the current studies, transgenic mice were created with a chimeric gene consisting of 16.5 kb of the mouse VIP gene fused to the β‐galactosidase reporter. In situ hybridization analysis in adult mice indicated that reporter gene expression was correctly targeted to neurons in the esophagus, stomach, small intestine, and colon. No expression was observed in the brain, including regions that contain abundant VIP‐expressing cells, such as the thalamus, amygdala, cerebral cortex, hippocampus, and suprachiasmatic nucleus. Analysis of transgene expression in neonatal and embryonic day 13.5 mice revealed a near perfect correlation between VIP and β‐galactosidase gene expression in cranial cholinergic ganglia and the superior cervical ganglia, and lack of transgene expression in sensory ganglia and in nonneuronal tissue. Potential ectopic transgene expression was observed in neonates, in the cerebellar external granule layer and in a small subpopulation of neurons in the olfactory epithelium. We conclude that the 16.5 kb of VIP gene used in these studies contains sequences sufficient for directing expression specifically to VIP neurons in the PNS, and that sequences located elsewhere on the gene are required for proper CNS expression. The VIP gene sequences used here should be capable of targeting other gene products to specific populations of embryonic and adult peripheral neurons without causing significant expression in the CNS.