RhoE is spatiotemporally regulated in the postnatal mouse CNS.

Rnd proteins are a family of small GTPases that have been involved in axon path finding and CNS development by their control of actin cytoskeleton dynamics. Rnd proteins are constitutively activated and, subsequently, their functions determined by their localization and expression levels. In this work we have analyzed by Western blot and immunohistochemistry the levels and localization of Rnd3/RhoE during mouse postnatal development. CNS was found to be the main tissue for RhoE protein expression, which was detected in all regions of the adult brain and spinal cord, with the highest levels in the olfactory bulb and cortex. RhoE protein levels were considerably higher in all the regions of the CNS the first 2-3 weeks of postnatal development, undergoing later a decrease that led to low levels in the adult. Immunohistochemical detection of RhoE at postnatal day 21 showed an intense and widespread labelling throughout the CNS. RhoE immunoreactivity was detected in the granular and mitral cells and anterior olfactory nuclei of the olfactory bulb and in all cerebral layers. In the striatum, diencephalon, mesencephalon, pons, medulla oblongata and spinal cord, RhoE was widely distributed with higher intensity in the motoneurones and in some brainstem nuclei such as the red nucleus or the reticulotegmental nucleus. The pyramidal cells of CA1-3 and the polymorph layer, but not the granular cells of the dentate gyrus in the hippocampus were strongly labelled. At earlier stages the labelling was nearly similar; however, a prominent labelling was detected in the cells of the rostral migratory stream and in the external granule cells of the cerebellum. Our results suggest that RhoE can play important roles in the postnatal development and maturation of the CNS, especially in the migratory processes affecting the neurones.

Expression and regulation of insulin and the glucose transporter GLUT8 in the testes of diabetic rats.

Diabetes induces several malfunctions in male germ cells. The aim of this study was to analyze the levels and localization of the glucose transporter GLUT8 and insulin in the testes of rats induced to a diabetic status by a single dose of streptozotocin. One month after inducing diabetes, the GLUT8 immunoreactivity in diabetic rats was mainly located associated to the acrosomic system of spermatids, and at low levels in Leydig cells. Neither the immunohistochemical localization of this transporter nor its levels showed any difference when compared to control rats. Furthermore, it was observed that control rat testes expressed insulin, which was diffusely located in the cytoplasm of both Leydig cells and early elongated spermatids and concentrated in a cytoplasmic compartment in the more mature spermatids. Testicular insulin levels measured by western blot were reduced by more than half in diabetic rats, although the distribution of the hormone was unchanged. These results indicate that i) insulin is produced by testicular cells, ii) insulin is depleted by streptozotocin-induced diabetes, and iii) that insulin depletion and hyperglycemia do not regulate the expression of GLUT8 in testes. These results also suggest that testicular production of insulin could play a role in regulating spermatogenesis and/or glucose metabolism in these organs.

Motoneuron survival is enhanced in the absence of neuromuscular junction formation in embryos.

Approximately half of the motoneurons produced during development die before birth or shortly after birth. Although it is believed that survival depends on a restricted supply of a trophic sustenance produced by the synaptic target tissue (i.e., muscle), it is unclear whether synapse formation per se is involved in motoneuron survival. To address this issue, we counted cranial motoneurons in a set of mutant mice in which formation of neuromuscular junctions is dramatically impaired (i.e., null mutants for agrin, nerve-derived agrin, rapsyn, and MuSK). We demonstrate that in the absence of synaptogenesis, there is an 18-34% increase in motoneuron survival in the facial, trochlear, trigeminal motor, and hypoglossal nuclei; the highest survival occurred in the MuSK-deficient animals in which synapse formation is most severely compromised. There was no change in the size of the mutant motoneurons as compared with control animals, and the morphology of the mutant motoneurons appeared normal. We postulate that the increased axonal branching observed in these mutants leads to a facilitated "access" of the motoneurons to muscle-derived trophic factors at sites other than synapses or that inactivity increases the production of such factors. Finally, we examined motoneurons in double mutants of CNTFRalpha(-/-) (in which there is a partial loss of motoneurons) and MuSK(-/-) (in which there is an increased survival of motoneurons). The motoneuron numbers in the double mutants parallel those of the single MuSK-deficient mice, indicating that synapse disruption can even overcome the deleterious effect of CNTFRalpha ablation.

Soluble TNF receptors partially protect injured motoneurons in the postnatal CNS.

There is accumulating evidence that cytokines are involved in the functioning of the brain and the spinal cord. However, it has been controversial whether they exert a neurotoxic or a neuroprotective effect. To address this question in vivo, we have examined the survival of injured motoneurons in a line of transgenic mice that overexpress the soluble form of tumour necrosis factor receptor-1 (sTNFR1). In these animals, all of the circulating TNF and lymphotoxin-alpha are neutralized by the continuous expression of the soluble receptor. Following axotomy of the facial nerve in 7-day-old control mice, we observed a loss of approximately 90% of the motoneurons at two weeks survival. In the transgenic mice under the same conditions, the percentage of motoneuron survival was increased two-fold (515 vs. 224) and varied as a function of the level of the circulating receptor. These results indicate that neutralization of endogenous TNF and lymphotoxin-alpha by means of overexpression of the soluble receptor can decrease cell death of injured motoneurons and suggest that these cytokines may play an important role in neuronal degeneration in the CNS following a lesion.

IAP family proteins delay motoneuron cell death in vivo.

Neuronal apoptosis inhibitory protein (NAIP), and human inhibitors of apoptosis 1 and 2 (HIAP1 and HIAP2) are three members of the mammalian family of antiapoptosis proteins called 'inhibitors of apoptosis' (IAP). These molecules can prevent apoptosis in vitro and the over-expression of NAIP can decrease ischemic damage in the hippocampus. The goal of our experiments was to determine whether administration of NAIP, HIAP1 and HAIP2 could rescue motoneurons following axotomy of a peripheral nerve. In young rats, an adenoviral gene transfer technique was used to deliver and express these proteins in motoneurons; a fluorescent tracer was simultaneously added as a means for quantitatively assessing the rescue of fluorescently labelled motoneurons in serial sections of the lumbar spinal cord. Control experiments using adenoviral vectors (adv) expressing the lacZ gene showed that 14% of the sciatic motoneuron pool could be transfected indicating the existence of a subpopulation of spinal motoneurons susceptible to this class of viral vectors. The administration of an adv-NAIP, adv-HIAP1 and adv-HIAP2 rescued 30-40% of motoneurons at one week after sciatic axotomy. The efficiency of these proteins was similar to that of two neurotrophic factors, ciliary neurotrophic factor and brain-derived neurotrophic factor, administrated by the same viral technique. The effect of the IAP proteins on motoneuron survival decreased with time but was still present after 4 weeks postaxotomy; the duration of the response was dependent upon the viral titre. These experiments demonstrate that IAP family proteins can prevent motoneuron cell death in vivo and may offer a new therapeutic approach for motoneuron diseases.

NGF-induced motoneuron cell death depends on the genetic background and motoneuron sub-type.

Nerve growth factor (NGF) promotes the survival of several neuronal populations, but recently it has also been shown to induce neuronal cell death. Here we report the effects of NGF on lesioned motoneurons. We have analyzed facial and sciatic motoneurons in newborn and adult BALB/c and C57BL/6 mice, in addition to mice deficient in the low-affinity p75 receptor for the neurotrophins (p75NTR). NGF application did not alter survival of lesioned facial motoneurons in any of the strains examined independent of the age of the animals. Only in the adult C57BL/6 mouse strain where the sciatic nerve had been crushed prior to factor application did NGF induce cell death of axotomized sciatic motoneurons. Our results illustrate the importance of the genetic background and the motoneuron sub-type in studies related to cell death and survival of motoneurons in relation to NGF and p75NTR.

Expression of the genes for alpha-type and beta-type calcitonin gene-related peptide during rat embryogenesis.

Throughout rat embryogenesis we analysed the expression patterns of the three mature transcripts generated from the two calcitonin gene-related peptide genes: calcitonin, alpha-calcitonin gene-related peptide, and beta-calcitonin gene-related peptide messenger RNAs. In addition, we examined in parallel the distribution of calcitonin gene-related peptide and calcitonin immunoreactivity. Of the three transcripts, beta-calcitonin gene-related peptide messenger RNA was first detected in sensory ganglia on embryonic day 14, and by embryonic day 15 was seen to a lesser degree in motor neurons and autonomic ganglia. Starting at embryonic day 16, however, the highest levels of beta-calcitonin gene-related peptide messenger RNA were found in motor neurons rather than sensory ganglia. Alpha-calcitonin gene-related peptide messenger RNA was first detected on embryonic day 16 in both sensory ganglia and motor neurons, but at lower levels than beta-calcitonin gene-related peptide, particularly in the motor neurons of the spinal cord. By embryonic day 20, transcripts for alpha- and beta-calcitonin gene-related peptide were expressed in distinct brain regions. High levels of alpha-calcitonin gene-related peptide messenger RNA were detected in hypoglossal, facial, and parabrachial nuclei, and moderate levels in the trigeminal motor and ambiguus nuclei. By contrast, beta-calcitonin gene-related peptide messenger RNA was detected at low levels in hypoglossal, ambiguus, facial, and parabrachial nuclei, and at high levels in the trigeminal nucleus. In the oculomotor-trochlear nucleus, beta-calcitonin gene-related peptide messenger RNA was the sole isotype expressed. Low levels of messenger RNA for both calcitonin gene-related peptide transcripts were appreciated in the inferior olive. Outside the nervous system, alpha-calcitonin gene-related peptide messenger RNA was weakly expressed in the thyroid gland and beta-calcitonin gene-related peptide messenger RNA in the thymus. Throughout embryogenesis, calcitonin gene-related peptide immunoreactivity usually followed the expression of either alpha- or beta-calcitonin gene-related peptide messenger RNA. Calcitonin messenger RNA and protein were detected only in the thyroid gland from embryonic day 18 onward. This work shows that of the three mature transcripts produced by the two calcitonin gene-related peptide genes, beta-calcitonin gene-related peptide messenger RNA is the predominant transcript produced early in rat embryogenesis. However, by perinatal stages alpha-calcitonin gene-related peptide shows the highest expression in the brain and spinal cord. In autonomic ganglia, beta-calcitonin gene-related peptide is either the sole or the predominant transcript. Unlike the chick embryo in which calcitonin messenger RNA is expressed early in the CNS, in rat it was only expressed outside the nervous system in the thyroid gland during the last days of embryogenesis.

The two mature transcripts of the chick calcitonin gene are expressed within the central nervous system during embryogenesis.

Calcitonin mRNA and calcitonin gene-related peptide (CGRP) mRNA both are generated from the calcitonin gene because of tissue-specific alternative splicing of the primary transcript. It is currently established that, of the two mature transcripts, calcitonin mRNA is far the predominant transcript produced in thyroid C-cells whereas only CGRP mRNA is produced in the nervous system. However, here we provide evidence that the two splicing forms of the chick calcitonin primary transcript are found within the developing central nervous system, although displaying specific patterns of expression. While CGRP mRNA is first expressed in motor neurons at rather advanced stages of embryogenesis, calcitonin mRNA is expressed in the floor plate and dorsal rhombencephalon from earliest stages.

Expression of the genes for alpha-type and beta-type calcitonin gene-related peptide during postnatal rat brain development.

In this study we have analysed the expression of the genes for both alpha-type and beta-type calcitonin gene-related peptide (CGRP) during postnatal development of the rat brain and compared it with the expression of CGRP-like immunoreactivity. At birth both alpha-type and beta-type CGRP messenger RNA were present in the parabrachial nucleus, inferior olive and motor nuclei (except for abducens nucleus), and only alpha-type CGRP messenger RNA in some posterior thalamic nuclei. As development advanced, new nuclei started to express either only alpha-CGRP gene (superior olive, parabigeminal, sagulum, and some hypothalamic and cranial thalamic nuclei) or both genes (abducens nucleus). In the inferior olive both genes were transiently expressed. Beta-CGRP messenger RNA disappeared by postnatal day 10 and alpha-CGRP messenger RNA by postnatal day 20. During the whole postnatal development beta-CGRP gene expression predominated over that of alpha-CGRP in the trigeminal and eye motor nuclei, while in the remainder nuclei alpha-CGRP messenger RNA was either the predominant isoform or the sole one. CGRP-like immunoreactivity, which does not distinguish between alpha-type and beta-type CGRP, was detected in those nuclei containing either alpha-CGRP messenger RNA or beta-CGRP messenger RNA. However, no CGRP messenger RNA was detected in areas such as superior colliculus, lateral pontine nucleus, pars reticulata of the substantia nigra, perifornical area, or zona incerta in which CGRP-like immunoreactivity was prominent. CGRP-like immunoreactivity, but not CGRP messenger RNA, was also transiently detected by postnatal day 5 in some cells of the globus pallidus. In the adult brain, the levels of alpha- and beta-CGRP messenger RNA as well as those of CGRP-like immunoreactivity were considerably reduced. This fact, similar to that of other growth- and development-associated factors, suggests a role for CGRP as a neuron-derived neurotrophic factor. The transient expression in neurons of the inferior olive, matching the period when climbing fibres and cerebellar cortex are developing, seems to support such an idea. The results of this study show that those nuclei expressing beta-CGRP gene also express alpha-CGRP gene. However, there are a number of nuclei that only express alpha-CGRP gene. On the other hand, CGRP-like immunoreactivity is detected in some nuclei which express no CGRP messenger RNA. It suggests that such nuclei express any CGRP-related protein (identified by the antibodies against CGRP) or, if they really contain CGRP protein, this is produced from undetectable amounts (using our in situ hybridization histochemistry procedure) of CGRP messenger RNA or it comes from other nuclei that connect with them in which CGRP protein is synthesized and then transferred.

Differential expression of alpha-CGRP and beta-CGRP genes within hypoglossal motoneurons in response to axotomy.

In this study we have analysed, by in situ hybridization, the expression of the genes for both alpha-CGRP and beta-CGRP in hypoglossal motor nuclei following transection of the left hypoglossal nerve. Our results show that the gene for alpha-CGRP displays a peculiar sequence of regulation (a successive up-down-up-recovery sequence) within ipsilateral hypoglossal motoneurons in response to axotomy. It is initially up-regulated, then down-regulated (displaying mRNA levels below basal), and later again up-regulated before recovery. By contrast, the gene for beta-CGRP displays a successive and distinct up-down-recovery sequence of regulation (it does not display a second increase in mRNA production). The first up-regulation of the alpha-CGRP gene occurs just during the early period of perineuronal glial reaction and the second up-regulation just during the period of delayed astrocyte reaction and muscle reinnervation. Because alpha-CGRP is a neuron-derived factor for many types of cells, including astrocytes and skeletal myocytes, our results suggest that the pleiotropic alpha-CGRP may be a motoneuron-derived trophic signal for both glial and skeletal muscle cells in order to maintain the motoneuron itself and, in consequence, might be of therapeutic interest in treating degenerative disease of motoneurons. beta-CGRP might be redundant within the hypoglossal motoneurons.