Martha C. Bohn

Ontogeny and distribution of glial cell line-derived neurotrophic factor (GDNF) mRNA in rat

Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-beta family isolated from the rat glial tumor cell line, B49. In embryonic dopaminergic (DA) neurons in vitro, GDNF promotes survival, high-affinity dopamine uptake, and neurite outgrowth. We have used a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific to GDNF to study the developmental expression of GDNF mRNA in central nervous system (CNS) and peripheral organs of embryonic rat on gestational days E11.5, E13.5 and E18, neonatal rat on postnatal days P0 and P10, and adult rat. GDNF mRNA is expressed throughout the CNS, with highest levels in P0 spinal cord and in P0 and P10 striatum. Lower levels are present in the brainstem (including the ventral mesencephalon, which contains the DA neurons of the substantia nigra), cerebellum, diencephalon, and telencephalon, as well as in primary cultures of cerebellar granule cells prepared from P7 cerebellum and astrocytes prepared from P1 cortex. The cerebellum has an unusual temporal pattern of expression, high at birth and in the adult, but undetectable at P10. GDNF mRNA is also expressed in many peripheral tissues at higher levels than in brain. These include embryonic limb bud, kidney and gut; neonatal kidney, gut, lung and testis; and adult lung, liver and ovary. In addition to the predicted RT-PCR product, we also observed a minor band which was shown to be identical to GDNF in the mature peptide sequence, but which has a 78 base pair deletion in the preproprotein sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Astrocytes retrovirally transduced with BDNF elicit behavioral improvement in a rat model of Parkinson's disease.

Neurotrophic factors that improve the survival of specific neuronal types during development and after exposure to various neuronal insults hold potential for treatment of neurodegenerative diseases. In particular, brain-derived neurotrophic factor (BDNF) has been shown to exert trophic and protective effects on dopaminergic neurons, the cell type known to degenerate in Parkinsons disease. To determine whether increased levels of biologically produced BDNF affect the function or regeneration of damaged dopaminergic neurons, the effects of grafting astrocytes transduced with the human BDNF gene into the striatum of the partially lesioned hemiparkinsonian rat were examined. Replication deficient retroviruses carrying either human prepro-BDNF or human alkaline phosphatase (AP) cDNA were used to transduce primary type 1 astrocytes purified from neonatal rat cortex. In vitro, BDNF mRNA was expressed by BDNF transduced astrocytes (BDNF astrocytes), but not control AP transduced astrocytes (AP astrocytes), as determined by reverse transcription polymerase chain reaction (RT-PCR). The modified astrocytes were injected into the right striatum 15 days after partial lesioning of the right substantia nigra with 6-hydroxydopamine. Transplantation of BDNF astrocytes, but not AP astrocytes, significantly attenuated amphetamine-induced rotation by 45% 32 days after grafting. Apomorphine-induced rotation increased over time in both groups, but was not significantly different in the BDNF-treated group. The modified BDNF astrocytes survived well with non-invasive growth in the brain for up to 42 days. Although BDNF mRNA positive cells were not detected within the graft site using in situ hybridization, alkaline phosphatase immunoreactive (IR) cells were present in control graft sites suggesting that the retroviral construct continued to be expressed at 42 days. Analysis of the density of tyrosine hydroxylase (TH)-IR fibers showed no effect of BDNF on TH-IR fiber density in the striatum on the lesioned side. These findings suggest that ex vivo gene therapy with BDNF ameliorates parkinsonian symptoms through a mechanism(s) other than one involving an effect of BDNF on regeneration or sprouting from dopaminergic neurons.

Adrenergic and non-adrenergic neurons in the C1 and C3 areas project to locus coeruleus: A fluorescent double labeling study

Following iontophoretic injections of the retrograde tracer Fluoro-gold into the rat locus coeruleus (LC), retrogradely labeled neurons were seen predominantly in the area of C1 adrenergic neurons in the ventrolateral medulla (nucleus paragigantocellularis; PGi) and in the area of C3 adrenergic neurons in the dorsomedial medulla (nucleus prepositus hypoglossi; PrH). Subsequent immunofluorescence for phenylethanolamine-N-methyltransferase indicated that adrenergic and non-adrenergic LC projecting neurons in both areas are interdigitated, and that 21% of LC afferent neurons in the PGi are adrenergic while only 4% of LC afferent neurons in the area of PrH are adrenergic.

Retinal ganglion cell survival is promoted by genetically modified astrocytes designed to secrete brain-derived neurotrophic factor (BDNF).

Genetically engineered cells carrying genes for neurotrophic factors have potential application for treatment of neurodegenerative diseases and injuries to the nervous system. Brain-derived neurotrophic factor (BDNF) promotes the survival of specific neurons, including retinal ganglion cells (RGC). To determine whether genetically engineered astrocytes might be used for delivering bioactive BDNF, we infected primary type 1 rat astrocytes with a retrovirus harboring a human prepro-BDNF cDNA and assayed the medium conditioned by these astrocytes for effects on survival of rat RGCs in vitro. High levels of BDNF mRNA were expressed by infected astrocytes, but not by control astrocytes as determined by RNase protection assay using a BDNF specific probe. To test for secretion of bioactive BDNF from the transgenic astrocytes, embryonic day 17 rat retinas were dissociated and grown in medium conditioned (CM) for 24 h by astrocytes infected with a replication deficient retrovirus carrying BDNF, NGF, or alkaline phosphatase (AP) cDNA. After 3 days, the number of Thy-1 immunoreactive RGCs was counted. BDNF astrocyte CM significantly enhanced RGC survival by 15-fold compared to the AP control. NGF astrocyte CM had no significant effect. The rate of BDNF secretion was estimated at 83-166 pg/10(5) cells/h. This study demonstrates that astrocytes can be genetically engineered to synthesize and secrete bioactive BDNF. These techniques may be applicable to rescuing neurons from degenerative processes and also for enhancing their survival following transplantation.

Selective Loss of α Motoneurons Innervating the Medial Gastrocnemius Muscle in a Mouse Model of Amyotrophic Lateral Sclerosis

Abstract Mutations in the superoxide dismutase gene 1 (SOD-1) are found in patients with familial amyotrophic lateral sclerosis (FALS). Overexpression of a mutated human SOD-1 gene in mice results in neurodegenerative disease as result of motoneuron loss in lumbar spinal cord (10). Using this mouse model of FALS, we have established a quantitative assay utilizing the retrograde tracer Fluorogold (FG) to determine the number of motoneurons innervating one skeletal muscle in mice with ongoing disease. In adult wild-type mice, the number of α motoneurons retrogradely labeled by an injection of FG into medial gastrocnemius muscle is 50 ± 7 and this number remains constant from 7 to 18 weeks of age. In mutant mice, the number of α motoneurons retrogradely labeled by FG is the same as in wild-type mice at 7 and 9 weeks, but then declines to 36% of that in normal mice at 18 weeks. This decline also correlates positively to severity of motor impairments in these mice as assessed by the hindlimb splay test. In contrast, the number of FG-labeled γ motoneurons remains relatively unchanged in both wild-type and mutant mice up to 18 weeks. At 18 weeks of age, this apparent α motoneuron denervation is paralleled by an average of 55% reduction of MG-muscle mass and 40% weaker performance in the hindlimb splay test. These data suggest that α motoneurons are the most vulnerable neuronal subtype in this mouse model of ALS and it is primarily their loss that leads to functional motor deficits. This quantitative bioassay also will be valuable for evaluating novel therapeutics for ALS.

Nerve growth factor released by transgenic astrocytes enhances the function of adrenal chromaffin cell grafts in a rat model of Parkinson's disease.

Previous studies have demonstrated that astrocytes genetically modified to express recombinant nerve growth factor (NGF) support the survival and neuronal transdifferentiation of intrastriatal adrenal chromaffin cell grafts at 2 weeks post-transplantation [15]. The present study was performed to determine whether these effects would be maintained at longer times post-transplantation and, if so, whether the co-grafts would reduce rotational behavior in the unilateral 6-hydroxydopamine-lesioned rat. In the present study, we have demonstrated that primary type I rat astrocytes infected with a replication-defective retrovirus conferring expression of a mouse beta-NGF cDNA sequence secrete NGF at a rate that is approximately 40-fold higher than that of controls (i.e., 8.0 vs. 0.2 pg NGF/h/10(5) cells, respectively). The genetically modified astrocytes were also found to express recombinant NGF following intrastriatal transplantation, as indicated by a 23% increase in striatal NGF content compared with controls, measured at 4 weeks post-transplantation. When NGF-producing astrocytes and adrenal chromaffin cells were co-grafted into the dopamine-denervated striatum of the unilateral 6-hydroxydopamine-lesioned rat, the chromaffin cells displayed extensive neurite outgrowth and a 5-12-fold increase in survival compared to controls at 10 weeks post-grafting. These effects were paralleled by a 60% reduction of apomorphine-induced rotational behavior, suggesting a partial normalization of striatal function. These results suggest that genetically modified astrocytes promote the prolonged survival and function of adrenal chromaffin cell grafts in a rat model of Parkinsons disease.

Development of mRNAs for glucocorticoid and mineralocorticoid receptors in rat hippocampus

The hippocampus plays an important role in mediating glucocorticoid effects on the brain. Glucocorticoids are also implicated in neurogenesis and age-related neuronal death in the hippocampus. The effects of glucocorticoids in the hippocampus are elicited through two receptors with high-affinity for corticosterone, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). In this study, we used a sensitive RNase protection assay to quantify the ontogeny of GR mRNA and MR mRNA in hippocampus from embryonic day 18 (E18) to postnatal day 60 (P60). GR mRNA and MR mRNA are expressed at approximately equal levels in the E18 hippocampus. However, by birth, the level of MR mRNA is three-fold that of GR mRNA and remains elevated up to P60. The levels of both mRNAs increase gradually during the period of postnatal neurogenesis after which they markedly increase to adult levels. In addition, the levels of hippocampal MR mRNA are the same in male and female rats, whereas the levels of GR mRNA are significantly higher in the P60 female rat hippocampus, but not in younger female rats. Our data on the development of mRNA levels do not parallel the levels of glucocorticoid and mineralocorticoid receptors as reported in a number of binding studies. Therefore, our studies, when considered together with previous reports, suggest that posttranscriptional mechanisms play a major role in regulating the levels of glucocorticoid-binding sites in the hippocampus.

Recombinant Adenovirus: A Gene Transfer Vector for Study and Treatment of CNS Diseases☆

Gene transfer to the CNS with recombinant adenoviral vectors is a relatively recent event. In initial reports it was clearly demonstrated that adenoviral vectors can transfer genetic material to multiple cell types within the CNS. The relative ease in generating recombinant adenovirus (Ad) led to feasibility studies in the CNS with application to animal models of inherited disease, neurodegenerative diseases (e.g., Parkinsons and amyotrophic lateral sclerosis), and cerebrovascular disease. In combination with Ad gene transfer to peripheral tissues, these experiments have identified specific limitations and directed further research to improve vector design, formulation, and delivery.

Regulation of phenylethanolamine N-methyltransferase (PNMT) mRNA in the rat adrenal medulla by corticosterone.

In the adrenal medulla of adult rat, physiological levels of glucocorticoid hormones are required to maintain the catalytic activity of the epinephrine‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT). The present study was undertaken to determine whether glucocorticoid regulation of PNMT occurs at the level of mRNA coding for PNMT. Adult male Sprague‐Dawley rats were hypophysectomized (HPX) and killed after 2 weeks; pellets of corticosterone were implanted for 1, 3 or 7 days prior to killing. Determinations were made of plasma corticosterone levels, adrenal PNMT activity and PNMT mRNA levels by Northern gel analysis. HPX resulted in a decrease in plasma corticosterone to undetectable levels and decreases in PNMT activity and PNMT mRNA levels to 1 and 18% of the levels observed in sham rats, respectively. Corticosterone replacement produced high prolonged plasma levels of corticosterone which were 10 times those of sham rats, and significantly increased levels of PNMT activity and mRNA. However, corticosterone replacement failed to restore PNMT activity and mRNA levels fully.

In Vitro Studies of Glucocorticoid Effects on Neurons and Astrocytes

Studies using immunocytochemistry and RNase protection assay demonstrate that glucocorticoid and mineralocorticoid receptors (GR, MR) and their corresponding mRNAs are co-expressed in hippocampal neurons cultured in serum-free, defined medium and at lower levels in cultured astrocytes. Addition of serum or medium conditioned by astrocytes increases the levels of MR mRNA, but has little effect on the levels of GR mRNA. Cellular levels of both GR mRNA and MR mRNA are upregulated by growth of embryonic hippocampal neurons in corticosterone. This is in distinct contrast to regulation of receptor expression in vivo where mRNAs for these receptors are downregulated in the rat hippocampus by corticosterone treatment of the adult adrenalectomized rat. However, in cultured astrocytes, GR and MR mRNAs are also downregulated by corticosterone. To begin to define the role of glucocorticoids in gene expression in astrocytes, we have used giant two-dimensional (2D) gel electrophoresis to separate astrocyte cellular proteins and translation products synthesized in vitro from astrocyte poly A+ RNA. Analysis of approximately 1,500 in vitro translation products by giant 2D gel electrophoresis reveals 11 protein inductions and 1 repression that occur at the level of mRNA in the absence of protein synthesis following treatment of astrocytes with corticosterone. Interestingly, these changes appear to be mediated by GR, but not by MR. The in vitro studies described here are relevant to identifying the role of GR and MR in gene expression in specific cell types in the hippocampus.