Gustavo R. Morel

Insulin‐like growth factor‐I gene therapy increases hippocampal neurogenesis, astrocyte branching and improves spatial memory in female aging rats

In rats, learning and memory performance decline during aging, which makes this rodent species a suitable model to evaluate therapeutic strategies of potential value for correcting age‐related cognitive deficits. Some of these strategies involve neurotrophic factors like insulin‐like growth factor‐I (IGF‐I), a powerful neuroprotective molecule in the brain. Here, we implemented 18‐day long intracerebroventricular (ICV) IGF‐I gene therapy in 28 months old Sprague–Dawley female rats, and assessed spatial memory performance in the Barnes maze. We also studied hippocampal morphology using an unbiased stereological approach. Adenovectors expressing the gene for rat IGF‐I or the reporter DsRed were used. Cerebrospinal fluid (CSF) samples were taken and IGF‐I levels determined by radioimmunoassay. At the end of the study, IGF‐I levels in the CSF were significantly higher in the experimental group than in the DsRed controls. After treatment, the IGF‐I group showed a significant improvement in spatial memory accuracy as compared with DsRed counterparts. In the dentate gyrus (DG) of the hippocampus, the IGF‐I group showed a higher number of immature neurons than the DsRed controls. The treatment increased hippocampal astrocyte branching and reduced their number in the hippocampal stratum radiatum. We conclude that the ependymal route is an effective approach to increase CSF levels of IGF‐I and that this strategy improves the accuracy of spatial memory in aging rats. The favorable effect of the treatment on DG neurogenesis and astrocyte branching in the stratum radiatum may contribute to improving memory performance in aging rats.

The Thymus-Neuroendocrine Axis : Physiology, Molecular Biology, and Therapeutic Potential of the Thymic Peptide Thymulin

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. It consists of a nonapeptide component coupled to the ion zinc, which confers biological activity to the molecule. After its discovery in the early 1970s, thymulin was characterized as a thymic hormone involved in several aspects of intrathymic and extrathymic T cell differentiation. Subsequently, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, a growing core of information, to be reviewed here, points to thymulin as a hypophysotropic peptide. In recent years, interest has arisen in the potential use of thymulin as a therapeutic agent. Thymulin was shown to possess anti‐inflammatory and analgesic properties in the brain. Furthermore, an adenoviral vector harboring a synthetic gene for thymulin, stereotaxically injected in the rat brain, achieved a much longer expression than the adenovirally mediated expression in the brain of other genes, thus suggesting that an anti‐inflammatory activity of thymulin prevents the immune system from destroying virus‐transduced brain cells. Other studies suggest that thymulin gene therapy may also be a suitable therapeutic strategy to prevent some of the endocrine and metabolic alterations that typically appear in thymus‐deficient animal models. The present article briefly reviews the literature on the physiology, molecular biology, and therapeutic potential of thymulin.

Estrogen inhibits tuberoinfundibular dopaminergic neurons but does not cause irreversible damage

Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons.

Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats.

Insulin-like growth factor-I (IGF-I) is a powerful neuroprotective molecule in the brain and spinal cord. We have previously shown that intracerebroventricular (i.c.v.) IGF-I gene therapy is an effective strategy to increase IGF-I levels in the cerebrospinal fluid (CSF). Since aging in rats is associated with severe motor function deterioration, we implemented i.c.v. IGF-I gene therapy in very old rats (30-31 months) and assessed the beneficial impact on motor performance. We used recombinant adenovectors (RAds) expressing either green fluorescent protein (GFP) or rat IGF-I. Injection in the lateral or fourth ventricle led to high transgene expression in the ependymal cell layer in the brain and cervical spinal cord. RAd-IGF-I-injected rats but not RAd-GFP-injected controls, showed significantly increased levels of CSF IGF-I. Motor tests showed the expected age-related decline in aged rats. Seventeen-day IGF-I gene therapy induced a significant improvement in motor performance in the aged but not in the young animals. These results show that IGF-I is an effective restorative molecule in the aging brain and spinal cord. The data also reveal that the ependymal route constitutes a promising approach for implementing protective IGF-I gene therapy in the aging CNS.

Cognitive impairment and morphological changes in the dorsal hippocampus of very old female rats.

The hippocampus, a medial temporal lobe structure necessary for the formation of spatial memory, is particularly affected by both normal and pathologic aging. In previous studies, we observed a significant age-related increase in dopaminergic neuron loss in the hypothalamus and the substantia nigra of female rats, which becomes more conspicuous at extreme ages. Here, we extend our studies by assessing spatial memory in 4-6 month-old (young), 26-month-old (old) and 29-32-month-old (senile) Sprague-Dawley female rats as well as the age-related histopathological changes in their dorsal hippocampus. Age changes in spatial memory performance were assessed with a modified version of the Barnes maze test. We employed two probe trials (PTs), one and five days after training, respectively, in order to evaluate learning ability as well as short-term and longer-term spatial memory retention. A set of relevant hippocampal cell markers was also quantitated in the animals by means of an unbiased stereological approach. The results revealed that old rats perform better than senile rats in acquisition trials and young rats perform better than both aging groups. However, during short-term PT both aging groups showed a preserved spatial memory while in longer-term PT, spatial memory showed deterioration in both aged groups. Morphological analysis showed a marked decrease (94-97%) in doublecortin neuron number in the dentate gyrus in both aged groups and a reduction in glial fibrillary acidic protein-positive cell number in the stratum radiatum of aging rats. Astroglial process length and branching complexity decreased in aged rats. We conclude that while target-seeking activity and learning ability decrease in aged females, spatial memory only declines in the longer-term tests. The reduction in neuroblast number and astroglial arborescence complexity in the dorsal hippocampus are likely to play a role in the cognitive deficits of aging rats.

Peripheral and mesencephalic transfer of a synthetic gene for the thymic peptide thymulin.

Thymulin is a thymic peptide with antiinflammatory activity in the brain. We constructed a recombinant adenoviral vector, RAd-FTS, expressing a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin and used it for peripheral and central gene transfer in rats. Thymulin concentration in serum and brain tissue was determined by bioassay. Reporter gene expression in the substantia nigra (SN) was quantitated by enzymohistochemistry or fluorescence microscopy using an appropriate image analysis software. A single intramuscular injection (10(8) plaque forming units (pfu)/animal) of RAd-FTS in thymectomized rats (nondetectable serum thymulin) induced supraphysiologic serum thymulin levels for at least 110 days (123+/-22 fg/ml versus 598+/-144 fg/ml in intact and vector-injected rats, respectively). Stereotaxic intranigral injection of RAd-FTS induced steady expression levels of met-FTS for at least 90 days, whereas expression of adenovirally transferred reporter genes coding for green fluorescent protein fused to HSV thymidine kinase (GFP-TK)(fus) or E.coli beta-galactosidase (beta-gal), declined drastically within a month (% transgene expression in the SN on post-injection day 30 relative to day 2 was: 18, <1 and 125%, for beta-gal, (GFP-TK)(fus) and met-FTS, respectively). We conclude that RAd-FTS constitutes a suitable biotechnological tool for the assessment of peripheral and central thymulin gene therapy in animal models of nigral dopaminergic neurodegeneration induced by pro-inflammatory agents.

The Neuroendocrine System as a Model to Evaluate Experimental Gene Therapy

The implementation of experimental gene therapy in animal models of neurological diseases is an area of growing interest. Although the neuroendocrine system offers unique advantages for the assessment of in vivo gene therapy, little work has been done in this model. Here we review the core of documented studies in which in vivo gene therapy has been implemented in the neuroendocrine system of rodent models. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotrophic cell proliferation. Stereotaxic injection of an adenoviral vector expressing Insulin-like Growth Factor-I (IGF-I) was able to correct their chronic hyperprolactinemia and restore tuberoinfundibular dopaminergic (TIDA) neuron numbers. In young and old F-344 male rats, Glial Cell Line-derived Neurotrophic Factor (GDNF) gene delivery in the hypothalamus induced body weight loss. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.

Therapeutic potential of IGF-I on hippocampal neurogenesis and function during aging

ABSTRACT In rats, learning and memory performance decline during normal aging, which is paralleled by a severe reduction of the levels of neurogenesis in the hippocampal dentate gyrus (DG). A promising therapeutic strategy to restore neurogenesis in the hippocampus of old rats and their spatial memory involves the use of insulin-like growth factor-I (IGF-I). The peptide exerts pleiotropic effects in the brain, regulating multiple cellular processes. Thus, 4-week intracerebroventricular (ICV) perfusion of IGF-I significantly restored spatial memory and hippocampal neurogenesis in old male rats. Similar results were achieved by ICV IGF-I gene therapy in aging female rats. Thus, the treatment seemed to increase the number of immature neurons in the DG of 28 mo old rats, which was paralleled by an increase in the accuracy of the animals to remember specific patterns, which is known as pattern separation memory. The DG is thought to be the main hippocampal structure involved in pattern separation memory and there is evidence that the level of neurogenesis in the DG is directly related to pattern separation performance in rodents. Summing up, IGF-I emerges as a promising restorative molecule for increasing hippocampal neurogenesis and memory accuracy in aged individuals and possibly, in neurodegenerative pathologies.

Gene Therapy and Cell Reprogramming For the Aging Brain: Achievements and Promise

In the central nervous system, cholinergic and dopaminergic (DA) neurons are among the cells most susceptible to the deleterious effects of age. Thus, the basal forebrain cholinergic system is known to undergo moderate neurodegenerative changes during normal aging as well as severe atrophy in Alzheimers disease (AD). Parkinsons disease (PD), a degeneration of nigro-striatal DA neurons is the most conspicuous reflection of the vulnerability of DA neurons to age. Overall, there is growing evidence that a progressive decline in cognitive function and central DA activity represents basic features of normal aging both in humans and laboratory rodents. Spontaneous or environmental neurotoxin-mediated exacerbation of these processes contributes to the symptoms of AD and PD, respectively. In this context, neurotrophic factors that can prevent or delay the decline in cognitive function and central DA activity are of clinical interest. Among them, Insulin-like Growth Factor I and Glial cell line-Derived Neurotrophic Factor are emerging as powerful neuroprotective molecules. This article discusses the experimental evidence supporting the neuroprotective relevance of these and related factors in the aging brain. The availability of induced pluripotent stem cells offers a new promise for the treatment of pathologies associated with the loss of specific cell types as for instance, nigral DA neurons (in PD) or basal forebrain cholinergic neurons (BFCN) in the early stages of AD. Recent studies documenting the use of cell reprogramming for the generation of multipotent neuronal precursors as well as functional BFCN and DA neurons are reviewed.

Insulin-like growth factor-I gene therapy reverses morphologic changes and reduces hyperprolactinemia in experimental rat prolactinomas

BackgroundThe implementation of gene therapy for the treatment of pituitary tumors emerges as a promising complement to surgery and may have distinct advantages over radiotherapy for this type of tumors. Up to now, suicide gene therapy has been the main experimental approach explored to treat experimental pituitary tumors. In the present study we assessed the effectiveness of insulin-like growth factor I (IGF-I) gene therapy for the treatment of estrogen-induced prolactinomas in rats.ResultsFemale Sprague Dawley rats were subcutaneously implanted with silastic capsules filled with 17-β estradiol (E2) in order to induce pituitary prolactinomas. Blood samples were taken at regular intervals in order to measure serum prolactin (PRL). As expected, serum PRL increased progressively and 23 days after implanting the E2 capsules (Experimental day 0), circulating PRL had undergone a 3–4 fold increase. On Experimental day 0 part of the E2-implanted animals received a bilateral intrapituitary injection of either an adenoviral vector expressing the gene for rat IGF-I (RAd-IGFI), or a vector (RAd-GFP) expressing the gene for green fluorescent protein (GFP). Seven days post vector injection all animals were sacrificed and their pituitaries morphometrically analyzed to evaluate changes in the lactotroph population. RAd-IGFI but not RAd-GFP, induced a significant fall in serum PRL. Furthermore, RAd-IGFI but not RAd-GFP significantly reversed the increase in lactotroph size (CS) and volume density (VD) induced by E2 treatment.ConclusionWe conclude that IGF-I gene therapy constitutes a potentially useful intervention for the treatment of prolactinomas and that bioactive peptide gene delivery may open novel therapeutic avenues for the treatment of pituitary tumors.