Osteoporosis and Alzheimer´s disease (or Alzheimer´s disease and Osteoporosis).

Alzheimer's disease (AD) and osteoporosis are two diseases that mainly affect elderly people, with increases in the occurrence of cases due to a longer life expectancy. Several epidemiological studies have shown a reciprocal association between both diseases, finding an increase in incidence of osteoporosis in patients with AD, and a higher burden of AD in osteoporotic patients. This epidemiological relationship has motivated the search for molecules, genes, signaling pathways and mechanisms that are related to both pathologies. The mechanisms found in these studies can serve to improve treatments and establish better patient care protocols.

Hippocampal DNA Methylation, Epigenetic Age, and Spatial Memory Performance in Young and Old Rats.

In humans and rats, aging is associated with a progressive deterioration of spatial learning and memory. These functional alterations are correlated with morphological and molecular changes in the hippocampus. Here, we assessed age-related changes in DNA methylation (DNAm) landscape in the rat hippocampus and the correlation of spatial memory with hippocampal DNAm age in 2.6- and 26.6-month-old rats. Spatial memory performance was assessed with the Barnes maze test. To evaluate learning ability and spatial memory retention, we assessed the time spent by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. The rat pan-tissue clock was applied to DNAm data from hippocampal tissue. An enrichment pathway analysis revealed that neuron fate commitment, brain development, and central nervous system development were processes whose underlying genes were enriched in hypermethylated CpGs in the old rats. In the old rat hippocampi, the methylation levels of CpG proximal to transcription factors associated with genes Pax5, Lbx1, Nr2f2, Hnf1b, Zic1, Zic4, Hoxd9; Hoxd10, Gli3, Gsx1 and Lmx1b, and Nipbl showed a significant regression with spatial memory performance. Regression analysis of different memory performance indices with hippocampal DNAm age was significant. These results suggest that age-related hypermethylation of transcription factors related to certain gene families, such as Zic and Gli, may play a causal role in the decline in spatial memory in old rats. Hippocampal DNAm age seems to be a reliable index of spatial memory performance in young and old rats.

Therapeutic potential of glial cell line-derived neurotrophic factor and cell reprogramming for hippocampal-related neurological disorders.

Hippocampus serves as a pivotal role in cognitive and emotional processes, as well as in the regulation of the hypothalamus-pituitary axis. It is known to undergo mild neurodegenerative changes during normal aging and severe atrophy in Alzheimer's disease. Furthermore, dysregulation in the hippocampal function leads to epilepsy and mood disorders. In the first section, we summarized the most salient knowledge on the role of glial cell-line-derived neurotrophic factor and its receptors focused on aging, cognition and neurodegenerative and hippocampal-related neurological diseases mentioned above. In the second section, we reviewed the therapeutic approaches, particularly gene therapy, using glial cell-line-derived neurotrophic factor or its gene, as a key molecule in the development of neurological disorders. In the third section, we pointed at the potential of regenerative medicine, as an emerging and less explored strategy for the treatment of hippocampal disorders. We briefly reviewed the use of partial reprogramming to restore brain functions, non-neuronal cell reprogramming to generate neural stem cells, and neural progenitor cells as source-specific neuronal types to be implanted in animal models of specific neurodegenerative disorders.

A regulatable adenovector system for GDNF and GFP delivery in the rat hippocampus.

Spatial memory performance declines in both normal aging and Alzheimer's disease. This cognitive deficit is related to hippocampus dysfunction. Gene therapy using neurotrophic factors like Glial cell line-derived neurotrophic factor (GDNF) emerges as a promising approach to ameliorate age-related cognitive deficits. We constructed a two vector regulatable system (2VRS) which consists of a recombinant adenoviral vector (RAd) harboring a Tet-Off bidirectional promoter flanked by GDNF and Green Fluorescent Protein (GFP) genes. A second adenovector, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are cotransduced by the 2VRS, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is silenced. We tested the 2VRS in CHO-K1 cells where we observed a dose-dependent GFP expression that was completely inhibited by DOX (1 mg/ml). The 2VRS injected in the hippocampal CA1 region transduced both neurons and astrocytes and was efficiently inhibited by DOX added to the drinking water. In order to assess GDNF biological activity we injected 2VRS and its Control (CTRL) vector in the hypothalamus and monitored body weight for one month. The results showed that GDNF retards weight recovery 6 days more than CTRL. In conclusion, our 2VRS demonstrated optimal GFP expression and showed a bioactive effect of transgenic GDNF in the brain.

Cryopreservation of a Human Brain and Its Experimental Correlate in Rats.

Several countries have established self-help cryonics groups whose mission is to cryopreserve human bodies or brains after legal death and ship them to cryonics organizations. The objective of this study was to report the first case of human brain cryopreservation in Argentina and complementary experiments in rats. After legal death, the body of a 78-year-old Caucasian woman was transported to a funeral home where her head was submitted to intracarotid perfusion with 5 L cold physiologic saline followed by the same volume of cold saline containing 13% dimethyl sulfoxide and 13% glycerol. The brain was removed, temporarily frozen at -80°C, and shipped to a U.S. cryostasis facility. Three groups of rats were intracardially perfused with fixative but not frozen (Reference group), vitrification solution VM1 (Control group), or the cryoprotection solution used in the patient (Experimental group). Control and Experimental brains were stored at -80°C and subsequently assessed by immunohistochemistry for the adult neuron marker (NeuN), the immature neuron marker doublecortin (DCX), the dopaminergic neuron marker tyrosine hydroxylase, and the presynaptic marker synaptophysin (SYN). The number of NeuN-positive neurons remained unchanged in the experimental brain cortex, whereas the number of immature DCX neurons in the hippocampus fell markedly in the cryoprotected brains. The results were highly variable for hypothalamic dopaminergic neurons. Confocal microscopy for SYN revealed that cryopreservation did not affect the synaptic network in the hippocampus. To our knowledge, this is the first report correlating a human cryoprotection procedure with results in complementary experiments in laboratory animals.

Mesenchymal Stem Cells Therapy Improved the Streptozotocin-Induced Behavioral and Hippocampal Impairment in Rats.

Sporadic Alzheimer's disease (sAD) is the most prevalent neurodegenerative pathology with no effective therapy until date. This disease promotes hippocampal degeneration, which in turn affects multiple cognitive domains and daily life activities. In this study, we hypothesized that long-lasting therapy with mesenchymal stem cells (MSC) would have a restorative effect on the behavioral alterations and cognitive decline typical of sAD, as they have shown neurogenic and immunomodulatory activities. To test this, we chronically injected intravenous human MSC in a sAD rat model induced by the intracerebroventricular injection of streptozotocin (STZ). During the last 2 weeks, we performed open field, Barnes maze, and marble burying tests. STZ-treated rats displayed a poor performance in all behavioral tests. Cell therapy increased exploratory behavior, decreased anxiety, and improved spatial memory and marble burying behavior, the latter being representative of daily life activities. On the hippocampus, we found that STZ promotes neuronal loss in the Cornus Ammoni (CA1) field and decreased neurogenesis in the dentate gyrus. Also, STZ induced a reduction in hippocampal volume and presynaptic protein levels and an exacerbated microgliosis, relevant AD features. The therapy rescued CA1 neurodegeneration but did not reverse the decrease of immature neurons, suggesting that the therapy effect varied among hippocampal neuronal populations. Importantly, cell therapy ameliorated microgliosis and restored hippocampal atrophy and some presynaptic protein levels in the sAD model. These findings, by showing that intravenous injection of human MSC restores behavioral and hippocampal alterations in experimental sAD, support the potential use of MSC therapy for the treatment of neurodegenerative diseases.

Umbilical Cord Cell Therapy Improves Spatial Memory in Aging Rats.

There is a growing interest in the potential of adult stem cells for implementing regenerative medicine in the brain. We assessed the effect of intracerebroventricular (icv) administration of human umbilical cord perivascular cells (HUCPVCs) on spatial memory of senile (27 mo) female rats, using intact senile counterparts as controls. Approximately one third of the animals were injected in the lateral ventricles with a suspension containing 4.8 X 105 HUCPVC in 8 µl per side. The other third received 4.8 X 105 transgenic HUCPVC overexpressing Insulin-like growth factor-1 (IGF-1) and the last third of the rats received no treatment. Spatial memory performance was evaluated using a modified version of the Barnes maze test. In order to evaluate learning ability as well as spatial memory retention, we assessed the time spent (permanence) by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. Fluorescence microscopy revealed the prescence of Dil-labeled HUCPVC in coronal sections of treated brains. The HUCPVC were located in close contact with the ependymal cells with only a few labeled cells migrating into the brain parenchyma. After treatment with naïve or IGF-1 transgenic HUCPVC, permanence in GS1 and GS3 increased significantly whereas there were no changes in the intact animals. We conclude that HUCPVC injected icv are effective to improve some components of spatial memory in senile rats. The ready accessibility of HUCPVC constitutes a significant incentive to continue the exploration of their therapeutic potential on neurodegenerative diseases.

Mesenchymal stem cell therapy improves spatial memory and hippocampal structure in aging rats.

There is a growing interest in the potential of mesenchymal stem cells (MSCs) for implementing regenerative medicine in the brain as they have shown neurogenic and immunomodulatory activities. We assessed the effect of intracerebroventricular (icv) administration of human bone marrow-derived MSCs (hBM-MSCs) on spatial memory and hippocampal morphology of senile (27 months) female rats, using 3-months-old counterparts as young controls. Half of the animals were injected in the lateral ventricles (LV) with a suspension containing 5 × 105hBM-MSCs in 8 µl per side. The other half received no treatment (senile controls). Spatial memory performance was assessed with a modified version of the Barnes maze test. We employed one probe trial, one day after training in order to evaluate learning ability as well as spatial memory retention. Neuroblast (DCX) and microglial (Iba-1 immunoreactive) markers were also immunohistochemically quantitated in the animals by means of an unbiased stereological approach. In addition, hippocampal presynaptic protein expression was assessed by immunoblotting analysis. After treatment, the senile MSC-treated group showed a significant improvement in spatial memory accuracy and extended permanence in a one- and 3-hole goal sectors as compared with senile controls. The MSC treatment increased the number of neuroblasts in the hippocampal dentate gyrus, reduced the number of reactive microglial cells, and restored presynaptic protein levels as compared to senile controls. We conclude that icv injected hBM-MSCs are effective in improving spatial memory in senile rats and that the strategy improves some functional and morphologic brain features typically altered in aging rats.

Regulatable adenovector harboring the GFP and Yamanaka genes for implementing regenerative medicine in the brain.

Biological rejuvenation by partial cell reprogramming is an emerging avenue of research. In this context, regulatable pluripotency gene expression systems are the most widely used at present. We have constructed a regulatable bidirectional adenovector expressing the humanized green fluorescent protein (GFP) and oct4, sox2, klf4, and c-myc genes (known as the Yamanaka genes or OSKM). The OSKM genes are arranged as a bicistronic tandem (hSTEMCCA tandem), which is under the control of a Tet-Off bidirectional promoter that also controls the expression of the gFP gene. Separately, a constitutive cassette expresses the regulatory protein tTA. Vector DNA was transfected in HEK293 Cre cells, which were additionally infected with the helper adenovector H14, unable to package its DNA due to the Cre recombinase produced by the HEK293 Cre cells. The newly generated vector was expanded by six iterated coinfections of the above cells which were lysed at the end of the process and the adenovector purified by ultracentrifugation in a CsCl gradient. The titer of the initial preparation was 1.2 × 1012 physical viral particles/ml. As expected, GFP fluorescence in vector-transduced rat fibroblast cultures declined with the dose of doxycycline (DOX) present in the medium. Immunocytochemical analysis of transduced cells confirmed the expression of the four Yamanaka genes. Additionally, 3 days after vector injection in the hypothalamus of rats, a significant level of fluorescence was observed in the region. Addition of 2 mg/ml DOX to the drinking water reduced the GFP expression. This adenovector constitutes a promising tool for implementing nonintegrative partial cell reprogramming.

Intracerebroventricular streptozotocin induces impaired Barnes maze spatial memory and reduces astrocyte branching in the CA1 and CA3 hippocampal regions.

Sporadic Alzheimer's disease (SAD) is the most common form of dementia; therefore, there is an urgent need for a model that recapitulates the main pathologic hallmarks of this disease. The intracerebroventricular (icv) injection of streptozotocin (icv-STZ) in rats constitutes a promising model, and thus, icv-STZ rats develop insulin-resistant brain state and cognitive impairments. Even though a great piece of studies has hitherto described this system as a model for SAD, further behavioral and morphometric studies are still needed to fully characterize it. In this study, using Sprague Dawley rats, we evaluated short-term effects on behavior and hippocampus morphometry of the icv-STZ injection at two doses: 1 (STZ1) and 3 mg/kg (STZ3). We found that, following icv-STZ injection, STZ3 animals, but not STZ1, exhibited impairments in spatial reference learning and memory (Barnes maze test) and in recognition memory (object recognition test). Furthermore, the results from behavioral and morpho-histochemical data are compatible. STZ3 rats displayed Stratum Radiatum volume reduction and a decreased NeuN immunoreactivity (neuron loss) in hippocampal CA1 region, together with an increased immunoreactivity for microglial (Iba1) and astroglial (GFAP) markers (neuroinflammation). Sholl analysis revealed the vulnerability of hippocampal astrocytes to STZ in CA1 and CA3. Thus, both doses induced a reduction in process length and in the number of main processes, accompanied by a frank decrease in branching complexity. The present study provides important knowledge of this AD rat model. Overall, we found that the only high STZ dose induced severe and acute neurodegenerative lesions, associated with an inflammation process.

Rejuvenating Effect of Long-Term Insulin-Like Growth Factor-I Gene Therapy in the Hypothalamus of Aged Rats with Dopaminergic Dysfunction.

The aging female rat constitutes an interesting model of spontaneous and progressive age-related dopaminergic dysfunction as it allows assessing new therapeutic strategies for Parkinson's disease. Insulin-like growth factor I (IGF-I) is emerging as a powerful neuroprotective molecule, strongly induced in the central nervous system after different insults. We constructed a helper-dependent recombinant adenoviral vector (HDRAd-IGFI) harboring the gene for rat IGF-I. This was used to implement long-term IGF-I gene therapy in the hypothalamus of aged female rats, which display hypothalamic dopaminergic (DA) dysfunction and, as a consequence, chronic hyperprolactinemia. Rejuvenating long-term IGF-I gene therapy was implemented in young (3 months) and aged (24 months) female rats, which received a single intrahypothalamic injection of 4 × 109 viral particles of either HD-RAd-IGFI or HD-RAd-DsRed (control vector) and were sacrificed 119 days postinjection. In the young animals, neither vector modified serum prolactin (PRL) levels, but in the RAd-IGFI-injected aged rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase (TH)-positive cells in the hypothalamus of experimental compared with control aged animals (5874 ± 486 and 3390 ± 498, respectively). Our results indicate that IGF-I gene therapy in aged female rats is highly effective in rejuvenating the hypothalamic DA neuron groups.

IGF-I Gene Therapy in Aging Rats Modulates Hippocampal Genes Relevant to Memory Function.

In rats, learning and memory performance decline during normal aging, which makes this rodent species a suitable model to evaluate therapeutic strategies. In aging rats, insulin-like growth factor-I (IGF-I), is known to significantly improve spatial memory accuracy as compared to control counterparts. A constellation of gene expression changes underlie the hippocampal phenotype of aging but no studies on the effects of IGF-I on the hippocampal transcriptome of old rodents have been documented. Here, we assessed the effects of IGF-I gene therapy on spatial memory performance in old female rats and compared them with changes in the hippocampal transcriptome. In the Barnes maze test, experimental rats showed a significantly higher exploratory frequency of the goal hole than controls. Hippocampal RNA-sequencing showed that 219 genes are differentially expressed in 28-month-old rats intracerebroventricularly injected with an adenovector expressing rat IGF-I as compared with placebo adenovector-injected counterparts. From the differentially expressed genes, 81 were down and 138 upregulated. From those genes, a list of functionally relevant genes, concerning hippocampal IGF-I expression, synaptic plasticity as well as neuronal function was identified. Our results provide an initial glimpse at the molecular mechanisms underlying the neuroprotective actions of IGF-I in the aging brain.

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

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.

Identification of a conserved gene signature associated with an exacerbated inflammatory environment in the hippocampus of aging rats.

There have been a few descriptive studies in aged rodents about transcriptome changes in the hippocampus, most of them in males. Here, we assessed the age changes in spatial memory performance and hippocampal morphology in female rats and compared those changes with changes in the hippocampal transcriptome. Old rats displayed significant deficits in spatial memory. In both age groups, hole exploration frequency showed a clear peak at hole 0 (escape hole), but the amplitude of the peak was significantly higher in the young than in the old animals. In the hippocampus, there was a dramatic reduction in neurogenesis, whereas reactive microglial infiltrates revealed an inflammatory hippocampal state in the senile rats. Hippocampal RNA-sequencing showed that 210 genes are differentially expressed in the senile rats, most of them being downregulated. Our RNA-Seq data showed that various genes involved in the immune response, including TYROBP, CD11b, C3, CD18, CD4, and CD74, are overexpressed in the hippocampus of aged female rats. Enrichment analysis showed that the pathways overrepresented in the senile rats matched those of an exacerbated inflammatory environment, reinforcing our morphologic findings. After correlating our results with public data of human and mouse hippocampal gene expression, we found an 11-gene signature of overexpressed genes related to inflammatory processes that was conserved across species. We conclude that age-related hippocampal deficits in female rats share commonalities between human and rodents. Interestingly, the 11-gene signature that we identified may represent a cluster of immune and regulatory genes that are deregulated in the hippocampus and possibly other brain regions during aging as well as in some neurodegenerative diseases and low-grade brain tumors. Our study further supports neuroinflammation as a promising target to treat cognitive dysfunction in old individuals and some brain tumors. © 2017 Wiley Periodicals, Inc.

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.

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 Alzheimer's disease (AD). Parkinson's 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.

Role of copper and cholesterol association in the neurodegenerative process.

Age is one of the main factors involved in the development of neurological illnesses, in particular, Alzheimer, and it is widely held that the rapid aging of the world population is accompanied by a rise in the prevalence and incidence of Alzheimer disease. However, evidence from recent decades indicates that Cu and Cho overload are emerging causative factors in neurodegeneration, a hypothesis that has been partially investigated in experimental models. The link between these two variables and the onset of Alzheimer disease has opened up interesting new possibilities requiring more in-depth analysis. The aim of the present study was therefore to investigate the effect of the association of Cu + Cho (CuCho) as a possible synergistic factor in the development of an Alzheimer-like pathology in Wistar rats. We measured total- and nonceruloplasmin-bound Cu and Cho (free and sterified) contents in plasma and brain zones (cortex and hippocampus), markers of oxidative stress damage, inflammation, and programmed cell death (caspase-3 and calpain isoforms). The ratio beta-amyloid (1-42)/(1-40) was determined in plasma and brain as neurodegenerative biomarker. An evaluation of visuospatial memory (Barnes maze test) was also performed. The results demonstrate the establishment of a prooxidative and proinflammatory environment after CuCho treatment, hallmarked by increased TBARS, protein carbonyls, and nitrite plus nitrate levels in plasma and brain zones (cortex and hippocampus) with a consequent increase in the activity of calpains and no significant changes in caspase-3. A simultaneous increase in the plasma A ß 1-42/A ß 1-40 ratio was found. Furthermore, a slight but noticeable change in visuospatial memory was observed in rats treated with CuCho. We conclude that our model could reflect an initial stage of neurodegeneration in which Cu and Cho interact with one another to exacerbate neurological damage.

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.

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.

Potential of gene therapy for restoration of endocrine thymic function in thymus-deficient animal models.

The aim of the present article is to discuss the potential of gene therapy for thymic hormones as a novel therapeutic strategy to treat dyshomeostatic conditions associated with congenital athymia or hypofunction of the endocrine thymus. Recent studies using an adenoviral vector harboring a synthetic gene for the thymic peptide thymulin are reviewed. This adenoviral vector was injected intramuscularly in thymectomized and nude mice as well as in thymectomized rats. Transduced myocytes acted as an ectopic source of thymulin thus restoring circulating thymulin levels to normal values. This restorative effect was long lasting (several months) even though an adenoviral vector was used. In the rat brain, adenovirally-mediated delivery of the synthetic gene for thymulin achieved longer expression than in the case of adenovirally-delivered reporter genes, which is consistent with the reported antiinflammatory activity of thymulin in the brain. Furthermore, neonatal thymulin gene therapy in nude female mice was able to prevent the pituitary and ovarian alterations that typically occur in this mutant after puberty. Neonatal thymulin gene therapy in nude mice was able to prevent some of the alterations in lipid metabolism that develop during adult life in congenitally athymic mice. We conclude that the availability of the above biotechnological tools should boost basic studies on the molecular biology of thymulin and should also allow an assessment of the potential of gene therapy to restore circulating thymulin levels in thymodeficient animal models and eventually, in humans.