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.

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 thymulin-lactotropic axis in rodents: thymectomy, immunoneutralization and gene transfer studies.

OBJECTIVES: There is clear evidence on the existence of a thymus-pituitary axis which seems to be particularly important during perinatal life. In particular, the thymic peptide thymulin has been shown to be a relevant player in thymus-pituitary communication. Our goal was to explore the effect of thymulin on circulating prolactin (PRL) levels in different animal models. To this end we undertook a series of experiments in rats and mice, implementing adult thymectomy, thymulin immunoneutralization in normal C57BL/6 mice and neonatal thymulin gene therapy in nude mice. METHODS: We assessed the impact of the above manipulations on PRL secretion and lactotrope morphology by measuring serum PRL by radioimmunoassay and by performing morphometric analysis of the lactotropic cell population in the anterior pituitary gland. RESULTS: Adult thymectomy in female rats slightly increased serum PRL, an effect that was partially reversed by thymulin gene therapy. In mice, thymulin immunoneutralization from birth to age 32 days reduced serum PRL both in males and females. Thymulin immunoneutralization induced a significant (p < 0.01) decrease in lactotrope cell density (CD) and volume density (VD) without changes in cell size (CS). Neonatal thymulin gene therapy markedly increased serum thymulin (p < 0.01) and lactotrope CD, CS and VD in nude mice of both sexes. CONCLUSIONS: Our findings suggest a modulatory effect of thymulin on the lactotrope cell population and on serum PRL, particularly during early life.

Thymulin gene therapy prevents the histomorphometric changes induced by thymulin deficiency in the thyrotrope population of mice.

There is evidence of the existence of a bidirectional relationship between the thymus gland and the thyroid axis. Since the thymic peptide thymulin possesses hypophysiotropic activity, we undertook the task of assessing the histomorphometric changes induced by thymulin deficiency on the thyrotrope population of normal mice and the action of neonatal thymulin gene therapy on the thyrotropin (TSH)-cells of nude mice. C57BL/6 mice were subjected to immunoneutralization of circulating thymulin from postnatal day 1 to the end of the study (postnatal day 32) by intraperitoneal injections of rabbit anti-factor thymulin serum (α-FTS) and normal rabbit serum in controls. Also, neonatal thymulin gene therapy was implemented in athymic nude mice using an adenoviral vector expressing a gene for thymulin (RAd-FTS). On postnatal day 1, heterozygous (nu/+) and homozygous (nu/nu) pups received a single bilateral intramuscular (i.m.) injection of either RAd-FTS or RAd-GFP (the latter being the control vector). The pituitaries were immunostained for TSH. Thymulin immunoneutralization severely reduced serum thymulin (p < 0.01). We detected a significant (p < 0.05) decrease in cell size (CS) and volume density (VD) with a nonsignificant decrease in cell density (CD) in C57BL/6 in both males and females. A single neonatal i.m. injection of RAd-FTS markedly increased the circulating levels of serum thymulin in the athymic mice and increased the CD (p < 0.05), CS (p < 0.01) and VD (p < 0.01) of the thyrotrope population in nu/nu mice. Thyroid histology was not affected. Our results suggest a possible modulating effect of thymulin on the thyrotrope population.

Thymulin-based gene therapy and pituitary function in animal models of aging.

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. After its discovery and initial characterization in the 1970s, 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 hypophysiotropic peptide. Additionally, thymulin was shown to possess anti-inflammatory and analgesic properties in the brain. In recent years, a synthetic DNA sequence coding for a biologically active analog of thymulin, metFTS, was constructed and cloned in different adenoviral vectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be downregulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies suggest that thymulin gene therapy may be a suitable therapeutic strategy to prevent some of the endocrine and reproductive alterations that typically appear in congenitally athymic (nude) mice, taken as a suitable model of neuroendocrine and reproductive aging. The present article briefly reviews the literature on the physiology of the thymulin-pituitary axis as well as on the new molecular tools available to exploit the therapeutic potential of thymulin.

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.

Intracerebroventricular Delivery of Human Umbilical Cord Mesenchymal Stem Cells as a Promising Therapy for Repairing the Spinal Cord Injury Induced by Kainic Acid.

Spinal cord injury (SCI) is a common pathological condition that leads to permanent or temporal loss of motor and autonomic functions. Kainic acid (KA), an agonist of kainate receptors, a type of ionotropic glutamate receptor, is widely used to induce experimental neurodegeneration models of CNS. Mesenchymal Stem Cells (MSC) therapy applied at the injured nervous tissue have emerged as a promising therapeutic treatment. Here we used a validated SCI experimental model in which an intraparenchymal injection of KA into the C5 segment of rat spinal cord induced an excitotoxic lesion. Three days later, experimental animals were treated with an intracerebroventricular injection of human umbilical cord (hUC) MSC whereas control group only received saline solution. Sensory and motor skills as well as neuronal and glial reaction of both groups were recorded. Differences in motor behavior, neuronal counting and glial responses were observed between hUC-MSC-treated and untreated rats. According to the obtained results, we suggest that hUC-MSC therapy delivered into the fourth ventricle using the intracerebroventricular via can exert a neuroprotective or neurorestorative effect on KA-injected animals.

Adenovirus-Mediated Transduction of Insulin-Like Growth Factor 1 Protects Hippocampal Neurons from the Toxicity of Aß Oligomers and Prevents Memory Loss in an Alzheimer Mouse Model.

Alzheimer's disease (AD) is the main cause of dementia in the elderly. Although activation of brain insulin signaling has been shown to be neuroprotective, to preserve memory in AD models, and appears beneficial in patients, the role of insulin-like growth factor 1 (IGF1) remains incompletely understood. We found reduced active/inactive IGF1 ratio and increased IGF1R expression in postmortem hippocampal tissue from AD patients, suggesting impaired brain IGF1 signaling in AD. Active/inactive IGF-1 ratio was also reduced in the brains of mouse models of AD. We next investigated the possible protective role of IGF1 in AD models. We used a recombinant adenoviral vector, RAd-IGF1, to drive the expression of IGF1 in primary hippocampal neuronal cultures prior to exposure to AßOs, toxins that accumulate in AD brains and have been implicated in early synapse dysfunction and memory impairment. Cultures transduced with RAd-IGF1 showed decreased binding of AßOs to neurons and were protected against AßO-induced neuronal oxidative stress and loss of dendritic spines. Significantly, in vivo transduction with RAd-IGF1 blocked memory impairment caused by intracerebroventricular (i.c.v.) infusion of AßOs in mice. Our results demonstrate altered active IGF1 and IGF1R levels in AD hippocampi, and suggest that boosting brain expression of IGF1 may comprise an approach to prevent neuronal damage and memory loss in AD.

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.

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.

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.

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.

A new adenovector system for implementing thymulin gene therapy for inflammatory disorders.

Thymulin is a thymic peptide possessing anti-inflammatory effects. In order to manipulate thymulin expression in gene therapy studies, we built a bidirectional regulatable two-vector Tet-Off system and the corresponding control system. The experimental two-vector system, ETV, consists of a recombinant adenovector (RAd) harboring an expression cassette centered on a Tet-Off bidirectional promoter flanked by a synthetic gene for thymulin and the gene for humanized Green Fluorescent Protein (hGFP). The second adenovector of this system, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are co-transduced by the two adenovector components, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is deactivated. The control two-vector system, termed CTV, is similar to ETV but only expresses hGFP. In CHO-K1, BHK, and C2C12 cells, ETV and CTV induced a dose-dependent hGFP expression. In CHO-K1 cells, transgene expression was almost completely inhibited by DOX (1mg/ml). After intracerebroventricular injection of ETV in rats, thymulin levels increased significantly in the cerebrospinal fluid and there was high hGFP expression in the ependymal cell layer. When injected intramuscularly the ETV system induced a progressive increase in serum thymulin levels, which were inhibited when DOX was added to the drinking water. We conclude that our regulatable two-adenovector system is an effective molecular tool for implementing short and long-term anti-inflammatory thymulin gene therapy in animal models of acute or chronic inflammation.

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.

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.

Partial prevention of hepatic lipid alterations in nude mice by neonatal thymulin gene therapy.

During adult life athymic (nude) male mice display not only a severe T-cell-related immunodeficiency but also endocrine imbalances and a moderate hyperglycemia. We studied the impact of congenital athymia on hepatic lipid composition and also assessed the ability of neonatal thymulin gene therapy to prevent the effects of athymia. We constructed a recombinant adenoviral vector, RAd-metFTS, expressing a synthetic DNA sequence encoding met-FTS, an analog of the thymic peptide facteur thymique sérique (FTS), whose Zn-bound biologically active form is known as thymulin. On postnatal day 1-2 homozygous (nu/nu) nude and heterozygous (nu/+) mice were injected with 10(8) pfu of RAd-metFTS or RAd-betagal (control vector) intramuscularly. The animals were processed at 52 d of age. Serum thymulin, glycemia, hepatic phospholipid FA composition and free and esterified cholesterol were determined. Adult homozygous male nudes were significantly (P < 0.01) hyperglycemic when compared with their heterozygous counterparts (2.04 vs. 1.40 g/L, respectively). The relative percentage of 16:0, 18:1 n-9, and 18:1n-7 FA was lower, whereas that of 18:0, 20:4n-6, and 22:6n-3 FA was higher, in hepatic phospholipid (PL) of nu/nu animals as compared with their nu/+ counterparts. Some of these alterations, such as that in the relative content of 22:6n-3 in liver PL and the unsaturation index, were completely or partially prevented by neonatal thymulin gene therapy. We conclude that the thymus influences lipid metabolism and that thymulin is involved in this modulatory activity.

Regenerative Medicine for the Aging Brain.

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. In this context, cell reprogramming offers novel therapeutic possibilities for the treatment of these devastating diseases. In effect, the generation of induced pluripotent stem cells (iPSCs) from somatic cells demonstrated that adult mammalian cells can be reprogrammed to a pluripotent state by the overexpression of a few embryonic transcription factors (TF). This discovery fundamentally widened the research horizon in the fields of disease modeling and regenerative medicine. Although it is possible to re-differentiate iPSCs to specific somatic cell types, the tumorigenic potential of contaminating iPSCs that failed to differentiate, increases the risk for clinical application of somatic cells generated by this procedure. Therefore, reprogramming approaches that bypass the pluripotent stem cell state are being explored. A method called lineage reprogramming has been recently documented. It consists of the direct conversion of one adult cell type into another by transgenic expression of multiple lineage-specific TF or microRNAs. Another approach, termed direct reprogramming, features several advantages such as the use of universal TF system and the ability to generate a rejuvenated multipotent progenitor cell population, able to differentiate into specific cell types in response to a specific differentiation factors. These novel approaches offer 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 in the early stages of AD. The above topics are reviewed here.

Physiology and therapeutic potential of the thymic peptide thymulin.

Thymulin is a thymic hormone exclusively produced by the epithelial cells of the thymus. After its discovery and initial characterization in the '70s, it was demonstrated that the production and secretion of thymulin are strongly influenced by the neuro-endocrine system. Conversely, a growing body of evidence, to be reviewed here, suggests that thymulin is a hypophysiotropic peptide. Additionally, a substantial body of information pointing to thymulin and a synthetic analog as anti-inflammatory and analgesic peptides in the central nervous system brain and other organs will be also reviewed. In recent years, a synthetic DNA sequence encoding a biologically active analog of thymulin, metFTS, was constructed and cloned in a number of adenovectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be down-regulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies indicate that gene therapy for thymulin may be an effective therapeutic strategy to prevent some of the hormonal and reproductive abnormalities that typically appear in congenitally athymic (nude) mice, used as a suitable model of neuroendocrine and reproductive aging. Summing up, this article briefly reviews the publications on the physiology of the thymulin-neuroendocrine axis and the anti-inflammatory properties of the molecule and its analog. The availability of novel 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.

Magnetic field-assisted gene delivery: achievements and therapeutic potential.

The discovery in the early 2000's that magnetic nanoparticles (MNPs) complexed to nonviral or viral vectors can, in the presence of an external magnetic field, greatly enhance gene transfer into cells has raised much interest. This technique, called magnetofection, was initially developed mainly to improve gene transfer in cell cultures, a simpler and more easily controllable scenario than in vivo models. These studies provided evidence for some unique capabilities of magnetofection. Progressively, the interest in magnetofection expanded to its application in animal models and led to the association of this technique with another technology, magnetic drug targeting (MDT). This combination offers the possibility to develop more efficient and less invasive gene therapy strategies for a number of major pathologies like cancer, neurodegeneration and myocardial infarction. The goal of MDT is to concentrate MNPs functionalized with therapeutic drugs, in target areas of the body by means of properly focused external magnetic fields. The availability of stable, nontoxic MNP-gene vector complexes now offers the opportunity to develop magnetic gene targeting (MGT), a variant of MDT in which the gene coding for a therapeutic molecule, rather than the molecule itself, is delivered to a therapeutic target area in the body. This article will first outline the principle of magnetofection, subsequently describing the properties of the magnetic fields and MNPs used in this technique. Next, it will review the results achieved by magnetofection in cell cultures. Last, the potential of MGT for implementing minimally invasive gene therapy will be discussed.