Corticosteroids impair epithelial regeneration in immune-mediated intestinal damage.

Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal (GI) graft-versus-host disease (GVHD). CST is intended to target the immune system, but the glucocorticoid receptor (GR) is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids (CS) directly targeted intestinal epithelium, potentially worsening immune-mediated GI damage. CS administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory (SR) GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with SR, GR-deficient T cells or IFN-γ. These findings correlated with CS-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, IL-22 administration enhanced STAT3 activity and overcame CS-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing a rationale for clinical approaches combining such tissue-targeted therapies with immunosuppression.

A tissue-intrinsic IL-33/EGF circuit promotes epithelial regeneration after intestinal injury.

Intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but mechanisms regulating ISCs and their niche after damage remain poorly understood. Utilizing radiation injury to model intestinal pathology, we report here that the Interleukin-33 (IL-33)/ST2 axis, an immunomodulatory pathway monitored clinically as an intestinal injury biomarker, regulates intrinsic epithelial regeneration by inducing production of epidermal growth factor (EGF). Three-dimensional imaging and lineage-specific RiboTag induction within the stem cell compartment indicated that ISCs expressed IL-33 in response to radiation injury. Neighboring Paneth cells responded to IL-33 by augmenting production of EGF, which promoted ISC recovery and epithelial regeneration. These findings reveal an unknown pathway of niche regulation and crypt regeneration whereby the niche responds dynamically upon injury and the stem cells orchestrate regeneration by regulating their niche. This regenerative circuit also highlights the breadth of IL-33 activity beyond immunomodulation and the therapeutic potential of EGF administration for treatment of intestinal injury.

A phase 2 study of interleukin-22 and systemic corticosteroids as initial treatment for acute GVHD of the lower GI tract.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic transplantation. In experimental models, interleukin-22 promotes epithelial regeneration and induces innate antimicrobial molecules. We conducted a multicenter single-arm phase 2 study evaluating the safety and efficacy of a novel recombinant human interleukin-22 dimer, F-652, used in combination with systemic corticosteroids for treatment of newly diagnosed lower gastrointestinal acute GVHD. The most common adverse events were cytopenias and electrolyte abnormalities, and there were no dose-limiting toxicities. Out of 27 patients, 19 (70%; 80% confidence interval, 56%-79%) achieved a day-28 treatment response, meeting the prespecified primary endpoint. Responders exhibited a distinct fecal microbiota composition characterized by expansion of commensal anaerobes, which correlated with increased overall microbial α-diversity, suggesting improvement of GVHD-associated dysbiosis. This work demonstrates a potential approach for combining immunosuppression with tissue-supportive strategies to enhance recovery of damaged mucosa and promote microbial health in patients with gastrointestinal GVHD. This trial was registered at www.clinicaltrials.gov as NCT02406651.

The tissue protective functions of interleukin-22 can be decoupled from pro-inflammatory actions through structure-based design.

Interleukin-22 (IL-22) acts on epithelial cells to promote tissue protection and regeneration, but can also elicit pro-inflammatory effects, contributing to disease pathology. Here, we engineered a high-affinity IL-22 super-agonist that enabled the structure determination of the IL-22-IL-22Rα-IL-10Rß ternary complex to a resolution of 2.6 Å. Using structure-based design, we systematically destabilized the IL-22-IL-10Rß binding interface to create partial agonist analogs that decoupled downstream STAT1 and STAT3 signaling. The extent of STAT bias elicited by a single ligand varied across tissues, ranging from full STAT3-biased agonism to STAT1/3 antagonism, correlating with IL-10Rß expression levels. In vivo, this tissue-selective signaling drove tissue protection in the pancreas and gastrointestinal tract without inducing local or systemic inflammation, thereby uncoupling these opposing effects of IL-22 signaling. Our findings provide insight into the mechanisms underlying the cytokine pleiotropy and illustrate how differential receptor expression levels and STAT response thresholds can be synthetically exploited to endow pleiotropic cytokines with enhanced functional specificity.

T Cell Recruitment to the Intestinal Stem Cell Compartment Drives Immune-Mediated Intestinal Damage after Allogeneic Transplantation.

The key sites within the gastrointestinal (GI) tract where T cells mediate effector responses and the impact of these responses on intestinal stem cells (ISCs) remain unclear. Using experimental bone marrow transplantation to model immune-mediated GI damage and 3D imaging to analyze T cell localization, we found that the ISC compartment is the primary intestinal site targeted by T cells after transplantation. Recruitment to the crypt base region resulted in direct T cell engagement with the stem cell compartment and loss of crypt base columnar ISCs, which expressed both MHC classes I and II. Vasculature expressing the adhesion molecule MAdCAM-1 clustered near the crypt base, preferentially regulating crypt compartment invasion and ISC reduction without affecting T cell migration to villi. These findings indicate that allogeneic T cells rapidly access the stem cell niche after transplantation, and this targeted recruitment to the stem cell compartment results in ISC loss during immune-mediated GI damage.

Extrathymically Generated Regulatory T Cells Establish a Niche for Intestinal Border-Dwelling Bacteria and Affect Physiologic Metabolite Balance.

The mammalian gut microbiota provides essential metabolites to the host and promotes the differentiation and accumulation of extrathymically generated regulatory T (pTreg) cells. To explore the impact of these cells on intestinal microbial communities, we assessed the composition of the microbiota in pTreg cell-deficient and -sufficient mice. pTreg cell deficiency led to heightened type 2 immune responses triggered by microbial exposure, which disrupted the niche of border-dwelling bacteria early during colonization. Moreover, impaired pTreg cell generation led to pervasive changes in metabolite profiles, altered features of the intestinal epithelium, and reduced body weight in the presence of commensal microbes. Absence of a single species of bacteria depleted in pTreg cell-deficient animals, Mucispirillum schaedleri, partially accounted for the sequelae of pTreg cell deficiency. These observations suggest that pTreg cells modulate the metabolic function of the intestinal microbiota by restraining immune defense mechanisms that may disrupt a particular bacterial niche.

RIG-I/MAVS and STING signaling promote gut integrity during irradiation- and immune-mediated tissue injury.

The molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) during acute tissue damage are poorly understood. We describe a protective role for IFN-I and the RIG-I/MAVS signaling pathway during acute tissue damage in mice. Mice lacking mitochondrial antiviral-signaling protein (MAVS) were more sensitive to total body irradiation- and chemotherapy-induced intestinal barrier damage. These mice developed worse graft-versus-host disease (GVHD) in a preclinical model of allogeneic hematopoietic stem cell transplantation (allo-HSCT) than did wild-type mice. This phenotype was not associated with changes in the intestinal microbiota but was associated with reduced gut epithelial integrity. Conversely, targeted activation of the RIG-I pathway during tissue injury promoted gut barrier integrity and reduced GVHD. Recombinant IFN-I or IFN-I expression induced by RIG-I promoted growth of intestinal organoids in vitro and production of the antimicrobial peptide regenerating islet-derived protein 3 γ (RegIIIγ). Our findings were not confined to RIG-I/MAVS signaling because targeted engagement of the STING (stimulator of interferon genes) pathway also protected gut barrier function and reduced GVHD. Consistent with this, STING-deficient mice suffered worse GVHD after allo-HSCT than did wild-type mice. Overall, our data suggest that activation of either RIG-I/MAVS or STING pathways during acute intestinal tissue injury in mice resulted in IFN-I signaling that maintained gut epithelial barrier integrity and reduced GVHD severity. Targeting these pathways may help to prevent acute intestinal injury and GVHD during allogeneic transplantation.

Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease.

The effect of alterations in intestinal microbiota on microbial metabolites and on disease processes such as graft-versus-host disease (GVHD) is not known. Here we carried out an unbiased analysis to identify previously unidentified alterations in gastrointestinal microbiota-derived short-chain fatty acids (SCFAs) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amount of only one SCFA, butyrate, were observed only in the intestinal tissue. The reduced butyrate in CD326(+) intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored after local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate-producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate disease severity.

Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.

DNA polymerase-ß mediates the neurogenic effect of ß-amyloid protein in cultured subventricular zone neurospheres.

ß-Amyloid protein (Aß) is thought to be responsible for neuronal apoptosis in Alzheimer's disease (AD). Paradoxically, Aß can also promote neurogenesis, both in vitro and in vivo, by inducing neural progenitor cells (NPCs) to differentiate into neurons. However, the mechanisms of Aß-induced neurogenesis are unknown. Here we examined the role of DNA polymerase-ß (DNA pol-ß), a DNA repair enzyme that is required for proper neurogenesis during brain development and is also responsible for Aß-induced neuronal apoptosis. In neurospheres obtained from the adult mouse subventricular zone (SVZ), the knockdown of DNA pol-ß or its pharmacological blockade showed that the enzyme functioned both to repress proliferation of early nestin(+) progenitor cells and to promote the maturation of TuJ-1(+) neuronal cells. In neurospheres challenged with oligomers of synthetic Aß(42) , the expression levels of DNA pol-ß were rapidly increased. DNA pol-ß knockdown prevented the Aß(42) -promoted differentiation of nestin(+) progenitor cells into nestin(+) /Dlx-2(+) neuroblasts. Moreover, when neurospheres were seeded to allow full differentiation of their elements, blockade of DNA pol-ß prevented Aß(42) -induced differentiation of progenitors into MAP-2(+) neurons. Thus, our data demonstrate that Aß(42) arrests the proliferation of a subpopulation of nestin(+) cells via the induction of DNA pol-ß, thereby allowing for their differentiation toward the neuronal lineage. Our findings reveal a novel role of DNA pol-ß in Aß(42) -induced neurogenesis and identify DNA pol-ß as a key mechanistic link between the neurogenic effect of Aß(42) on NPCs and the proapoptotic effect of Aß(42) on mature neurons.

Electrically guiding migration of human induced pluripotent stem cells.

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small, physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode, with a stimulation threshold of <30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary, whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF, while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition, a method to aid expansion and survival of stem cells, significantly increased the motility, but reduced directionality of iPS cells in an EF by 70-80%. Thus, our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells.

Apoptosis inducing factor deficiency causes reduced mitofusion 1 expression and patterned Purkinje cell degeneration.

Alteration in mitochondrial dynamics has been implicated in many neurodegenerative diseases. Mitochondrial apoptosis inducing factor (AIF) plays a key role in multiple cellular and disease processes. Using immunoblotting and flow cytometry analysis with Harlequin mutant mice that have a proviral insertion in the AIF gene, we first revealed that mitofusion 1 (Mfn1), a key mitochondrial fusion protein, is significantly diminished in Purkinje cells of the Harlequin cerebellum. Next, we investigated the cerebellar pathology of Harlequin mice in an age-dependent fashion, and identified a striking process of progressive and patterned Purkinje cell degeneration. Using immunohistochemistry with zebrin II, the most studied compartmentalization marker in the cerebellum, we found that zebrin II-negative Purkinje cells first started to degenerate at 7 months of age. By 11 months of age, almost half of the Purkinje cells were degenerated. Subsequently, most of the Purkinje cells disappeared in the Harlequin cerebellum. The surviving Purkinje cells were concentrated in cerebellar lobules IX and X, where these cells were positive for heat shock protein 25 and resistant to degeneration. We further showed that the patterned Purkinje cell degeneration was dependent on caspase but not poly(ADP-ribose) polymerase-1 (PARP-1) activation, and confirmed the marked decrease of Mfn1 in the Harlequin cerebellum. Our results identified a previously unrecognized role of AIF in Purkinje cell degeneration, and revealed that AIF deficiency leads to altered mitochondrial fusion and caspase-dependent cerebellar Purkinje cell loss in Harlequin mice. This study is the first to link AIF and mitochondrial fusion, both of which might play important roles in neurodegeneration.

Does microglial dysfunction play a role in autism and Rett syndrome?

Autism spectrum disorders (ASDs) including classic autism is a group of complex developmental disabilities with core deficits of impaired social interactions, communication difficulties and repetitive behaviors. Although the neurobiology of ASDs has attracted much attention in the last two decades, the role of microglia has been ignored. Existing data are focused on their recognized role in neuroinflammation, which only covers a small part of the pathological repertoire of microglia. This review highlights recent findings on the broader roles of microglia, including their active surveillance of brain microenvironments and regulation of synaptic connectivity, maturation of brain circuitry and neurogenesis. Emerging evidence suggests that microglia respond to pre- and postnatal environmental stimuli through epigenetic interface to change gene expression, thus acting as effectors of experience-dependent synaptic plasticity. Impairments of these microglial functions could substantially contribute to several major etiological factors of autism, such as environmental toxins and cortical underconnectivity. Our recent study on Rett syndrome, a syndromic autistic disorder, provides an example that intrinsic microglial dysfunction due to genetic and epigenetic aberrations could detrimentally affect the developmental trajectory without evoking neuroinflammation. We propose that ASDs provide excellent opportunities to study the influence of microglia on neurodevelopment, and this knowledge could lead to novel therapies.

Group I metabotropic glutamate receptors control proliferation, survival and differentiation of cultured neural progenitor cells isolated from the subventricular zone of adult mice.

Neural progenitor cells (NPCs) are found in the subventricular zone (SVZ) of the adult brain, a specialized neurogenic niche that might provide a substrate for brain repair after injury. The incomplete knowledge of how NPCs in the niche respond to local signals limits the use of cultured NPCs in the development of cell transplantation strategies. We show that neurospheres obtained from the SVZ of the adult mouse expressed functional mGlu1 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of mGlu5 receptors promoted the apoptotic death of progenitors undergoing differentiation into neurons (PSA/NCAM+ cells for the most part), whereas blockade of mGlu1 receptors reduced the proliferation rate of NPCs, and promoted their differentiation towards the neuronal lineage. We conclude that endogenous activation of mGlu5 receptors might support specifically the survival of neuronal-restricted precursors, whereas endogenous activation of mGlu1 receptors might sustain the proliferation of earlier progenitors. Moreover, mGlu1 receptor antagonists increased the survival of NPCs, suggesting that endogenously activated mGlu1 receptors might play a role in the natural cell loss regulating the number or the type of progenitors.

TGF-beta1 targets the GSK-3beta/beta-catenin pathway via ERK activation in the transition of human lung fibroblasts into myofibroblasts.

Transforming growth factor-beta1 (TGF-beta1) is known to induce the transition of human lung fibroblasts to myofibroblasts, a primary event in the pathogenesis of idiopathic pulmonary fibrosis. The molecular pathways involved in myofibroblast transformation are only partially identified. We found that a 24-h treatment with TGF-beta1 (10 ng/ml) induced alpha-smooth actin (SMA) expression and collagen production in human lung fibroblasts. These effects were abrogated by PD98059, a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway. TGF-beta1 treatment activated the MAPK pathway, as shown by an increased phosphorylation of extracellular-regulated kinases (ERK)1/2 after 30 min of exposure. TGF-beta1 also increased the expression of the Ser-9-phosphorylated inactive form of glycogen synthase kinase-3beta (GSK-3beta), an effect that was largely attenuated by PD98059. A nuclear translocation of beta-catenin in human lung fibroblasts was observed 2h after TGF-beta1 addition both by confocal microscopy and nuclear protein analysis. At this time, TGF-beta1 also increased the total levels of beta-catenin, an effect that was prevented by PD98059. Similarly to TGF-beta1, the GSK-3beta inhibitor lithium chloride (10mM), increased the total levels of beta-catenin and promoted alpha-SMA expression and collagen production. This study demonstrates that TGF-beta1 induces alpha-SMA expression and collagen production in human lung fibroblasts via ERK1/2 activation, GSK-3beta inhibition and nuclear beta-catenin translocation. The evidence that the silencing of beta-catenin by siRNAs was able to prevent the induction of alpha-SMA expression in TGF-beta1-treated fibroblasts further supports the hypothesis of a contribution of the GSK-3beta/beta-catenin pathway in the pathogenesis of idiopathic pulmonary fibrosis.

Astrocyte-like cells as a main target for estrogen action during neuronal differentiation.

Neurospheres from the subventricular zone of adult mice were used as an experimental model to analyse the early differential effects of 17beta-estradiol (17beta-E2). Both floating and differentiating neurospheres expressed estrogen receptors (ERs) alpha and beta. The initial phases of differentiation coincided with a peak of ERalpha expression as by Western blot analysis. Treatment with 10 nM 17beta-E2 induced a significant increase in the glial fibrillary acidic protein (GFAP)-positive population and a greater expression of GFAP, an effect sensitive to the estrogen receptor antagonist ICI 182,780. The GFAP-positive cell population induced by 17beta-E2 was characterized by a highly differentiated phenotype and intense immunostaining as by immunocytochemistry and flow cytometry. These cells co-expressed ERalpha and were positive to BrdU. 17beta-E2 also affected neuronal differentiation by rapidly and transiently increasing the percentage of polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive progenitors, and by accelerating the appearance of a mature neuronal phenotype, as evaluated by microtubule-associated protein 2 (MAP2) staining. Our results point to a key role for ERalpha during initial phases of differentiation of brain cells and to an effect of 17beta-E2 that sequentially involves both glia and neurons.

The nature of the cell cycle in neurons: focus on a "non-canonical" pathway of DNA replication causally related to death.

The mechanism whereby a reactivation of cell cycle in neurons causes cell death is beginning to be identified. In cellular models of Alzheimer's disease, activation of a non-canonical pathway of DNA replication contributes to neuronal death. This pathway involves the repair enzyme DNA polymerase-beta, which is highly expressed in neurons of the Alzheimer's brain at early stages of the disease. Loading of DNA polymerase-beta into the replication forks generates a death signal, which involves the tumor suppressor p53. The increasing knowledge of the main actors of the unscheduled DNA replication in neurons will pave the way for novel therapeutic interventions in Alzheimer's disease and other neurodegenerative disorders.

DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid.

Cultured neurons exposed to synthetic beta-amyloid (Abeta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments on cross-linked nucleoprotein fragments from Abeta-treated neurons, we demonstrate that DNA pol-beta coimmunoprecipitates with cell division cycle 45 (Cdc45) and with DNA primase in short nucleoprotein fragments. This indicates that DNA pol-beta is loaded into neuronal DNA replication forks after Abeta treatment. In response to Abeta the canonical DNA-synthesizing enzyme DNA pol-delta also was loaded into neuronal replication forks, but at later times than DNA pol-beta. Methoxyamine, an inhibitor of the apurinic/apyrimidinic endonuclease that allows for the recruitment of DNA pol-beta during the process of base excision repair (BER), failed to affect coimmunoprecipitation between DNA pol-beta and Cdc45, indicating that DNA pol-beta loading to the replication forks is independent of DNA breaks. However, methoxyamine reduced DNA replication and ensuing apoptosis in neurons exposed to Abeta, suggesting that an efficient BER process allows DNA replication to proceed up to the threshold for death. These data demonstrate that DNA pol-beta is an essential component of the DNA replication machinery in Abeta-treated neurons and additionally support the hypothesis of a close association of cell cycle events with neuronal death in Alzheimer's disease (AD). Accordingly, by investigating the neuronal expression of DNA pol-beta, along with phosphorylated retinoblastoma protein and neurofibrillary changes in AD brain, we show an early involvement of DNA pol-beta in the pathogenesis of AD.

Nicergoline, a drug used for age-dependent cognitive impairment, protects cultured neurons against beta-amyloid toxicity.

Nicergoline, a drug used for the treatment of Alzheimer's disease and other types of dementia, was tested for its ability to protect neurons against beta-amyloid toxicity. Pure cultures of rat cortical neurons were challenged with a toxic fragment of beta-amyloid peptide (betaAP(25-35)) and toxicity was assessed after 24 h. Micromolar concentrations of nicergoline or its metabolite, MDL, attenuated betaAP(25-35)-induced neuronal death, whereas MMDL (another metabolite of nicergoline), the alpha1-adrenergic receptor antagonist, prazosin, or the serotonin 5HT-2 receptor antagonist, methysergide, were inactive. Nicergoline increased the basal levels of Bcl-2 and reduced the increase in Bax levels induced by beta-amyloid, indicating that the drug inhibits the execution of an apoptotic program in cortical neurons. In mixed cultures of rat cortical cells containing both neurons and astrocytes, nicergoline and MDL were more efficacious than in pure neuronal cultures in reducing beta-amyloid neurotoxicity. Experiments carried out in pure cultures of astrocytes showed that a component of neuroprotection was mediated by a mechanism of glial-neuronal interaction. The conditioned medium of cultured astrocytes treated with nicergoline or MDL for 72-96 h (collected 24 h after drug withdrawal) was neuroprotective when transferred to pure neuronal cultures challenged with beta-amyloid. In cultured astrocytes, nicergoline increased the intracellular levels of transforming-growth factor-beta and glial-derived neurotrophic factor, two trophic factors that are known to protect neurons against beta-amyloid toxicity. These results raise the possibility that nicergoline reduces neurodegeneration in the Alzheimer's brain.