Sunitinib-mediated inhibition of STAT3 in skeletal muscle and spinal cord does not affect the disease in a mouse model of ALS.

Variability in disease onset and progression is a hallmark of amyotrophic lateral sclerosis (ALS), both in sporadic and genetic forms. Recently, we found that SOD1-G93A transgenic mice expressing the same amount of mutant SOD1 but with different genetic backgrounds, C57BL/6JOlaHsd and 129S2/SvHsd, show slow and rapid muscle wasting and disease progression, respectively. Here, we investigated the different molecular mechanisms underlying muscle atrophy. Although both strains showed similar denervation-induced degradation of muscle proteins, only the rapidly progressing mice exhibited early and sustained STAT3 activation that preceded atrophy in gastrocnemius muscle. We therefore investigated the therapeutic potential of sunitinib, a tyrosine kinase inhibitor known to inhibit STAT3 and prevent cancer-induced muscle wasting. Although sunitinib treatment reduced STAT3 activation in the gastrocnemius muscle and lumbar spinal cord, it did not preserve spinal motor neurons, improve neuromuscular impairment, muscle atrophy and disease progression in the rapidly progressing SOD1-G93A mice. Thus, the effect of sunitinib is not equally positive in different diseases associated with muscle wasting. Moreover, given the complex role of STAT3 in the peripheral and central compartments of the neuromuscular system, the present study suggests that its broad inhibition may lead to opposing effects, ultimately preventing a potential positive therapeutic action in ALS.

HSPB8 frameshift mutant aggregates weaken chaperone-assisted selective autophagy in neuromyopathies.

Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases.Abbreviations : ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.

Identification of HSPB8 modulators counteracting misfolded protein accumulation in neurodegenerative diseases.

AIMS: The small Heat Shock Protein B8 (HSPB8) is the core component of the Chaperone-Assisted Selective Autophagy (CASA) complex. This complex selectively targets, transports, and tags misfolded proteins for their recognition by autophagy receptors and insertion into the autophagosome for clearance. CASA is essential to maintain intracellular proteostasis, especially in heart, muscle, and brain often exposed to various types of cell stresses. In neurons, HSPB8 protects against neurotoxicity caused by misfolded proteins in several models of neurodegenerative diseases; by facilitating autophagy, HSPB8 assists misfolded proteins degradation also counteracting proteasome overwhelming and inhibition. MATERIALS AND METHODS: To enhance HSPB8 protective activity, we screened a library of approximately 120,000 small molecules to identify compounds capable of increasing HSPB8 gene transcription, translation, or protein stability. KEY FINDINGS: We found 83 active compounds active in preliminary dose-response assays and further classified them in 19 chemical classes by medicinal chemists' visual inspection. Of these 19 prototypes, 14 induced HSPB8 mRNA and protein levels in SH-SY5Y cells. Out of these 14 compounds, 3 successfully reduced the aggregation propensity of a disease-associated mutant misfolded superoxide dismutase 1 (SOD1) protein in a flow cytometry-based aggregation assay (Flow cytometric analysis of Inclusions and Trafficking (FloIT)) and induced the expression (mRNA and protein) of some autophagy receptors. Notably, the 3 hits were inactive in HSPB8-depleted cells, confirming that their protective activity is mediated by and requires HSPB8. SIGNIFICANCE: These compounds may be highly relevant for a therapeutic approach in several human disorders, including neurodegenerative diseases, in which enhancement of CASA exerts beneficial activities.

Pathogenic variants of Valosin-containing protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells.

AIM: Mutations in the valosin-containing protein (VCP) gene cause various lethal proteinopathies that mainly include inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). Different pathological mechanisms have been proposed. Here, we define the impact of VCP mutants on lysosomes and how cellular homeostasis is restored by inducing autophagy in the presence of lysosomal damage. METHODS: By electron microscopy, we studied lysosomal morphology in VCP animal and motoneuronal models. With the use of western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence and filter trap assay, we evaluated the effect of selected VCP mutants in neuronal cells on lysosome size and activity, lysosomal membrane permeabilization and their impact on autophagy. RESULTS: We found that VCP mutants induce the formation of aberrant multilamellar organelles in VCP animal and cell models similar to those found in patients with VCP mutations or with lysosomal storage disorders. In neuronal cells, we found altered lysosomal activity characterised by membrane permeabilization with galectin-3 redistribution and activation of PPP3CB. This selectively activated the autophagy/lysosomal transcriptional regulator TFE3, but not TFEB, and enhanced both SQSTM1/p62 and lipidated MAP1LC3B levels inducing autophagy. Moreover, we found that wild type VCP, but not the mutants, counteracted lysosomal damage induced either by trehalose or by a mutant form of SOD1 (G93A), also blocking the formation of its insoluble intracellular aggregates. Thus, chronic activation of autophagy might fuel the formation of multilamellar bodies. CONCLUSION: Together, our findings provide insights into the pathogenesis of VCP-related diseases, by proposing a novel mechanism of multilamellar body formation induced by VCP mutants that involves lysosomal damage and induction of lysophagy.

Retinoic Acid Downregulates HSPB8 Gene Expression in Human Breast Cancer Cells MCF-7.

Breast cancer (BC) is a serious and widespread disease for which different treatments have been developed. In addition to the classic therapies, the treatment with retinoic acid (RA) is still being clinically investigated. RA reduces cancer cells proliferation and migration, but its molecular mechanism of action is not clear. In tumor development, autophagy promotes cancer cell survival and prevents apoptosis. Small heat shock protein B8 (HSPB8) acts together with its co-chaperone BCL-2 associated athanogene 3 (BAG3) stimulating BC proliferation and migration. We analyzed whether direct correlations exist between RA and HSPB8 or BAG3 and how this may play a role in BC. We measured HSPB8 and BAG3 gene expression in MCF-7 BC cells and we analyzed the potential correlation between the antiproliferative and antimigratory effect of RA with the expression level of HSPB8. We found that in MCF-7 cells RA reduces both HSPB8 and BAG3 gene expression and it alters the mitotic spindle organization. Notably, the effects of RA on HSPB8 levels are exerted at both transcriptional and translational levels. RA effects are possibly mediated by miR-574-5p that targets the HSPB8 transcript. Our results suggest that therapeutic doses of RA can efficiently counteract the adverse effects of HSPB8 in BC progression.

Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration.

Macroautophagy/autophagy, a defense mechanism against aberrant stresses, in neurons counteracts aggregate-prone misfolded protein toxicity. Autophagy induction might be beneficial in neurodegenerative diseases (NDs). The natural compound trehalose promotes autophagy via TFEB (transcription factor EB), ameliorating disease phenotype in multiple ND models, but its mechanism is still obscure. We demonstrated that trehalose regulates autophagy by inducing rapid and transient lysosomal enlargement and membrane permeabilization (LMP). This effect correlated with the calcium-dependent phosphatase PPP3/calcineurin activation, TFEB dephosphorylation and nuclear translocation. Trehalose upregulated genes for the TFEB target and regulator Ppargc1a, lysosomal hydrolases and membrane proteins (Ctsb, Gla, Lamp2a, Mcoln1, Tpp1) and several autophagy-related components (Becn1, Atg10, Atg12, Sqstm1/p62, Map1lc3b, Hspb8 and Bag3) mostly in a PPP3- and TFEB-dependent manner. TFEB silencing counteracted the trehalose pro-degradative activity on misfolded protein causative of motoneuron diseases. Similar effects were exerted by trehalase-resistant trehalose analogs, melibiose and lactulose. Thus, limited lysosomal damage might induce autophagy, perhaps as a compensatory mechanism, a process that is beneficial to counteract neurodegeneration. Abbreviations: ALS: amyotrophic lateral sclerosis; AR: androgen receptor; ATG: autophagy related; AV: autophagic vacuole; BAG3: BCL2-associated athanogene 3; BECN1: beclin 1, autophagy related; CASA: chaperone-assisted selective autophagy; CTSB: cathepsin b; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; EGFP: enhanced green fluorescent protein; fALS, familial amyotrophic lateral sclerosis; FRA: filter retardation assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLA: galactosidase, alpha; HD: Huntington disease; hIPSCs: human induced pluripotent stem cells; HSPA8: heat shock protein A8; HSPB8: heat shock protein B8; IF: immunofluorescence analysis; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LGALS3: lectin, galactose binding, soluble 3; LLOMe: L-leucyl-L-leucine methyl ester; LMP: lysosomal membrane permeabilization; Lys: lysosomes; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NDs: neurodegenerative diseases; NSC34: neuroblastoma x spinal cord 34; PBS: phosphate-buffered saline; PD: Parkinson disease; polyQ: polyglutamine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform; RT-qPCR: real-time quantitative polymerase chain reaction; SBMA: spinal and bulbar muscular atrophy; SCAs: spinocerebellar ataxias; siRNA: small interfering RNA; SLC2A8: solute carrier family 2, (facilitated glucose transporter), member 8; smNPCs: small molecules neural progenitors cells; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STUB1: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TFEB: transcription factor EB; TPP1: tripeptidyl peptidase I; TREH: trehalase (brush-border membrane glycoprotein); WB: western blotting; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.

Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases.

Motoneuron diseases, like spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS), are associated with proteins that because of gene mutation or peculiar structures, acquire aberrant (misfolded) conformations toxic to cells. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Here, using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrate that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome, rather than by autophagy and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner.

The small heat shock protein B8 (HSPB8) modulates proliferation and migration of breast cancer cells.

Breast cancer (BC) is one of the major causes of cancer death in women and is closely related to hormonal dysregulation. Estrogen receptor (ER)-positive BCs are generally treated with anti hormone therapy using antiestrogens or aromatase inhibitors. However, BC cells may become resistant to endocrine therapy, a process facilitated by autophagy, which may either promote or suppress tumor expansion. The autophagy facilitator HSPB8 has been found overexpressed in some BC. Here we found that HSPB8 is highly expressed and differentially modulated by natural or synthetic selective ER modulators (SERMs), in the triple-positive hormone-sensitive BC (MCF-7) cells, but not in triple-negative MDA-MB-231 BC cells. Specific SERMs induced MCF-7 cells proliferation in a HSPB8 dependent manner whereas, did not modify MDA-MB-231 cell growth. ER expression was unaffected in HSPB8-depleted MCF-7 cells. HSPB8 over-expression did not alter the distribution of MCF-7 cells in the various phases of the cell cycle. Conversely and intriguingly, HSPB8 downregulation resulted in an increased number of cells resting in the G0/G1 phase, thus possibly reducing the ability of the cells to pass through the restriction point. In addition, HSPB8 downregulation reduced the migratory ability of MCF-7 cells. None of these modifications were observed, when another small HSP (HSPB1), also expressed in MCF-7 cells, was downregulated. In conclusion, our data suggest that HSPB8 is involved in the mechanisms that regulate cell cycle and cell migration in MCF-7 cells.

The Role of the Protein Quality Control System in SBMA.

Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked disease associated with the expansion of the CAG triplet repeat present in exon 1 of the androgen receptor (AR) gene. This results in the production of a mutant AR containing an elongated polyglutamine tract (polyQ) in its N-terminus. Interestingly, the ARpolyQ becomes toxic only after its activation by the natural androgenic ligands, possibly because of aberrant androgen-induced conformational changes of the ARpolyQ, which generate misfolded species. These misfolded ARpolyQ species must be cleared from motoneurons and muscle cells, and this process is mediated by the protein quality control (PQC) system. Experimental evidence suggested that failure of the PQC pathways occurs in disease, leading to ARpolyQ accumulation and toxicity in the target cells. In this review, we summarized the overall impact of mutant and misfolded ARpolyQ on the PQC system and described how molecular chaperones and the degradative pathways (ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), and the unfolded protein response (UPR), which activates the endoplasmic reticulum-associated degradation (ERAD)) are differentially affected in SBMA. We also extensively and critically reviewed several molecular and pharmacological approaches proposed to restore a global intracellular activity of the PQC system. Collectively, these data suggest that the fine and delicate equilibrium existing among the different players of the PQC system could be restored in a therapeutic perspective by the synergic/additive activities of compounds designed to tackle sequential or alternative steps of the intracellular defense mechanisms triggered against proteotoxic misfolded species.

Synergic prodegradative activity of Bicalutamide and trehalose on the mutant androgen receptor responsible for spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease due to a CAG triplet-repeat expansion in the androgen receptor (AR) gene, which is translated into an elongated polyglutamine (polyQ) tract in AR protein (ARpolyQ). ARpolyQ toxicity is activated by the AR ligand testosterone (or dihydrotestosterone), and the polyQ triggers ARpolyQ misfolding and aggregation in spinal cord motoneurons and muscle cells. In motoneurons, testosterone triggers nuclear toxicity by inducing AR nuclear translocation. Thus, (i) prevention of ARpolyQ nuclear localization, combined with (ii) an increased ARpolyQ cytoplasmic clearance, should reduce its detrimental activity. Using the antiandrogen Bicalutamide (Casodex(®)), which slows down AR activation and nuclear translocation, and the disaccharide trehalose, an autophagy activator, we found that, in motoneurons, the two compounds together reduced ARpolyQ insoluble forms with higher efficiency than that obtained with single treatments. The ARpolyQ clearance was mediated by trehalose-induced autophagy combined with the longer cytoplasmic retention of ARpolyQ bound to Bicalutamide. This allows an increased recognition of misfolded species by the autophagic system prior to their migration into the nucleus. Interestingly, the combinatory use of trehalose and Bicalutamide was also efficient in the removal of insoluble species of AR with a very long polyQ (Q112) tract, which typically aggregates into the cell nuclei. Collectively, these data suggest that the combinatory use of Bicalutamide and trehalose is a novel approach to facilitate ARpolyQ clearance that has to be tested in other cell types target of SBMA (i.e. muscle cells) and in vivo in animal models of SBMA.

Proteasomal and autophagic degradative activities in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA or Kennedy's disease) is a fatal neurodegenerative disease characterized by the selective loss of motor neurons in the bulbar region of the brain and in the anterior horns of the spinal cord. The disease has been associated to an expansion of a CAG triplet repeat present in the first coding exon of the androgen receptor (AR) gene. SBMA was the first identified member of a large class of neurodegenerative diseases now known as CAG-related diseases, which includes Huntington's disease (HD), several types of spinocerebellar ataxia (SCAs), and dentatorubral and pallidoluysian atrophy (DRPLA). The expanded CAG tract is translated to an aberrantly long polyglutamine tract (ARpolyQ) in the N-terminal region of the AR protein. The elongated polyQ tract seems to confer a neurotoxic gain-of-function to the mutant AR, possibly via the generation of aberrant conformations (misfolding). Protein misfolding is thought to be a trigger of neurotoxicity, since it perturbs a wide variety of motor neuronal functions. The first event is the accumulation of the ARpolyQ into ubiquitinated aggregates in a ligand (testosterone) dependent manner. The mutant ARpolyQ also impairs proteasome functions. The autophagic pathway may be activated to compensate these aberrant events by clearing the mutant ARpolyQ from motor neuronal cells. This review illustrates the mechanisms at the basis of ARpolyQ degradation via the proteasomal and autophagic systems.

Gangliosides influence EGFR/ErbB2 heterodimer stability but they do not modify EGF-dependent ErbB2 phosphorylation.

Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM(3) to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/-)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and beta-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM(3) have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM(3) added in combination with [D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, beta-casein gene expression appeared strongly down-regulated, and beta-casein mRNA levels were partially restored by exogenous GM(3) treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM(3) as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.

Neuritin (cpg15) enhances the differentiating effect of NGF on neuronal PC12 cells.

Neuritin is a small, highly conserved GPI-anchored protein involved in neurite outgrowth. We have analyzed the involvement of neuritin in NGF-induced differentiation of PC12 cells by investigating the time-course of neuritin expression, the effects of its overexpression or silencing, and the possible mechanisms of its regulation and action. Real-time PCR analysis has shown that neuritin gene is upregulated by NGF in PC12 cells hours before neurite outgrowth becomes appreciable. PC12 cells transfected with a plasmid expressing neuritin display a significant increase in the response to NGF: 1) in the levels of SMI312 positive phosphorylated neurofilament proteins (markers for axonal processes) and tyrosine hydroxylase; 2) in the percentage of cells bearing neurites; as well as 3) in the average length of neurites when compared to control cells. On the contrary, neuritin silencing significantly reduces neurite outgrowth. These data suggest that neuritin is a modulator of NGF-induced neurite extension in PC12 cells. We also showed that neuritin potentiated the NGF-induced differentiation of PC12 cells without affecting TrkA or EGF receptor mRNAs expression. Moreover, the S-methylisothiourea (MIU), a potent inhibitor of inducible nitric oxide synthases, partially counteracts the NGF-mediated neuritin induction. These data suggest that NGF regulates neuritin expression in PC12 cells via the signaling pathway triggered by NO. This study reports the first evidence that neuritin plays a role in modulating neurite outgrowth during the progression of NGF-induced differentiation of PC12 cells. PC12 cells could be considered a valuable model to unravel the mechanism of action of neuritin on neurite outgrowth. (c) 2007 Wiley-Liss, Inc.

Effects of progesterone derivatives, dihydroprogesterone and tetrahydroprogesterone, on the subependymal layer of the adult rat.

Indirect evidence suggests that in the subependymal layer (SEL) steroid hormones could be partially involved in the modulation of neurogenesis, but little or nothing is known about a direct effect of these molecules on this cellular system. The possible effect of progesterone (P) and/or its neuroactive metabolites, dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), on the two cellular components of the SEL (i.e., proliferating/migrating neuroblasts and protoplasmic astrocytes) has been analyzed in adult male rat. P, DHP, and THP were administered by intraventricular injections and after 2 days the SEL was analyzed by immunohistochemistry by using anti-glial fibrillary acidic protein (GFAP) and anti-vimentin antibodies, to label the glial compartment, anti-polysialylated form of the neural cell adhesion molecule (PSA-NCAM), anti-Stathmin, and anti-beta III Tubulin antibodies to label the migrating neuroblasts. Furthermore, the newly formed cells were identified by using intraventricular injections of 5-bromo-2'-deoxyuridine (BrdU) detected immunohistochemically. Our results demonstrate that DHP and THP treatments drastically decrease the number of BrdU-labeled cells within the SEL. THP, DHP, and to a lesser extent P, administrations also induce molecular and structural modifications of the SEL glial compartment. On the whole, the present results indicate that neuroactive derivatives of P (i.e., DHP and THP) exert direct effects on adult neurogenesis, strongly affecting both neuroblasts and astrocytes of the SEL.

Steroid hormones and growth factors act in an integrated manner at the levels of hypothalamic astrocytes: a role in the neuroendocrine control of reproduction.

Several growth factors (e.g., transforming growth factors beta and alpha, basic fibroblast growth factor), produced by hypothalamic astrocytes, participate in the control of hypothalamic gonadotrophin-releasing hormone (GnRH) neurons. On this basis, we have hypothesized that steroid hormones, like estrogens and progestagens, influence the GnRH neurons by modulating in glial cells the synthesis and the release of these growth factors. Data reported here indicate that the expression of transforming growth factor beta 1 is modulated in hypothalamic astrocytes by a progesterone derivative (i.e., dihydroprogesterone), while estrogens modulate that of basic fibroblast growth factor. Moreover, it is interesting to highlight that the effect of estrogens on basic fibroblast growth factor is mediated by another growth factor (i.e., transforming growth factor alpha). Altogether, the present findings support the concept that steroid hormones and growth factors act in an integrated manner at the level of hypothalamic astrocytes, thus adding a further piece of knowledge in the understanding of the mechanisms controlling GnRH neurons.

Neurogenesis in the subependymal layer of the adult rat: a role for neuroactive derivatives of progesterone.

The subependymal layer (SEL) of the adult mammalian brain provides a continuous supply of newborn cells that migrate to the olfactory bulb (OB) where they differentiate into interneurons. These newly generated cells migrate tangentially to the OB within a dense meshwork of astrocytic cells, organized to form tangentially oriented channels (glial tubes). The central nervous system is able to synthesize a variety of steroids. Among these, we analyzed the effects of progesterone (P) and its neuroactive metabolites dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), administered by intraventricular injection, on the SEL of the adult rat. We found that THP and DHP, but not their precursor P, modify glial tubes organization and decrease immunoreactivity for glial associated proteins in SEL astrocytes. Moreover P metabolites reduce the proliferative activity within the SEL.

Growth factors and steroid hormones: a complex interplay in the hypothalamic control of reproductive functions.

The mechanisms through which LHRH-secreting neurons are controlled still represent a crucial and debated field of research in the neuroendocrine control of reproduction. In the present review, we have specifically considered two potential signals reaching these hypothalamic neurons: steroid hormones and growth factors. Examples of the relevant physiological role of the interactions between these two families of biologically acting molecules have been provided. In many cases, these interactions occur at the level of hypothalamic astrocytes, which are presently accepted as functional partners of the LHRH-secreting neurons. On the basis of the observations here summarized, we have formulated the hypothesis that a functional co-operation of steroid hormones and growth factors occurring in the hypothalamic astrocytic compartment represents a key factor in the neuroendocrine control of reproductive functions.