TGF-beta1 inhibits caspase-3 activation and neuronal apoptosis in rat hippocampal cultures

The effect of TGF-beta1 on apoptosis varies depending on the cell type, the kind of stimulus and the experimental conditions. The present study attempted to identify whether TGF-beta1 can prevent neuronal apoptosis and interrupt caspase-3 activation in rat primary hippocampal cultures after staurosporine treatment. TGF-beta1 at the concentration of 1 and 10 ng/ml significantly reduced neuronal damage as detected by trypan blue exclusion. Nuclear staining with Hoechst 33258 and TUNEL-staining further demonstrated that TGF-beta1 at the same concentration range effectively diminished neuronal apoptosis 24 h after staurosporine treatment, whereas 0.1 ng/ml of TGF-beta1 did not. Furthermore, TGF-beta1 (1 and 10 ng/ml) markedly inhibited the activation of caspase-3 induced by staurosporine as demonstrated by both caspase-3 activity assay and Western blotting. This study provides evidence that TGF-beta1 is able to efficiently inhibit caspase-3 activation, and thereby protects cultured hippocampal neurons against apoptosis.     

Transforming growth factor-beta1 as a regulator of the serpins/t-PA axis in cerebral ischemia.

The tissue type plasminogen activator (t-PA) is a serine protease that is involved in neuronal plasticity and cell death induced by excitotoxins and ischemia in the brain. t-PA activity in the central nervous system is regulated through the activation of serine protease inhibitors (serpins) such as the plasminogen activator inhibitor (PAI-1), the protease nexin-1 (PN-1), and neuroserpin (NSP). Recently we demonstrated in vitro that PAI-1 produced by astrocytes mediates the neuroprotective effect of the transforming growth factor-beta1 (TGF-beta1) in NMDA-induced neuronal cell death. To investigate whether serpins may be involved in neuronal cell death after cerebral ischemia, we determined, by using semiquantitative RT-PCR and in situ hybridization, that focal cerebral ischemia in mice induced a dramatic overexpression of PAI-1 without any effect on PN-1, NSP, or t-PA. Then we showed that although the expression of PAI-1 is restricted to astrocytes, PN-1, NSP, and t-PA are expressed in both neurons and astrocytes. Moreover, by using semiquantitative RT-PCR and Western blotting, we observed that only the expression of PAI-1 was modulated by TGF-beta1 treatment via a TGF-beta-inducible element contained in the PAI-1 promoter (CAGA box). Finally, we compared the specificity of TGF-beta1 action with other members of the TGF-beta family by using luciferase reporter genes. These data show that TGF-beta and activin were able to induce the overexpression of PAI-1 in astrocytes, but that bone morphogenetic proteins, glial cell line-derived neutrophic factor, and neurturin did not. These results provide new insights into the regulation of the serpins/t-PA axis and the mechanism by which TGF-beta may be neuroprotective.     

Roles of Thapsigargin-Sensitive Ca2+ Stores in the Survival of Developing Cultured Neurons

The roles of the intracellular calcium pool involved in regulating the Ca2+ profile and the neuronal survival rate during development were studied by using thapsigargin (TG), a specific inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase in cultured cerebellar granule neurons. Measuring the neuronal [Ca2+]i directly in the culture medium, we found a bell-shaped curve for [Ca2+]i versus cultured days in cerebellar granule neurons maintained in medium containing serum and 25 mM K+. The progressive increase in [Ca2+]i of the immature granule neurons (1-4 days in vitro) was abolished by TG, which resulted in massive neuronal apoptosis. When the [K+] was lowered from 25 to 5 mM, neither the progressively increasing [Ca2+]i nor the survival of immature granule neurons was significantly changed over 24-h incubation. Similarly, TG caused a dramatic decrease in the [Ca2+]i and survival rate of these immature neurons when switched to 5 mM K+ medium. Following maturation, the granule neurons became less sensitive to TG for both [Ca2+]i and neuronal survival. However, TG can protect mature granule neurons from the detrimental effect of switching to a 5 mM K+ serum-free medium by decreasing [Ca2+]i to an even lower level than in the respective TG-free group. Based on these findings, we propose that during the immature stage, TG-sensitive ER Ca2+-ATPase plays a pivotal role in the progressive increase of [Ca2+]i, which is essential for the growth and maturation of cultured granule neurons.     

VEGF signalling controls GnRH neuron survival via NRP1 independently of KDR and blood vessels

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells that are born in the nasal placode during embryonic development and migrate through the nose and forebrain to the hypothalamus, where they regulate reproduction. Many molecular pathways that guide their migration have been identified, but little is known about the factors that control the survival of the migrating GnRH neurons as they negotiate different environments. We previously reported that the class 3 semaphorin SEMA3A signals through its neuropilin receptors, NRP1 and NRP2, to organise the axons that guide migrating GnRH neurons from their birthplace into the brain. By combining analysis of genetically altered mice with in vitro models, we show here that the alternative neuropilin ligand VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signalling pathways through NRP1. We also demonstrate that survival signalling relies on neuronal, but not endothelial, NRP1 expression and that it occurs independently of KDR, the main VEGF receptor in blood vessels. Therefore, VEGF164 provides survival signals directly to developing GnRH neurons, independently of its role in blood vessels. Finally, we show that the VEGF164-mediated neuronal survival and SEMA3A-mediated axon guidance cooperate to ensure that migrating GnRH neurons reach the brain. Thus, the loss of both neuropilin ligands leads to an almost complete failure to establish the GnRH neuron system.     

GDNF - a stranger in the TGF-beta superfamily?

Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.     

Mitochondrial quality control: insights on how Parkinson’s disease related genes PINK1, parkin,and Omi/HtrA2 interact to maintain mitochondrial homeostasis

Alterations in mitochondrial homeostasis have been implicated in the etiology of Parkinson disease (PD) as demonstrated by human tissue studies, cell culture and in vivo genetic and toxin models. Mutations in the genes encoding PTEN-induced kinase 1 (PINK1), Omi/HtrA2 and parkin contribute to rare forms of parkinsonian neurodegeneration. Recently, each of these proteins has been shown to play a normal role in regulating mitochondrial structure, function, fission-fusion dynamics, or turnover (autophagy and biogenesis), promoting neuronal survival. Here, we review the biochemical mechanisms of mitochondrial protection conferred by each of these PD associated gene products in neurons, neuronal cell lines and other cell types. Potential molecular interactions and mitoprotective signaling pathways involving these three PD associated gene products are discussed in the context of mitochondrial quality control, in response to increasing levels of mitochondrial damage. We propose that PINK1, Omi/HtrA2 and parkin participate at different levels in mitochondrial quality control, converging through some overlapping and some distinct steps to maintain a common phenotype of healthy mitochondrial networks.     

Loss of TGF-beta 1 leads to increased neuronal cell death and microgliosis in mouse brain

TGF-beta1 is a key regulator of diverse biological processes in many tissues and cell types, but its exact function in the developing and adult mammalian CNS is still unknown. We report that lack of TGF-beta1 expression in neonatal Tgfb1(-/-) mice results in a widespread increase in degenerating neurons accompanied by reduced expression of synaptophysin and laminin and a prominent microgliosis. Lack of TGF-beta1 also strongly reduces survival of primary neurons cultured from Tgfb1(-/-) mice. TGF-beta1 deficiency in adult Tgfb1(-/+) mice results in increased neuronal susceptibility to excitotoxic injury, whereas astroglial overexpression of TGF-beta1 protects adult mice against neurodegeneration in acute, excitotoxic and chronic injury paradigms. This study reveals a nonredundant function for TGF-beta1 in maintaining neuronal integrity and survival of CNS neurons and in regulating microglial activation. Because individual TGF-beta1 expression levels in the brain vary considerably between humans, this finding could have important implications for susceptibility to neurodegeneration.     

Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors

The generation of mice lacking specific components of the transforming growth factor-beta (TGF-beta) signal tranduction pathway shows that TGF-beta is a key player in the development and physiology of the cardiovascular system. Both pro- and anti-angiogenic properties have been ascribed to TGF-beta, for which the molecular mechanisms are unclear. Here we report that TGF-beta can activate two distinct type I receptor/Smad signalling pathways with opposite effects. TGF-beta induces phosphorylation of Smad1/5 and Smad2 in endothelial cells and these effects can be blocked upon selective inhibition of ALK1 or ALK5 expression, respectively. Whereas the TGF-beta/ALK5 pathway leads to inhibition of cell migration and proliferation, the TGF-beta/ALK1 pathway induces endothelial cell migration and proliferation. We identified genes that are induced specifically by TGF-beta-mediated ALK1 or ALK5 activation. Id1 was found to mediate the TGF-beta/ALK1-induced (and Smad-dependent) migration, while induction of plasminogen activator inhibitor-1 by activated ALK5 may contribute to the TGF-beta-induced maturation of blood vessels. Our results suggest that TGF-beta regulates the activation state of the endothelium via a fine balance between ALK5 and ALK1 signalling.     

Endoglin expression on human microvascular endothelial cells association with betaglycan and formation of higher order complexes with TGF-beta signalling receptors.

Transforming growth factor-beta (TGF-beta) plays an important role in angiogenesis and vascular function. Endoglin, a transmembrane TGF-beta binding protein, is highly expressed on vascular endothelial cells and is the target gene for the hereditary haemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. The specific function of endoglin responsible for HHT1 is believed to involve alterations in TGF-beta responses. The initial interactions on the cell surface between endoglin and TGF-beta receptors may be an important mechanism by which endoglin modulates TGF-beta signalling, and thereby responses. Here it is shown that on human microvascular endothelial cells, endoglin is co-expressed and is associated with betaglycan, a TGF-beta accessory receptor with which endoglin shares limited amino acid homology. This complex formation may occur in either a ligand-dependent or a ligand-independent manner. In addition, the occurrence of three higher order complexes containing endoglin, type II and/or type I TGF-beta receptors, on these cells is demonstrated. Our findings suggest that endoglin may modify TGF-beta signalling by interacting with both betaglycan and the TGF-beta signalling receptors at physiological receptor concentrations and ratios.     

TGF-beta and the regulation of neuron survival and death.

Transforming growth factor-betas (TGF-betas) constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation, and tissue remodeling. In the developing nervous system, TGF-beta2 and -beta3 occur in radial and astroglial cells as well as in many populations of postmitotic, differentiating neurons. TGF-beta1 is restricted to the choroid plexus and meninges. In addition to functions related to glial cell maturation and performances, TGF-beta2 and -beta3 are important regulators of neuron survival. In contrast to neurotrophic factors, as for example, neurotrophins, TGF-betas are most likely not neurotrophic by themselves. However, they can dramatically increase the potency of select neurotrophins, fibroblast growth factor-2, ciliary neurotrophic factor, and glial cell line-derived neurotrophic factor (GDNF). In the case of GDNF, we have shown that GDNF fails to promote the survival of highly purified neuron populations in vitro unless it is supplemented with TGF-beta. This also applies to the in vivo situation, where antibodies to all three TGF-beta isoforms fully prevent the trophic effect of GDNF on axotomized, target-deprived neurons. In addition to the TGF-beta isoforms -beta2 and -beta3, other members of the TGF-beta superfamily are expressed in the nervous system having important roles in embryonic patterning, cell migration, and neuronal transmitter determination. We have cloned and expressed a novel TGF-beta, named growth/differentiation factor-15 (GDF-15). GDF-15 is synthesized in the choroid plexus and released into the CSF, but also occurs in all regions investigated of the developing and adult brain. GDF-15 is a potent trophic factor for developing and 6-OHDA-lesioned midbrain dopaminergic neurons in vitro and in vivo, matching the potency of GDNF.     

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.     

Developmentally regulated expression of mouse HtrA3 and its role as an inhibitor of TGF-beta signaling

The expression of mouse HtrA1 is developmentally regulated and restricted in embryo tissues which depend largely on TGF-beta signaling for their differentiation. We examined whether mouse HtrA3, another HtrA family member very close to HtrA1, shows similar expression patterns. HtrA3 and -1 were expressed mostly in the same embryonic organs but exhibited complementary patterns in various tissues; the lens epithelial cells in day 12.5 embryo expressed HtrA3 whereas the ciliary body and pigment retina expressed HtrA1. In the vertebrae of day 14.5 embryo, HtrA3 was expressed in the tail region, but HtrA1 was predominantly expressed in the thoracic and lumbar regions. Similar to HtrA1, HtrA3 bound to various TGF-beta proteins and inhibited the signaling of BMP-4, -2 and TGF-beta 1. HtrA3 did not inhibit signaling originated from a constitutively active BMP receptor, indicating that the inhibition occurred upstream of the cell surface receptor. HtrA3 also showed proteolytic activities indistinguishable from those of HtrA1 toward beta-casein and some extracellular matrix (ECM) proteoglycans. The protease activity was absolutely required for the TGF-beta signal inhibition activity. All these data suggest that HtrA3 and -1 have the overlapping biological activities but can function in complementary fashion in certain types of tissues.     

Low Density Lipoprotein Receptor-related Protein 1 Promotes Anti-apoptotic Signaling in Neurons by Activating Akt Survival Pathway

The low density lipoprotein receptor-related protein 1 (LRP1) is a multi-ligand receptor abundantly expressed in neurons. Previous work has shown that brain LRP1 levels are decreased during aging and in Alzheimer disease. Although mounting evidence has demonstrated a role for LRP1 in the metabolism of apolipoprotein E/lipoprotein and amyloid-β peptide, whether LRP1 also plays a direct role in neuronal survival is not clear. Here, we show that LRP1 expression is critical for the survival of primary neurons under stress conditions including trophic withdrawal, the presence of apoptosis inducers, or amyloid-β-induced neurotoxicity. Using lentiviral short hairpin RNA to knock down endogenous LRP1 expression, we showed that a depletion of LRP1 leads to an activation of caspase-3 and increased neuronal apoptosis, an effect that was rescued by a caspase-3 inhibitor. A correlation between decreased Akt phosphorylation and the activation of caspase-3 was demonstrated in LRP1 knocked down neurons. Notably, LRP1 knockdown decreased insulin receptor levels in primary neurons, suggesting that decreased neuronal survival might be a consequence of an impaired insulin receptor signaling pathway. Correspondingly, both insulin receptor and phospho-Akt levels were decreased in LRP1 forebrain knock-out mice. These results demonstrate that LRP1 mediates anti-apoptotic function in neurons by regulating insulin receptor and the Akt survival pathway and suggest that restoring LRP1 expression in Alzheimer disease brain might be beneficial to inhibiting neurodegeneration.     

Regulation of later neurogenic stages of adult‐derived neural stem/progenitor cells by L‐type Ca2+ channels

In the adult hippocampus, new neurons are continuously generated and incorporated into the local circuitry in a manner dependent on the network activity. Depolarization evoked by neurotransmitters has been assumed to activate L‐type Ca²⁺ channels (LTCC) which regulate the intracellular Ca²⁺‐dependent signaling cascades. The process of neurogenesis contains several stages such as proliferation, fate determination, selective death/survival and maturation. Here, we investigated which stage of neurogenesis is under the regulation of LTCC using a clonal line of neural stem/progenitor cells, PZ5, which was derived from adult rat hippocampus. Although undifferentiated PZ5 cells were type 1‐like cells expressing both nestin and glial fibrillary acidic protein, they generated neuronal, astrocytic and oligodendrocytic populations in differentiation medium containing retinoic acid. Proliferation of undifferentiated PZ5 cells was dependent on neither the LTCC antagonist, nimodipine (Nimo) nor the LTCC agonists, Bay K 8644 (BayK) or FPL 64176 (FPL), whereas the fraction of neuronal population that expressed both βIII‐tubulin and MAP2 was reduced by Nimo but increased by BayK or FPL. At an earlier period of differentiation (e.g. day 4), the fraction of PZ5 cells expressing HuC/D, pan‐neuronal marker, was not affected either by the LTCC activation or inhibition. At a later period of differentiation (e.g. day 9), the fraction of dying neurons was decreased by LTCC activation and increased by LTCC inhibition. It is suggested that the LTCC activation facilitates the survival and maturation of immature neurons, and that its inhibition facilitates the neuronal death.     

TGF‐beta signalling in the adult neurogenic niche promotes stem cell quiescence as well as generation of new neurons

Members of the transforming growth factor (TGF)‐β family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF‐β signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF‐β1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF‐β1 under a tetracycline regulatable Ca‐Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF‐β1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF‐β1 signalling in adult NPCs. The results demonstrate that TGF‐β1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF‐β1 in ageing and neurodegenerative diseases, TGF‐β1 signalling presents a molecular target for future interventions in such conditions.     

Dendritic morphology and spine density is not altered in motor cortex and dentate granular cells in mice lacking the ganglioside biosynthetic gene B4galnt1 - A quantitative Golgi cox study

Gangliosides are characteristic plasma membrane constituents of vertebrate brain used as milestones of neuronal development. As neuronal morphology is a good indicator of neuronal differentiation, we analyzed how lack of the ganglioside biosynthetic gene Galgt1 whose product is critical for production of four major adult mammalian brain complex gangliosides (GM1, GD1a, GD1b and GT1b) affects neuronal maturation in vivo. To define maturation of cortical neurons in mice lacking B4galnt1 we performed a morphological analysis of Golgi-Cox impregnated pyramidal neurons in primary motor cortex and granular cells of dentate gyrus in 3, 21 and 150 days old B4galnt1-null and wild type mice. Quantitative analysis of basal dendritic tree on layer III pyramidal neurons in the motor cortex showed very immature dendritic picture in both mice at postnatal day 3. At postnatal day 21 both mice reached adult values in dendritic length, complexity and spine density. No quantitative differences were found between B4galnt1-null and wild type mice in pyramidal cells of motor cortex or granular cells of dentate gyrus at any examined age. In addition, the general structural and neuronal organization of all brain structures, qualitatively observed on Nissl and Golgi-Cox, were similar Our results demonstrate that neurons can develop normal dendritic complexity and length without presence of complex gangliosides in vivo. Therefore, behavioral differences observed in B4galnt1-null mice may be attributed to functional rather than morphological level of dendrites and spines of cortical pyramidal neurons.     

Pattern of expression of HtrA1 during mouse development.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFbeta family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFbeta proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.