Ablation of Vti1a/1b Triggers Neural Progenitor Pool Depletion and Cortical Layer 5 Malformation in Late-embryonic Mouse Cortex.

Cortical morphogenesis entails several neurobiological events, including proliferation and differentiation of progenitors, migration of neuroblasts, and neuronal maturation leading to functional neural circuitry. These neurodevelopmental processes are delicately regulated by many factors. Endosomal SNAREs have emerged as formidable modulators of neuronal growth, aside their well-known function in membrane/vesicular trafficking. However, our understanding of their influence on cortex formation is limited. Here, we report that the SNAREs Vti1a and Vti1b (Vti1a/1b) are critical for proper cortical development. Following null mutation of Vti1a/1b in mouse, the late-embryonic mutant cortex appeared dysgenic, and the cortical progenitors therein were depleted beyond normal. Notably, cortical layer 5 (L5) is distinctively disorganized in the absence of Vti1a/1b. The latter defect, coupled with an overt apoptosis of Ctip2-expressing L5 neurons, likely contributed to the substantial loss of corticospinal and callosal projections in the Vti1a/1b-deficient mouse brain. These findings suggest that Vti1a/1b serve key neurodevelopmental functions during cortical histogenesis, which when mechanistically elucidated, can lend clarity to how endosomal SNAREs regulate brain development, or how their dysfunction may have implications for neurological disorders.

TGF-ß Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons.

Calcium homeostasis is a cellular process required for proper cell function and survival, maintained by the coordinated action of several transporters, among them members of the Na+/Ca2+-exchanger family, such as SLC8A3. Transforming growth factor beta (TGF-ß) signaling defines neuronal development and survival and may regulate the expression of channels and transporters. We investigated the regulation of SLC8A3 by TGF-ß in a conditional knockout mouse with deletion of TGF-ß signaling from Engrailed 1-expressing cells, i.e., in cells from the midbrain and rhombomere 1, and elucidated the underlying molecular mechanisms. The results show that SLC8A3 is significantly downregulated in developing dopaminergic and dorsal raphe serotonergic neurons in mutants and that low SLC8A3 abundance prevents the expression of the anti-apoptotic protein Bcl-xL. TGF-ß signaling affects SLC8A3 via the canonical and p38 signaling pathway and may increase the binding of Smad4 to the Slc8a3 promoter. Expression of the lipid peroxidation marker malondialdehyde (MDA) was increased following knockdown of Slc8a3 expression in vitro. In neurons lacking TGF-ß signaling, the number of MDA- and 4-hydroxynonenal (4-HNE)-positive cells was significantly increased, accompanied with increased cellular 4-HNE abundance. These results suggest that TGF-ß contributes to the regulation of SLC8A3 expression in developing dopaminergic and dorsal raphe serotonergic neurons, thereby preventing oxidative stress.

Spatiotemporal Role of Transforming Growth Factor Beta 2 in Developing and Mature Mouse Hindbrain Serotonergic Neurons.

Transforming growth factor betas are integral molecular components of the signalling cascades defining development and survival of several neuronal groups. Among TGF-ß ligands, TGF-ß2 has been considered as relatively more important during development. We have generated a conditional knockout mouse of the Tgf-ß2 gene with knock-in of an EGFP reporter and subsequently a mouse line with cell-type specific deletion of TGF-ß2 ligand from Krox20 expressing cells (i.e., in cells from rhombomeres r3 and r5). We performed a phenotypic analysis of the hindbrain serotonergic system during development and in adulthood, determined the neurochemical profile in hindbrain and forebrain, and assessed behavioural performance of wild type and mutant mice. Mutant mice revealed significantly decreased number of caudal 5-HT neurons at embryonic day (E) 14, and impaired development of caudal dorsal raphe, median raphe, raphe magnus, and raphe obscurus neurons at E18, a phenotype that was largely restored and even overshot in dorsal raphe of mutant adult mice. Serotonin levels were decreased in hindbrain but significantly increased in cortex of adult mutant mice, though without any behavioural consequences. These results highlight differential and temporal dependency of developing and adult neurons on TGF-ß2. The results also indicate TGF-ß2 being directly or indirectly potent to modulate neurotransmitter synthesis and metabolism. The novel floxed TGF-ß2 mouse model is a suitable tool for analysing the in vivo functions of TGF-ß2 during development and in adulthood in many organs.

Correction: Reelin controls the positioning of brainstem serotonergic raphe neurons.

[This corrects the article DOI: 10.1371/journal.pone.0200268.].

Bid Expression Network Controls Neuronal Cell Fate During Avian Ciliary Ganglion Development.

Avian ciliary ganglion (CG) development involves a transient execution phase of apoptosis controlling the final number of neurons, but the time-dependent molecular mechanisms for neuronal cell fate are largely unknown. To elucidate the molecular networks regulating important aspects of parasympathetic neuronal development, a genome-wide expression analysis was performed during multiple stages of avian CG development between embryonic days E6 and E14. The transcriptome data showed a well-defined sequence of events, starting from neuronal migration via neuronal fate cell determination, synaptic transmission, and regulation of synaptic plasticity to growth factor associated signaling. In particular, we extracted a neuronal apoptosis network that characterized the cell death execution phase at E8/E9 and apoptotic cell clearance at E14 by combining the gene time series analysis with network synthesis from the chicken interactome. Network analysis identified TP53 as key regulator and predicted involvement of the BH3 interacting domain death agonist (BID). A virus-based RNAi knockdown approach in vivo showed a crucial impact of BID expression on the execution of ontogenetic programmed cell death (PCD). In contrast, Bcl-XL expression did not impact PCD. Therefore, BID-mediated apoptosis represents a novel cue essential for timing within CG maturation.

Reelin controls the positioning of brainstem serotonergic raphe neurons.

Serotonin (5-HT) acts as both a morphogenetic factor during early embryonic development and a neuromodulator of circuit plasticity in the mature brain. Dysregulation of serotonin signaling during critical periods is involved in developmental neurological disorders, such as schizophrenia and autism. In this study we focused on the consequences of defect reelin signaling for the development of the brainstem serotonergic raphe system. We observed that reelin signaling components are expressed by serotonergic neurons during the critical period of their lateral migration. Further, we found that reelin signaling is important for the normal migration of rostral, but not caudal hindbrain raphe nuclei and that reelin deficiency results in the malformation of the paramedian raphe nucleus and the lateral wings of the dorsal raphe nuclei. Additionally, we showed that serotonergic neurons projections to laminated brain structures were severely altered. With this study, we propose that the perturbation of canonical reelin signaling interferes with the orientation of tangentially, but not radially, migrating brainstem 5-HT neurons. Our results open the window for further studies on the interaction of reelin and serotonin and the pathogenesis of neurodevelopmental disorders.

TGF-ß Signaling Regulates Development of Midbrain Dopaminergic and Hindbrain Serotonergic Neuron Subgroups.

Molecular and functional diversity within midbrain dopaminergic (mDA) and hindbrain serotonergic (5-HT) neurons has emerged as a relevant feature that could underlie selective vulnerability of neurons in clinical disorders. We have investigated the role of transforming growth factor beta (TGF-ß) during development of mDA and 5-HT subgroups. We have generated TßRIIflox/flox::En1cre/+ mice where type II TGF-ß receptor is conditionally deleted from engrailed 1-expressing cells and have investigated the hindbrain serotonergic system of these mice together with Tgf-ß2-/- mice. The results show a significant decrease in the number of 5-HT neurons in TGF-ß2-deficient mice at embryonic day (E) 12 and a selective significant decrease in the hindbrain paramedian raphe 5-HT neurons at E18, compared to wild type. Moreover, conditional deletion of TGF-ß signaling from midbrain and rhombomere 1 leads to inactive TGF-ß signaling in cre-expressing cells, impaired development of mouse mDA neuron subgroups and of dorsal raphe 5-HT neuron subgroups in a temporal manner. These results highlight a selective growth factor dependency of individual rostral hindbrain serotonergic subpopulations, emphasize the impact of TGF-ß signaling during development of mDA and 5-HT subgroups, and suggest TGF-ßs as potent candidates to establish diversity within the hindbrain serotonergic system. Thus, the data contribute to a better understanding of development and degeneration of mDA neurons and 5-HT-associated clinical disorders.

TGFß-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism.

The optic fissure is a transient gap in the developing vertebrate eye, which must be closed as development proceeds. A persisting optic fissure, coloboma, is a major cause for blindness in children. Although many genes have been linked to coloboma, the process of optic fissure fusion is still little appreciated, especially on a molecular level. We identified a coloboma in mice with a targeted inactivation of transforming growth factor ß2 (TGFß2). Notably, here the optic fissure margins must have touched, however failed to fuse. Transcriptomic analyses indicated an effect on remodelling of the extracellular matrix (ECM) as an underlying mechanism. TGFß signalling is well known for its effect on ECM remodelling, but it is at the same time often inhibited by bone morphogenetic protein (BMP) signalling. Notably, we also identified two BMP antagonists among the downregulated genes. For further functional analyses we made use of zebrafish, in which we found TGFß ligands expressed in the developing eye, and the ligand binding receptor in the optic fissure margins where we also found active TGFß signalling and, notably, also gremlin 2b (grem2b) and follistatin a (fsta), homologues of the regulated BMP antagonists. We hypothesized that TGFß is locally inducing expression of BMP antagonists within the margins to relieve the inhibition from its regulatory capacity regarding ECM remodelling. We tested our hypothesis and found that induced BMP expression is sufficient to inhibit optic fissure fusion, resulting in coloboma. Our findings can likely be applied also to other fusion processes, especially when TGFß signalling or BMP antagonism is involved, as in fusion processes during orofacial development.

Neuronal Activity, TGFß-Signaling and Unpredictable Chronic Stress Modulate Transcription of Gadd45 Family Members and DNA Methylation in the Hippocampus.

Neuronal activity is altered in several neurological and psychiatric diseases. Upon depolarization not only neurotransmitters are released but also cytokines and other activators of signaling cascades. Unraveling their complex implication in transcriptional control in receiving cells will contribute to understand specific central nervous system (CNS) pathologies and will be of therapeutically interest. In this study we depolarized mature hippocampal neurons in vitro using KCl and revealed increased release not only of brain-derived neurotrophic factor (BDNF) but also of transforming growth factor beta (TGFB). Neuronal activity together with BDNF and TGFB controls transcription of DNA modifying enzymes specifically members of the DNA-damage-inducible (Gadd) family, Gadd45a, Gadd45b, and Gadd45g. MeDIP followed by massive parallel sequencing and transcriptome analyses revealed less DNA methylation upon KCl treatment. Psychiatric disorder-related genes, namely Tshz1, Foxn3, Jarid2, Per1, Map3k5, and Arc are transcriptionally activated and demethylated upon neuronal activation. To analyze whether misexpression of Gadd45 family members are associated with psychiatric diseases, we applied unpredictable chronic mild stress (UCMS) as established model for depression to mice. UCMS led to reduced expression of Gadd45 family members. Taken together, our data demonstrate that Gadd45 family members are new putative targets for UCMS treatments.

The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-ß2.

Functional activation of the neuronal K(+)-Cl(-) co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor ß2 (TGF-ß2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-ß2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl(-) extrusion. We also identify the signaling pathway from TGF-ß2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-ß2-mediated KCC2 trafficking and functional activation. TGF-ß2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-ß2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-ß2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission.

HSPB3 protein is expressed in motoneurons and induces their survival after lesion-induced degeneration.

The human small heat shock proteins (HSPBs) form a family of molecular chaperones comprising ten members (HSPB1-HSPB10), whose functions span from protein quality control to cytoskeletal dynamics and cell death control. Mutations in HSPBs can lead to human disease and particularly point mutations in HSPB1 and HSPB8 are known to lead to peripheral neuropathies. Recently, a missense mutation (R7S) in yet another member of this family, HSPB3, was found to cause an axonal motor neuropathy (distal hereditary motor neuropathy type 2C, dHMN2C). Until now, HSPB3 protein localization and function in motoneurons (MNs) have not yet been characterized. Therefore, we studied the endogenous HSPB3 protein distribution in the spinal cords of chicken and mouse embryos and in the postnatal nervous system (central and peripheral) of chicken, mouse and human. We further investigated the impact of wild-type and mutated HSPB3 on MN cell death via overexpressing these genes in ovo in an avian model of MN degeneration, the limb-bud removal. Altogether, our findings represent a first step for a better understanding of the cellular and molecular mechanisms leading to dHMN2C.

Gdf-15 deficiency does not alter vulnerability of nigrostriatal dopaminergic system in MPTP-intoxicated mice.

Growth/differentiation factor-15 (Gdf-15) is a member of the transforming growth factor-ß (Tgf-ß) superfamily and has been shown to be a potent neurotrophic factor for midbrain dopaminergic (DAergic) neurons both in vitro and in vivo. Gdf-15 has also been shown to be involved in inflammatory processes. The aim of this study was to identify the role of endogenous Gdf-15 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease (PD) by comparing Gdf-15 (+/+) and Gdf-15 (-/-) mice. At 4 days and 14 days post-MPTP administration, both Gdf-15 (+/+) and Gdf-15 (-/-) mice showed a similar decline in DAergic neuron numbers and in striatal dopamine (DA) levels. This was followed by a comparable restorative phase at 90 days and 120 days, indicating that the absence of Gdf-15 does not affect the susceptibility or the recovery capacity of the nigrostriatal system after MPTP administration. The MPTP-induced microglial and astrocytic response was not significantly altered between the two genotypes. However, pro-inflammatory and anti-inflammatory cytokine profiling revealed the differential expression of markers in Gdf-15 (+/+) and Gdf-15 (-/-) mice after MPTP administration. Thus, the MPTP mouse model fails to uncover a major role of endogenous Gdf-15 in the protection of MPTP-lesioned nigrostriatal DAergic neurons, in contrast to its capacity to protect the 6-hydroxydopamine-intoxicated nigrostriatal system.

Comparative Analysis of the Effects of Neurotrophic Factors CDNF and GDNF in a Nonhuman Primate Model of Parkinson's Disease.

Cerebral dopamine neurotrophic factor (CDNF) belongs to a newly discovered family of evolutionarily conserved neurotrophic factors. We demonstrate for the first time a therapeutic effect of CDNF in a unilateral 6-hydroxydopamine (6-OHDA) lesion model of Parkinson's disease in marmoset monkeys. Furthermore, we tested the impact of high chronic doses of human recombinant CDNF on unlesioned monkeys and analyzed the amino acid sequence of marmoset CDNF. The severity of 6-OHDA lesions and treatment effects were monitored in vivo using 123I-FP-CIT (DaTSCAN) SPECT. Quantitative analysis of 123I-FP-CIT SPECT showed a significant increase of dopamine transporter binding activity in lesioned animals treated with CDNF. Glial cell line-derived neurotrophic factor (GDNF), a well-characterized and potent neurotrophic factor for dopamine neurons, served as a control in a parallel comparison with CDNF. By contrast with CDNF, only single animals responded to the treatment with GDNF, but no statistical difference was observed in the GDNF group. However, increased numbers of tyrosine hydroxylase immunoreactive neurons, observed within the lesioned caudate nucleus of GDNF-treated animals, indicate a strong bioactive potential of GDNF.

Expression of Tgfß1 and Inflammatory Markers in the 6-hydroxydopamine Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by loss of midbrain dopaminergic (mDA) neurons in the substantia nigra (SN). Microglia-mediated neuroinflammation has been described as a common hallmark of PD and is believed to further trigger the progression of neurodegenerative events. Injections of 6-hydroxydopamine (6-OHDA) are widely used to induce degeneration of mDA neurons in rodents as an attempt to mimic PD and to study neurodegeneration, neuroinflammation as well as potential therapeutic approaches. In the present study, we addressed microglia and astroglia reactivity in the SN and the caudatoputamen (CPu) after 6-OHDA injections into the medial forebrain bundle (MFB), and further analyzed the temporal and spatial expression patterns of pro-inflammatory and anti-inflammatory markers in this mouse model of PD. We provide evidence that activated microglia as well as neurons in the lesioned SN and CPu express Transforming growth factor ß1 (Tgfß1), which overlaps with the downregulation of pro-inflammatory markers Tnfα, and iNos, and upregulation of anti-inflammatory markers Ym1 and Arg1. Taken together, the data presented in this study suggest an important role for Tgfß1 as a lesion-associated factor that might be involved in regulating microglia activation states in the 6-OHDA mouse model of PD in order to prevent degeneration of uninjured neurons by microglia-mediated release of neurotoxic factors such as Tnfα and nitric oxide (NO).

Growth/differentiation factor-15 deficiency compromises dopaminergic neuron survival and microglial response in the 6-hydroxydopamine mouse model of Parkinson's disease.

Growth/differentiation factor-15 (Gdf-15) is a member of the TGF-ß superfamily and a pleiotropic, widely distributed cytokine, which has been shown to play roles in various pathologies, including inflammation. Analysis of Gdf-15(-/-) mice has revealed that it serves the postnatal maintenance of spinal cord motor neurons and sensory neurons. In a previous study, exogenous Gdf-15 rescued 6-hydroxydopamine (6-OHDA) lesioned Gdf-15(+/+) nigrostriatal dopaminergic (DAergic) neurons in vitro and in vivo. Whether endogenous Gdf-15 serves the physiological maintenance of nigrostriatal DAergic neurons in health and disease is not known and was addressed in the present study. Stereotactic injection of 6-OHDA into the medial forebrain bundle (MFB) led to a significant decline in the numbers of DAergic neurons in both Gdf-15(+/+) and Gdf-15(-/-) mice over a time-period of 14days. However, this decrease was exacerbated in the Gdf-15(-/-) mice, with only 5.5% surviving neurons as compared to 24% in the Gdf-15(+/+) mice. Furthermore, the microglial response to the 6-OHDA lesion was reduced in Gdf-15(-/-) mice, with significantly lower numbers of total and activated microglia and a differential cytokine expression as compared to the Gdf-15(+/+) mice. Using in vitro models, we could demonstrate the importance of endogenous Gdf-15 in promoting DAergic neuron survival thus highlighting its relevance in a direct neurotrophic supportive role. Taken together, these results indicate the importance of Gdf-15 in promoting survival of DAergic neurons and regulating the inflammatory response post 6-OHDA lesion.

TGFß1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity.

Microglia-mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson's disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor ß1 (TGFß1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFß1 is able to block IFNγ-induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down-regulated upon TGFß1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up-regulated after TGFß1 treatment. Finally, we demonstrate that TGFß1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ-driven neurotoxicity in mixed neuron-glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFß1 as a key immunoregulatory factor for microglia by silencing IFNγ-mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ-induced neurotoxicity. Interferon γ (IFNγ) is a potent pro-inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor ß1 (TGFß1) is able to inhibit the IFNγ-mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ-induced genes as well as the signaling receptor IFNγR1, TGFß1 reduces the responsiveness of microglia towards IFNγ. In mixed neuron-glia cultures, TGFß1 protects midbrain dopaminergic (mDA) neurons from IFNγ-induced neurotoxicity.

RARß regulates neuronal cell death and differentiation in the avian ciliary ganglion.

Programmed cell death during chicken ciliary ganglion (CG) development is mostly discussed as an extrinsically regulated process, guided either by the establishment of a functional balance between preganglionic and postganglionic activity or the availability of target-derived neurotrophic factors. We found that the expression of the gene coding for the nuclear retinoic acid receptor ß (RARB) is transiently upregulated prior to and during the execution phase of cell death in the CG. Using retroviral vectors, the expression of RARB was knocked down during embryonic development in ovo. The knockdown led to a significant increase in CG neuron number after the cell death phase. BrdU injections and active caspase-3 staining revealed that this increase in neuron number was due to an inhibition of apoptosis during the normal cell death phase. Furthermore, apoptotic neuron numbers were significantly increased at a stage when cell death is normally completed. While the cholinergic phenotype of the neurons remained unchanged after RARB knockdown, the expression of the proneural gene Cash1 was increased, but somatostatin-like immunoreactivity, a hallmark of the mature choroid neuron population, was decreased. Taken together, these results point toward a delay in neuronal differentiation as well as cell death. The availability of nuclear retinoic acid receptor ß (RARß) and RARß-induced transcription of genes could therefore be a new intrinsic cue for the maturation of CG neurons and their predisposition to undergo cell death.

Eye morphogenesis driven by epithelial flow into the optic cup facilitated by modulation of bone morphogenetic protein.

The hemispheric, bi-layered optic cup forms from an oval optic vesicle during early vertebrate eye development through major morphological transformations. The overall basal surface, facing the developing lens, is increasing, while, at the same time, the space basally occupied by individual cells is decreasing. This cannot be explained by the classical view of eye development. Using zebrafish (Danio rerio) as a model, we show that the lens-averted epithelium functions as a reservoir that contributes to the growing neuroretina through epithelial flow around the distal rims of the optic cup. We propose that this flow couples morphogenesis and retinal determination. Our 4D data indicate that future stem cells flow from their origin in the lens-averted domain of the optic vesicle to their destination in the ciliary marginal zone. BMP-mediated inhibition of the flow results in ectopic neuroretina in the RPE domain. Ultimately the ventral fissure fails to close resulting in coloboma.

The combination of limb-bud removal and in ovo electroporation techniques: a new powerful method to study gene function in motoneurons undergoing lesion-induced cell death.

BACKGROUND: The chicken embryo is an important model organism for developmental biology studies. At present, many techniques on this model have been set up, from surgical procedures to molecular biology methods, to answer capital questions of cell biology. The study of the genes involved in motoneurons (MNs) survival and cell death is critical for a better understanding of the molecular mechanisms leading to MNs degenerative diseases, such as amyothophic lateral sclerosis (ALS) and motor peripheral neuropathies. NEW METHOD: Here, we describe the combination of a well known surgical procedure able to induce MNs cell death, the limb-bud removal (LBR), with a very popular method used in molecular biology to test gene function in living organisms, the in ovo electroporation (IOE). The aim of this work is to provide an effective method for the investigation of genes involved in MNs survival and cell death under lesion conditions. RESULTS: Our method allows the successful electroporation of the 40-50% of MNs on the side of LBR with a high survival rate early and late after procedure. COMPARISON WITH OTHER METHODS: This modified LBR technique combined with IOE allows a higher MN expression efficiency compared to an already published method. CONCLUSIONS: Our work opens the possibility of screening a multitude of genes involved in MNs survival or cell death in vivo with high reproducibility and efficiency on a flexible and inexpensive animal model. The LBR/IOE technique opens a new way for the optimization of subsequent studies on mammalian models of diseases affecting MNs survival.

TGFß1 increases microglia-mediated engulfment of apoptotic cells via upregulation of the milk fat globule-EGF factor 8.

Milk fat globule-epidermal growth factor-factor 8 (Mfge8) has been described as an essential molecule during microglia-mediated clearance of apoptotic cells via binding to phosphatidylserine residues and subsequent phagocytosis. Impaired uptake of apoptotic cells by microglia results in prolonged inflammatory responses and damage of healthy cells. Although the mechanisms of Mfge8-mediated engulfment of apoptotic cells are well understood, endogenous or exogenous factors that regulate Mfge8 expression remain elusive. Here, we describe that TGFß1 increases the expression of Mfge8 and enhances the engulfment of apoptotic cells by primary mouse microglia in a Mfge8-dependent manner. Further, apoptotic cells are capable of increasing microglial TGFß expression and release and shift the microglia phenotype toward alternative activation. Moreover, we provide evidence that Mfge8 expression is differentially regulated in microglia after classical and alternative activation and that Mfge8 is not able to exert direct antiinflammatory effects on LPS-treated primary microglia. Together, these results underline the importance of TGFß1 as a regulatory factor for microglia and suggest that increased TGFß1 expression in models of neurodegeneration might be involved in clearance of apoptotic cells via regulation of Mfge8 expression.