Increased expression of the homologue of enhancer-of-split 1 protects neurons from beta amyloid neurotoxicity and hints at an alternative role for transforming growth factor beta1 as a neuroprotector

Introduction Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of β-amyloid (Aβ) in the brain, which produces progressive neuronal loss and dementia. We recently demonstrated that the noxious effects of Aβ on cultured hippocampal neurons are in part provoked by the antagonism of nerve growth factor (NGF) signalling, which impairs the activation of nuclear factor κB (NF-κB) by impeding the tyrosine phosphorylation of I-κBα. As a result, the expression of the homologue of Enhancer-of split 1 (Hes1) gene is downregulated and ultimately, gamma-aminobutyric acid (GABA)-ergic connectivity is lost. Methods Hes1 activity was promoted in cultured hippocampal neurons by overexpressing a Hes1-encoding plasmid or by upregulating this gene by activating NF-κB through different approaches (overexpressing either the I-κB kinaseβ, or p65/RelA/NF-κB). Alternatively neurons were exposed to TGFβ1. Dendrite patterning, GABAergic connectivity and cell survival were analyzed by immunofluorescence microscopy. Hes1 expression was determined by real-time PCR. NF-κB activation was measured using the dual-luciferase reporter assay. Results The expression of Hes1 abolished the effects of Aβ on dendritic patterning and GABAergic input, and it prevented the death of the cultured neurons. TGFβ1, a known neuroprotector, could counteract the deleterious effects of Aβ by inducing NF-κB activation following the serine phosphorylation of I-κBα. Indeed, the number of GABAergic terminals generated by inducing Hes1 expression was doubled. Conclusion Our data define some of the mechanisms involved in Aβ-mediated cell death and they point to potential means to counteract this noxious activity.


Introduction
The amyloid hypothesis considers amyloid beta (Aβ) as the principal agent underlying the various manifestations of Alzheimer's disease (AD). Accordingly, most therapeutic approaches for AD treatment target the Aβ peptide, which induces the formation of amyloid deposits in the brain [1]. These strategies attempt to limit Aβ production and fibril formation, or to increase Aβ clearance from brain deposits. This latter approach has been the focus of much investigation, with active and passive methods to immunize against Aβ effectively reducing brain Aβ content. However, this reduction in amyloid content has not been conclusively associated with improvements in cognitive performance or a slowing in the progression of AD [2,3].
An alternative approach to AD therapy involves interfering with the signals transduced by Aβ to offset neuronal deterioration and death. While neuronal Aβ signaling pathways have been studied extensively, many of the underlying mechanisms remain elusive. Several studies have related Aβ neurotoxicity with NGF neurotrophy and indeed, some current therapeutic approaches for AD involve the use of NGF or other compounds that mimic its effects [4][5][6][7][8][9]. The cellular and molecular bases underlying the antagonism of NGF by Aβ have been described recently, at least in part. Accordingly, Aβ competes with nerve growth factor (NGF) binding to p75 NTR [10,11], thereby preventing the activation of NF-B by impairing the tyrosine phosphorylation and the subsequent degradation of I-Bα [12]. The inhibition of nuclear factor kappa-B (NF-B) mediated by Aβ results in the downregulation of the enhancer of split homolog 1 (Hes1) gene, which is known to influence dendrite patterning and gamma-aminobutyric acid (GABA)-ergic inputs [13,14].
We recently reported that Aβ impairs the initial steps of NGF signalling at the level of RhoA GTPase and protein tyrosine phosphatase 1B (PTP1B) [15]. Furthermore, we demonstrated that potentiation of NGF signalling (for example, by inhibiting RhoA GTPase and activating PTP1B) confers neuronal resistance to Aβ neurotoxicity. In the present study, we explored alternative means of activating NF-B and promoting Hes1 expression. We found that overexpression of I-β kinase (IKKβ) a kinase that phosphorylates I-Bα (an NF-B inhibitor), p65/ RelA or Hes1 abolished the effects of Aβ on dendritic patterning, GABAergic input and the survival of cultured hippocampal neurons. Furthermore, administration of transforming growth factor β1 (TGFβ1) produced similar effects, augmenting Hes1 expression and GABAergic input, and conferring resistance to Aβ toxicity. These results further our understanding of Aβ toxicity in AD and they open new perspectives for AD treatment using anti-amyloid approaches.

Amyloid b preparation and characterization
Amyloid β (1-42) was purchased from NeoMPS (Strasbourg, France) and to obtain oligomeric forms, the peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol and the solution was allowed to evaporate for 2 h at room temperature. The peptide film was dissolved in dimethyl sulfoxide (DMSO), sonicated in a water bath for 10 minutes and diluted to 100 μM in PBS. This solution was then briefly vortexed and incubated overnight at 4°C. Aliquots were stored at -20°C. Our Aβ preparations were resolved by 12% Bis-Tris gel electrophoresis and electrotransferred to polyvinylidene difluoride (PVDF) membranes, which were probed with a mouse monoclonal anti-β Amyloid antibody (clone BAM-10, diluted 1:2,000: Sigma Aldrich). After further incubation with an horseradish peroxidise (HRP)-conjugated anti-mouse antibody (1:10,000: Jackson Immuno Research, West Grove, USA), immunoreactive Ab species were visualised by chemiluminiscence. This analysis revealed that most Aβ forms were monomeric and dimeric with a less prominent trimeric and tetrameric component ( Figure S1A in Additional file 1). To kill neurons in culture, a concentration of 5 μM was established empirically (Figure S1B in Additional file 1).

Other chemicals
Recombinant TGFβ1 was purchased from Preprotech EC LTD (London, UK). The cell-permeable NF-B inhibitor peptide SN-50 and its inactive control, SN-50M, were obtained from Calbiochem (Darmstadt, Germany).

Cell culture
Primary hippocampal neuronal cultures were prepared as described previously [21], with some modifications. Briefly, hippocampi were dissected from embryonic day 17 (E17) CD1 mouse foetuses and dissociated into single cells following trypsin digestion (Worthington, Lakewood, USA) and DNase I treatment (Roche Applied Science). Neurons were plated on glass coverslips or in plastic dishes coated with poly-L-lysine (Sigma-Aldrich, Madrid, Spain), and then cultured in Neurobasal A supplemented with 2 mM GlutaMAX I, 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco BRL, Crewe, UK). After 7 days in vitro (DIV) the neurons were exposed to either TGFβ1 or Aβ.

Neuronal transfection
For fluorescence immunocytochemistry, cultured neurons (7 days in vitro, DIV) were transfected with different vectors using the Effectene Transfection Reagent (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions, with some modifications. Briefly, 0.6 μg of DNA was added to 120 μl of EC buffer and 3.5 μL of enhancer, and then left for 5 minutes at room temperature before 10 μL of Effectene was added. After 15 minutes incubation at room temperature, the final solution was added to a 35 mm cell culture dish containing hippocampal neurons, for 3 h before the medium was changed. Less than 0.5% of the neurons were transfected in each dish, permitting the morphology of the neurons to be analyzed without the interference of neighboring labeled neurons.
To determine Hes1 mRNA, 300,000 neurons were transfected with different vectors using Lipofectamine LTX (Invitrogen), following the protocol recommended by the manufacturer. The rate of transfection was 20-25% of the total number of cells.

Immunocytochemistry, image acquisition and morphometric analysis of labeled hippocampal neurons
Neurons were fixed for 30 minutes in 4% paraformaldehyde (PFA) in PBS 18 h after transfection, permeabilized for 15 minutes at room temperature with 0.5% Triton X-100 in PBS and blocked with 10% goat serum in PBS containing 0.1% Triton X-100. The neurons were then incubated with the primary and secondary antibodies. To verify that the labeling was caused specifically by the primary antibodies, it was either omitted or replaced by similarly diluted normal serum from the same species.
Our methods for the evaluation of dendritic morphology and presynaptic terminal identification in dissociated cell cultures have been described previously [14]. Briefly, labeled neurons were visualized by standard epifluorescence under a Plan-Neofluar 63× oil objective with a numerical aperture of 1.3 (Zeiss, Oberkochen, Germany). Terminal counts and analysis of dendrite morphology were performed manually. A circular region of interest (ROI) with a diameter of 50 μm was projected onto the EGFP-labeled neuron, its centre roughly coinciding with the centre of the soma. The dendrite length was expressed as the number of dendritic trees that exceeded the limit of the ROI (number of dendrites > 50 μm). In co-transfection experiments, only double-labeled cells were analysed, representing > 90% of the single-labeled cells. Synaptic terminals in contact with an EGFP-labeled neuron were identified by single immunofluorescence, when an antibody against VIAAT was used.

Cell survival
After treatment, neurons were fixed for 30 minutes in 4% paraformaldehyde (PFA) in PBS and the nuclei were immunostained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI: Sigma-Aldrich). Non-viable neurons were recognized by nuclear condensation and/or fragmented chromatin. In two independent experiments, the number of viable neurons was counted in triplicate in approximately 50 fields. In co-transfection experiments, only the nuclei of double-labeled cells were analysed.

Quantitative real time polymerase chain reaction (PCR)
After treatments, total RNA was extracted from cultures at 7 DIV using the Illustra RNAspin Mini kit (GE-Healthcare, Piscataway, NJ, USA) and first strand cDNA was prepared from the RNA using the First Strand Synthesis kit according to the manufacturer's instructions (Fermentas GmbH, St Leon-Rot, Germany). Quantitative PCR was performed using the ABI Prism 7000 Sequence Detector (Applied Biosystems, Weiterstadt, Germany). TaqMan probes and primers for Hes1 and the housekeeping gene GADPH (as a control) were selected as the Assay-on-Demand gene expression products (Applied Biosystems). All TaqMan probes were labeled with 6-carboxy fluorescein (FAM) and real time PCR was performed using the TaqMan Universal PCR Master Mix according to the manufacturer's instructions. Hes1 expression was normalized to GAPDH expression.

Analysis of reporter gene activity
For reporter gene studies, 12-well plates (Becton Dickinson) containing 200,000 hippocampal neurons were transfected using the Lipofectamine LTX (Invitrogen) transfection reagent according to the manufacturer's instructions. Transfection efficiency ranged from 20 to 25% in control experiments, as revealed by co-transfection with pEGFP-N1 (Biosciences/Clontech). Cells were co-transfected with 0.5 μg/well of a plasmid containing five tandem repeats of the B enhancer element upstream of the coding sequence of firefly-luciferase (pNF-B-luc; Clontech), along with 0.1 μg of a plasmid encoding Renilla-luciferase (phRL-TKluc: Promega, Madison, WI) as a transfection control for normalization in dual-luciferase assays. Dual-Luficerase reporter assays were performed according to the manufacturer's recommendations (Promega).

Statistical analysis
The data are presented as the mean ± standard error of the mean (SEM). Unpaired Student's t-tests were used to determine the significance, denoted as *P < 0.05, **P < 0.01, and ***P < 0.001. All experiments were repeated at least twice.

Hes1 in neuronal morphology, connectivity and survival
Earlier studies revealed that Hes1 mediates the effects of NGF on neuronal morphology and connectivity. NGF increases both dendrite length and GABAergic connectivity in cultured hippocampal neurons, effects that are abrogated by overexpression of Hes6 [13,14], a natural inhibitor of Hes1 [19]. We transfected a Hes1-expressing vector into cultured hippocampal neurons and analyzed the effects on dendrite morphology and synaptic connectivity. Overexpression of Hes1 increased the length of primary dendrites while decreasing their number ( Figure 1A, B), and it enhanced GABAergic connectivity, as evident by immunostaining for VIAAT-positive clusters ( Figure 1C). Interestingly, Hes1 transfection of neurons protected these cells from the neurotoxic effects of Aβ. Indeed, while oligomerization of Aβ ( Figure S1A in Additional file 1) decreased the length and increased the number of primary dendrites, albeit decreasing GABAergic input, these effects were completely reversed by Hes1 transfection ( Figure 1A, B and 1C). Moreover, Hes1 overexpression attenuated the effects of Aβ on neuronal survival. Using a concentration of 5 μM Aβ  in these studies to ensure that most cells would die during the experiment (performed over 90 h ( Figure S1B in Additional file 1), prior transfection with Hes1 rescued 50% of neurons from this Aβ-induced death ( Figure 1D). Taken together, these findings demonstrate that optimal expression of Hes1 counteracts the effects of Aβ on neurons at all the levels examined here.

Activation of NF-B blocks the effects of Ab on neuronal morphology and connectivity
Having previously revealed that NGF augments Hes1 by activating NF-B [13], the link between NF-B activation and Hes1 expression was confirmed here using an alternative experimental approach. Cultured hippocampal neurons were transfected with a plasmid expressing p65/ RelA, and Hes1 expression was assessed by quantitative PCR. Although only 20 to 25% of neurons were transfected, a significant increase (35 to 40%) in Hes1 mRNA was evident throughout the culture (Figure 2A). Transfected, myc-tagged p65/RelA was predominantly found in the nucleus, as expected given its capacity to promote Hes1 expression ( Figure 2B). Moreover, p65/RelA overexpression produced marked changes in dendrite arborisation, increasing the length and decreasing the number of primary dendrites ( Figure 2B). These effects were paralleled by a substantial increase in GABAergic connectivity ( Figure 2B, D) concomitant with an increase in Hes1 expression ( Figure 1). As seen following Hes1 transfection, p65/RelA transfection blocked the effects of Aβ on both dendrite morphology and connectivity, preventing the increase in dendrite length and the decrease in dendrite number induced by Aβ (Figure 2A, B and 2C).
Indeed, p65/RelA overexpression also prevented the decrease in VIAAT-positive terminals induced by Aβ. The anti-amyloid effects of p65/RelA overexpression on neuronal survival could not be studied, as p65/RelA overexpression induced neuronal death two days after transfection (that is, before Aβ had noxious effects on cultured neurons).
IKKb activation counteracts the effects of beta-amyloid NGF was shown to activate NF-B via tyrosine phosphorylation and the subsequent degradation of I-Bα [12]. The canonical NF-B activation pathway involves Ser (32,36) phosphorylation of I-Bα catalysed by the IKKs, and hence we tested whether increased activity of IKK proteins conferred amyloid resistance to cultured neurons. When cultured hippocampal neurons were transfected with a plasmid overexpressing IKKβ, again only 20 to 25% of neurons were transfected (data not shown). Nonetheless, the levels of Hes1 mRNA increased significantly (25 to 30%) throughout the entire culture ( Figure 3A). As expected, IKKβ overexpression produced similar alterations in neuronal morphology as Hes1 or p65/RelA transfection (longer and fewer dendrites) ( Figure 3B). In addition, IKKβ transfection conferred hippocampal neurons with resistance to Aβ ( Figure 3C, left panel). These effects were specific to IKKβ as transfection with IKKα produced no noticeable changes in dendrites (not shown) and conferred only very modest resistance to Aβ neurotoxicity ( Figure 3C, right panel).

Effects of TGFb1 on neuronal morphology, connectivity and survival
Based on the changes observed in dendrite morphology, and in neuronal connectivity and survival following Hes1 overexpression, we investigated the effects of an alternative means of activating NF-B using the cytokine TGFβ1 [22]. The role of TGFβ1 in neuronal polarity and axonal specification has been studied previously [23], and while TGFβ1-3 promote dendrite growth in retinal ganglion cells [24], the effects of TGFβ on neuronal plasticity remain unclear. We first studied the effects of TGFβ1 on dendritic patterning in cultured hippocampal neurons after 7 DIV. Exposure to TGFβ1 increased the number of primary dendrites while decreasing the number of remaining dendrites ( Figure 4A). Moreover, GABAergic connectivity was augmented in cultured hippocampal neurons treated with TGFβ1, as revealed by VIAAT immunostaining ( Figure 4A, right panel). Varicosities containing VIAAT also increased upon TGFβ1 administration. The effects of TGFβ1 were mediated by Hes1 as TGFβ1 had no such activity in neurons transfected with the Hes1 inhibitor Hes6 [19,20,25]. Impairment of Hes1 also prevented TGFβ1 from altering dendrite patterning and GABAergic connectivity.
Although the TGFβ1 transduction pathway that modulates neuronal plasticity is poorly understood (but see Discussion), TGFβ1 is known to activate NF-B in hippocampal neurons [22]. We found that this activation of NF-B by TGFβ1 may be involved in neuronal plasticity, and thus we analyzed the morphological changes induced  effect on TGFβ1 activity. Accordingly, the effects of TGFβ1 on neuronal plasticity are dependent upon serine phosphorylation of I-B and its capacity to activate NF-B. By contrast, overexpression of the tyrosine mutant form had no effect on TGFβ1 activity ( Figure 4B). In conjunction with previous findings [12], these results suggest that Hes1 can be activated by either NGF or TGFβ1. Both these factors activate NF-B, although the former degrades I-Bα by phosphorylation at tyrosine 42 [12] and the latter by phosphorylating serines 32 and 36.

Anti-amyloid effects of TGFb1
As Aβ prevents NGF-induced tyrosine phosphorylation and the subsequent degradation of I-Bα [12], we asked whether TGFβ1 could counteract the noxious effects of Aβ by activating NF-B after having promoted serine phosphorylation of I-Bα. Using a reporter gene luminescent assay, we demonstrated that TGFβ1 activated NF-B ( Figure 5A), more than doubling its activity in cultured neurons. Moreover, the reporter gene assay revealed a modest but significant decrease in NF-B activity produced by Aβ, which significantly failed to prevent TGFβ1-induced activation of NF-B. In accordance with this observation, we found that TGFβ1 increased neuronal Hes1 mRNA expression, while Aβ induced a significant reduction in Hes1 expression (Figure 5B). Indeed, exposure to TGFβ1 partially restored the low levels of Hes1 expression induced by Aβ. These results demonstrate that TGFβ1 opposes the effects of Aβ on NF-B activation and Hes1 expression. Based on the above findings, we examined the anti-amyloid effects of TGFβ1 on neuronal morphology, connectivity and survival ( Figure 5C-F). TGFβ1 blocked the effects of Aβ on dendrite length and number ( Figure 5C, D), and it prevented the Aβ-induced decrease in the number of GABAergic terminals ( Figure 5C, E). Finally, administration of TGFβ1 to cultured neurons protected about 50% of neurons from Aβ neurotoxicity Figure 5F). These . The I-Bα Y42F mutation that affects the tyrosine phosphorylation of the inhibitor had no effect on TGFβ1 activity. *P < 0.05, **P < 0.01, and ***P < 0.001. results emphasize the potential of TGFβ1 as a neuroprotective agent and reveal, at least in part, the molecular basis of this neuroprotective activity.

Discussion
The effect of NF-B activity on neuron survival Transcription factors, including NF-B, are implicated in experience-based synaptic regulation, and mouse models involving altered NF-B activity have revealed the importance of the various forms of this transcription factor in learning and memory (reviewed in [26]). NF-B may influence neuronal plasticity at multiple levels as it mediates neurite outgrowth [27] and participates in the development of dendritic spines [28]. NF-B also plays an important role in the dendritic development of Purkinje cells, since when it is inhibited with a lysyl oxidase peptide, serious deficits in dendritic arborisation are provoked [29]. Here, we show that p65/RelA transfection induces significant changes in the morphology of the dendrites emitted by cultured hippocampal neurons. These alterations were similar to those previously described for NGF, the effects of which are partially mediated by NF-B [13]. Overexpression of p65/RelA induces an increase in dendritic length and a decrease in dendrite number within 16 h. Perhaps more importantly, p65/RelA overexpression counteracts the effects of Aβ on dendrite morphology, decreasing dendrite length and increasing the number of primary dendrites. This finding provides an important indication of the anti-amyloid effects of p65/RelA overexpression.
NF-B exerts neuroprotective effects against some neurotoxic agents [30,31], including Aβ [32], and total abrogation of NF-B activation by pharmacological agents was followed by hippocampal neuron death ( Figure S2A in Additional file 2). However, with the exception of bcl-XL induction [33] and the suppression of apoptotic proteins such as Bax and Bim [34], the mechanisms underlying NF-B-induced neuroprotection remain elusive. In neurons, NF-B is required to maintain high GluR1 levels and neuronal hyperexcitability following the induction of longterm potentiation (LTP) [35]. However, increases in NF-B activity in response to enhanced excitatory transmission may accelerate the onset of the cognitive defect in a mouse model of Alzheimer's disease [36]. The present results show that p65/RelA promotes GABAergic connectivity in cultured hippocampal neurons, as revealed by the substantial increases in terminals containing VIAAT. We previously reported a substantial loss of VIAAT-labeled terminals shortly after Aβ administration in cultured hippocampal neurons [12] These effects were fully prevented by overexpressing p65/RelA, which may explain the basis underlying the anti-amyloid activity of NF-B.
Together with inhibitors of NF-B kinases, NF-B regulates many physiological responses, and activation of IKK in neurons should induce similar cellular changes to those elicited by p65/RelA overexpression. The canonical pathway of NF-B activation involves I-Bα phosphorylation via activation of the IKK complex (reviewed in [37]). Transfection of hippocampal neurons with a plasmid expressing IKKβ promoted dendritic growth while decreasing the number of primary dendrites. Furthermore, IKKβ transfection prevented Aβ from altering dendritic patterning. Most importantly, IKKβ overexpression protected a significant number of neurons from the deleterious effects of Aβ. Thus, canonical activation of NF-B conferred amyloid resistance to cultured hippocampal neurons.

The role of Hes1 in anti-amyloid neuroprotection
Hes1 is an important target of IKKβ/NF-B in terms of its influence on neuronal morphology and survival. Activation of NF-B by NGF increases Hes1 expression, whereas specific inhibition of this nuclear factor abrogates the activity of the neurotrophin [12,13] and it eventually caused cell death ( Figure S2A in Additional file 2). We found that overexpression of either IKKβ or p65/ RelA induces an increase in Hes1 expression in transfected hippocampal neurons. To further demonstrate that both the morphological and anti-amyloid effects of IKKβ and p65/RelA depend upon Hes1 expression, we transfected neurons with a vector that drives the overexpression of this gene. Hes1 overexpression induced the same morphological changes as seen following IKKβ or p65/RelA overexpression, including a large increase in GABAergic connectivity. As expected, Hes1 overexpression counteracted the effects of Aβ on cell morphology and GABAergic terminals, and strikingly, overexpression of Hes1 also rescued 50% of neurons from Aβ-induced death. Conversely, inhibition of Hes1 activity by overexpression of Hes6 induced cell death ( Figure S2B in Additional file 2). Together, these findings indicate that correct expression of Hes1 confers anti-amyloid resistance to cultured hippocampal neurons, strongly suggesting that strategies to increase Hes1 expression and activity may protect neurons from Aβ toxicity.

TGFb1 provides an alternative means of promoting Hes1 expression and inducing anti-amyloid activity
TGFβ1 has long been recognized as a neuroprotective agent (for a review see [38]) and indeed, neurodegeneration [39,40] and Aβ deposition [41] are enhanced in TGFβ1deficient mice. Furthermore, components of the hippocampal TGFβ pathway are altered in schizophrenia and psychiatric disorders [42]. Several TGFβ signalling pathways have been elucidated [43] and the canonical pathway involves the activation and nuclear localization of the Smad complex, where it modulates target gene transcription. However, our data suggest that the neuroprotective activity of TGFβ1 is not mediated by this canonical pathway but rather, by NF-B/Hes1. Administration of TGFβ1 to cultured neurons alters dendritic patterning and GABAergic connectivity in a manner consistent with Hes1 overexpression. Moreover, transfection with Hes6, an inhibitor of Hes1 transcriptional activity [19,20,25], abrogated all the effects of TGFβ1 on neuronal morphology and connectivity. Although Hes1 upregulation by TGFβ1 has been reported previously in fibroblasts [44], this is the first time the regulation of this bHLH gene by TGFβ1 has been described in hippocampal neurons.
Further assays of neuronal morphology and connectivity revealed the involvement of I-Bα in TGFβ1 signalling. Transfection with a serine mutant form of I-Bα abolishes the effects of TGFβ1 on both dendritic shape and on the number of GABAergic terminals. However, a tyrosine mutant form of I-Bα had no effect on TGFβ1 activity, indicating that serine phosphorylation of I-Bα preceded NF-B activation in this pathway. Direct biochemical measurements revealed that treatment of cultured neurons with TGFβ1 promoted NF-B activation and Hes1 expression. The activation of NF-B by TGFβ1 has been reported previously in cultured hippocampal neurons from rat embryos [22]. However, we also observed that TGFβ1 reversed the loss in NF-B activity and Hes1 expression induced by Aβ. Indeed, TGFβ1 also prevented the formation of VIAAT-positive clusters in response to Aβ, and it prevented Aβ from altering dendrite patterning. Most importantly, TGFβ1 rescued a significant portion of neurons from Aβ-induced death. These findings confirm the neuroprotective capacity of this cytokine and the underlying role of NF-B activation and Hes1 expression.
We showed previously that the action of Aβ is exerted in part by inhibiting early steps in the NGF signalling pathway, including the deactivation of RhoA and the activation of PTP1B, both events that are needed for NF-B activation and to promote Hes1 expression [15]. Accordingly, TGFβ1 can circumvent such effects by activating NF-B through an alternative mechanism that involves the serine phosphorylation of I-B, as shown here. However, when NF-B activation was pharmacologically impaired by SN-50 or when Hes1 activity was blocked by Hes6 overexpression, TGFβ1 activity was abrogated ( Figure S2A and S2B in Additional file 2). Together, these results suggest that on the one hand, NF-B activation and Hes1 activity are needed for cell survival, and on the other, that TGFβ1 exert its anti-amyloid activity by potentiating NF-B and Hes1 activities.

Modulation of GABAergic input in neuroprotection
Growth factors may control synaptic development and transmission in quantitative terms. Brain-derived neurotrophic factor (BDNF) upregulates glutamatergic input and downregulates the number of GABAergic synaptic terminals [45], while insulin promotes the postsynaptic accumulation of GABA A receptors by increasing Aktmediated phosphorylation of β subunits (reviewed in [46]). NGF also increases the expression of GABAergic terminals in cultured hippocampal neurons, an effect mediated by altering Hes1 expression [13]. Via the canonical pathway involving Smad4, TGFβ1 is a critical factor in use-dependent modulation of GABA A -mediated synaptic transmission and dendrite homeostasis [47].
In all experimental paradigms assayed here, including the transfection of cultured neurons with IKKβ, I-Bα or p65/RelA and the exposure of cultured neurons to TGFβ1, VIAAT immunocytochemistry indicated that there was a large increase in the expression of GABAergic terminals. These experimental approaches also consistently prevented Aβ from affecting GABAergic terminals, since exposure to Aβ decreases the number of GABAergic connections after 16 h and kills cells after 90 h. Thus, there is compelling evidence that Hes1 is an important element in the maintenance of GABAergic connectivity, although the mechanisms underlying this phenomenon remain unknown. The increase in GABAergic input promoted by NF-B/Hes1 may provide a negative feedback in the control of excitatory activity and consequently, protect neurons from excitotoxicity. The increase in inhibitory activity induced by NF-B Figure 6 Transforming growth factor b1 (TGFb1), nerve growth factor (NGF) and amyloid beta (Ab)-induced signaling in hippocampal neurons. Diagram outlining the signal transduction pathways downstream of TGFβ1 that promote Hes1 expression and neuronal survival. How NGF is impaired by Aβ is also illustrated. In contrast to NGF, the activation of NF-B and the increased expression of Hes1 induced by TGFβ1 are not inhibited by Aβ. activation or TGFβ1 administration may account for the neuroprotective effects observed. Indeed, TGFβ1 plays an important role in the excitatory/inhibitory balance of hippocampal transmission [47].

Conclusions
The findings presented here support the notion that neurons can be protected from the noxious effects of Aβ by modulating inhibitory transmission. Moreover, strategies that mildly activate NF-B and/or that enhance Hes1 expression could provide beneficial neuroprotection. Significantly, TGFβ1 signalling counteracts the inhibitory effect of Aβ on NGF signalling (summarized in Figure 6), possibly supplementing the attenuated activity of NGF in Alzheimer's disease and representing a potential target for the development of anti-amyloid therapies. . Lane (1) shows that the stock preparation mostly contained monomeric and dimeric species, with smaller quantities of trimeric and tetrameric forms. In lanes 2 to 5, Aβ was added to 35 mm culture dishes that containing one glass polylysine coated 2 × 2 cm coverslip and 2 mL of medium at the concentrations indicated. After a three-day incubation at 37°C, aliquots of the medium were taken and resolved by electrophoresis (supernatant, snt, lanes 2 and 3). Simultaneously, the glass coverslips were washed with LDS sample buffer and the material released was also separated in the same gels (immobilized on glass, imm, lanes 4 and 5). Note that the incubation of amyloid favoured the formation of higher molecular weight forms, although most species were small oligomers and the larger aggregates, including fibrils, only represented a small fraction of the amyloid. (B) Hippocampal neurons (7 days in vitro (DIV) and 30,000 cells/cm 2 ) were treated with Aβ as indicated. After 90 h, the cells were fixed and stained with 4',6-diamidino-2-phenylindole (DAPI) to asses the integrity of their nuclei. Note that Aβ (5 μM) produced a high rate of cell death, which justified the use of this concentration in further experiments. VF, microscope view field.

Additional material
Additional file 2: Figure showing the nuclear factor kappa B (NF-B) pathway and Hes1 activity are needed for the survival of neurons, while transforming growth factor b1 (TGFb1) is unable to rescue cells from death. E17 hippocampal neurons were plated at a density of 30,000 cells/cm 2 and cultured for 7 days in vitro (DIV). Neurons were (A) treated for 24 h with SN-50 (5 μM) or with its control peptide in the presence or absence of TGFβ1 (10 ng/ml). (B) Neurons were cotransfected with enhanced green fluorescent protein (EGFP) and a myctagged Hes6 vector for 48 h in the presence or absence of TGFβ1. The cells were fixed and labeled with anti-EGFP and anti-myc antibodies, while the integrity of their nuclei was assessed by 4',6-diamidino-2phenylindole (DAPI) staining. Note that the obliteration of either NF-B activation or Hes1 activity was followed by neuron death. The addition of TGFβ1 did not reverse these effects.