Marco F.M. Hoekman

The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation.

FoxO (forkhead box O; forkhead members of the O class) are transcription factors that function under the control of insulin/insulin-like signalling. FoxO factors have been associated with a multitude of biological processes, including cell-cycle, cell death, DNA repair, metabolism and protection from oxidative stress. Central to the regulation of FoxO factors is a shuttling system, which confines FoxO factors to either the nucleus or the cytosol. Shuttling of FoxO requires protein phosphorylation within several domains, and association with 14-3-3 proteins and the nuclear transport machinery. Description of the FoxO-shuttling mechanism contributes to the understanding of FoxO function in relation to signalling and gene regulation.

Sciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome

Peripheral nerve regeneration has been studied extensively in the sciatic nerve crush model, at the level of both function and gene expression. The crush injury allows full recovery of sensory and motor function in about 3 weeks as assessed by the foot reflex withdrawal test and De Medinacelli walking patterns. We used the recently developed CatWalk paradigm to study walking patterns in more detail in mice and rats. We found that, following the recovery of sensory function, the animals developed a state of mechanical allodynia, which retreated slowly over time. The motor function, although fully recovered with the conventional methods, was revealed to be still impaired because the animals did not put weight on their previously injured paw. The development of neuropathic pain following successful sensory recovery has not been described before in crush-lesioned animals and may provide an important new parameter to assess full sensory recovery.

Active immunisation of human interferon α transgenic mice with a human interferon α Kinoid induces antibodies that neutralise interferon α in sera from patients with systemic lupus erythematosus

Objectives Interferon α (IFNα) plays a central role in the pathogenesis of systemic lupus erythematosus (SLE) and is considered a target for its treatment. In the current study, the ability of active immunisation with a human (hu) IFNα2b Kinoid (IFN-K) to break B cell tolerance to IFNα and to induce huIFNα-neutralising antibodies in mice immunotolerant to huIFNα2b was assessed. Methods IFN-K was manufactured by crosslinking huIFNα2b to keyhole limpet haemocyanin (KLH). Transgenic mice expressing huIFNα2b received by intramuscular injection either saline or polymerised huIFNα2b as controls, or IFN-K, emulsified in ISA51vg adjuvant. Results All of the huIFNα2b-expressing mice immunised with IFN-K generated neutralising antibodies against huIFNα2b. In addition, these antibodies neutralised all 13 subtypes of huIFNα. They also neutralised IFNα activity in sera collected from 10 different patients with active SLE. However, the antibodies did not bind to huIFNγ or huIFNβ. Finally, cellular activation assays showed that immunisation with IFN-K did not induce memory T cells reactive to native huIFNα2b, whereas it did induce memory cells reactive to KLH. Conclusion These results show that active immunisation with IFN-K induces polyclonal antibodies that neutralise all subtypes of huIFNα as well as IFNα in sera from patients with SLE by breaking humoral but not cellular tolerance to IFNα. This suggests that immunisation with IFN-K is a promising new therapeutic strategy for the treatment of SLE.

Insulin-FOXO3 signaling modulates circadian rhythms via regulation of clock transcription

Circadian rhythms are responsive to external and internal cues, light and metabolism being among the most important. In mammals, the light signal is sensed by the retina and transmitted to the suprachiasmatic nucleus (SCN) master clock [1], where it is integrated into the molecular oscillator via regulation of clock gene transcription. The SCN synchronizes peripheral oscillators, an effect that can be overruled by incoming metabolic signals [2]. As a consequence, peripheral oscillators can be uncoupled from the master clock when light and metabolic signals are not in phase. The signaling pathways responsible for coupling metabolic cues to the molecular clock are being rapidly uncovered [3-5]. Here we show that insulin-phosphatidylinositol 3-kinase (PI3K)-Forkhead box class O3 (FOXO3) signaling is required for circadian rhythmicity in the liver via regulation of Clock. Knockdown of FoxO3 dampens circadian amplitude, an effect that is rescued by overexpression of Clock. Subsequently, we show binding of FOXO3 to two Daf-binding elements (DBEs) located in the Clock promoter area, implicating Clock as a transcriptional target of FOXO3. Transcriptional oscillation of both core clock and output genes in the liver of FOXO3-deficient mice is affected, indicating a disrupted hepatic circadian rhythmicity. Finally, we show that insulin, a major regulator of FOXO activity [6-9], regulates Clock levels in a PI3K- and FOXO3-dependent manner. Our data point to a key role of the insulin-FOXO3-Clock signaling pathway in the modulation of circadian rhythms.

Insulin inhibits extracellular regulated kinase 1/2 phosphorylation in a phosphatidylinositol 3-kinase (PI3) kinase-dependent manner in Neuro2a cells

Insulin signalling is well studied in peripheral tissue, but not in neuronal tissue. To gain more insight into neuronal insulin signalling we examined protein kinase B (PKB) and extracellular regulated kinase 1 and 2 (ERK1/2) regulation in serum‐deprived Neuro2a cells. Insulin phosphorylated PKB in a dose‐dependent manner but reduced phosphorylation of ERK1/2. Both processes were phosphatidylinositol 3‐kinase (PI3K) dependent. Interestingly, blockade of PI3K in combination with insulin induced phosphorylation of ERK1/2. The phosphorylation of ERK1/2 could be blocked with a specific inhibitor of mitogen‐activated protein/ERK kinase (MEK), suggesting that it was mediated through the highly conserved Ras–Raf–MEK–ERK1/2 pathway. Prolonged exposure to high concentrations of insulin resulted in a desensitized PI3K–PKB route. The insulin‐induced inhibition of ERK1/2 phosphorylation was also diminished when the PI3K–PKB route was desensitized. Blockade of PI3K in combination with insulin, however, still resulted in an unaltered MEK‐dependent phosphorylation of ERK1/2. We conclude that PI3K is an important integrator of insulin signalling in Neuro2a cells as it regulates activation of PKB and inhibition of ERK1/2, and is sensitive to the duration of the insulin stimulus.

Identification of forkhead transcription factors in cortical and dopaminergic areas of the adult murine brain.

The murine forkhead family of transcription factors consists of over 30 members, the vast majority of which is important in embryonic development. Implicated in processes such as proliferation, differentiation and survival, forkhead factors show highly restricted expression patterns. In search for forkhead genes expressed in specific neural systems, we identified multiple family members. We performed a detailed expression analysis for Foxj2, Foxk1 and the murine orthologue of the human ILF1 gene, which show a remarkable preference for complex cortical structures. In addition, a comprehensive examination of forkhead gene expression in dopamine neurons of the ventral tegmental area and substantia nigra pars compacta, revealed Ilf1 as a novel transcriptional regulator in midbrain dopamine neurons. These forkhead transcription factors may play a role in maintenance and survival of developing and adult neurons.

DEVELOPMENTAL REGULATION OF HOMEOBOX GENE EXPRESSION IN DORSAL ROOT GANGLION NEURONS IS NOT RECAPITULATED DURING REGENERATION OF THE CRUSHED SCIATIC NERVE

The adult peripheral nervous system is able to regenerate after injury. Regeneration is associated with the expression of new genes and proteins. Proteins abundant in developing axons increase in expression after injury, whereas proteins involved in neurotransmission are downregulated. It has been hypothesized that molecular mechanisms underlying regeneration-associated alterations in gene expression may be a recapitulation of developmental processes. These gene expression changes are likely to be regulated by changes in the gene expression of transcription factors. As homeobox genes play important roles in embryonic development of the nervous system, it makes them candidates for a regulatory role in the process of regeneration. Here we show that the relative mRNA expression levels of Isl1 decreased shortly after crush, but those of DRG11, Lmx1b, and Pax3 did not change after crush. These data indicate that the developmental expression patterns of the homeobox genes studied here are not recapitulated during regeneration of the dorsal root ganglia neurons. We conclude that developmental gene expression programs controlled by these homeobox genes are not directly involved in sciatic nerve regeneration.

Spatial and temporal lineage analysis of a Pitx3-driven Cre-recombinase knock-in mouse model.

Development and function of mesodiencephalic dopaminergic (mdDA) neurons has received a lot of scientific interest since these neurons are critically involved in neurological diseases as Parkinson and psychiatric diseases as schizophrenia, depression and attention deficit hyperactivity disorder (ADHD). The understanding of the molecular processes that lead to normal development and function of mdDA neurons has provided insight in the pathology and provided critical information on new treatment paradigms. In order to be able to study specific genetic ablation in mdDA neurons a new tools was developed that drives Cre-recombinase under the control of the Pitx3 locus. The Pitx3 gene is well known for its specific expression in mdDA neurons and is present at the onset of terminal differentiation. Analysis of newly generated Pitx3-Cre knock-in mice shows that Cre expression, measured through the activation of eYfp by removal of a “Stop” signal (LoxP-Stop-LoxP-eYfp reporter mouse), is present at the onset of terminal differentiation and mimics closely the native Pitx3 expression domain. In conclusion, we present here a new Cre-driver mouse model to be used in the restricted ablation of interesting genes in mdDA neurons in order to improve our understanding of the underlying molecular programming.

The Stellate Ganglion of the Squid Loligo pealeii as a Model for Neuronal Development: Expression of a POU Class VI Homeodomain Gene, Rpf-1

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FoxK2 is Required for Cellular Proliferation and Survival

FoxK2 is a forkhead transcription factor expressed ubiquitously in the developing murine central nervous system. Here we investigated the role of FoxK2 in vitro and focused on proliferation and cellular survival. Knockdown of FoxK2 results in a decrease in BrdU incorporation and H3 phosphorylation, suggesting attenuation of proliferation. In the absence of growth factors, FoxK2 knockdown results in a dramatic increase in caspase 3 activity and propidium iodide positive cells, indicative of cell death. Additionally, knockdown of FoxK2 results in an increase in the mRNA of Gadd45α, Gadd45γ, as well as an increase in the phosphorylation of the mTOR dependent kinase p70S6K. Rapamycin treatment completely blocked the increase in p70S6K and synergistically potentiated the decrease in H3 phosphorylation upon FoxK2 knockdown. To gain more insight into the proapoptotic effects upon FoxK2 knockdown we screened for changes in Bcl2 genes. Upon FoxK2 knockdown both Puma and Noxa were significantly upregulated. Both genes were not inhibited by rapamycin treatment, instead rapamycin increased Noxa mRNA. FoxK2 requirement in cellular survival is further emphasized by the fact that resistance to TGFβ‐induced cell death was greatly diminished after FoxK2 knockdown. Overall our data suggest FoxK2 is required for proliferation and survival, that mTOR is part of a feedback loop partly compensating for FoxK2 loss, possibly by upregulating Gadd45s, whereas cell death upon FoxK2 loss is induced in a Bcl2 dependent manner via Puma and Noxa. J. Cell. Physiol. 230: 1013–1023, 2015.