Motoneuron-Specific PTEN Deletion in Mice Induces Neuronal Hypertrophy and Also Regeneration after Facial Nerve Injury.

In postmitotic neurons, several tumor suppressor genes (TSGs), including p53, Rb, and PTEN, modulate the axon regeneration success after injury. Particularly, PTEN inhibition is a key driver of successful CNS axon regeneration after optic nerve or spinal cord injury. In contrast, in peripheral neurons, TSG influence in neuronal morphology, physiology, and pathology has not been investigated to the same depth. In this study, we conditionally deleted PTEN from mouse facial motoneurons (Chat-Cre/PtenloxP/loxP ) and analyzed neuronal responses in vivo with or without peripheral facial nerve injury in male and female mice. In uninjured motoneurons, PTEN loss induced somatic, axonal, and nerve hypertrophy, synaptic terminal enlargement and reduction in physiological whisker movement. Despite these morphologic and physiological changes, PTEN deletion positively regulated facial nerve regeneration and recovery of whisker movement after nerve injury. Regenerating PTEN-deficient motoneurons upregulated P-CREB and a signaling pathway involving P-Akt, P-PRAS40, P-mTOR, and P-4EBP1. In aged mice (12 months), PTEN deletion induced hair loss and facial hyperplasia of the epidermis. This suggests a time window in younger mice with PTEN loss stimulating axon growth after injury, however, at the risk of hyperplasia formation at later time points in the old animal. Overall, our data highlight a dual TSG function with PTEN loss impairing physiological neuron function but furthermore underscoring the positive effects of PTEN ablation in axon regeneration also for the PNS.SIGNIFICANCE STATEMENT Tumor suppressor genes (TSGs) restrict cell proliferation and growth. TSG inhibition, including p53 and PTEN, stimulates axon regeneration after CNS injury. In contrast, in PNS axon regeneration, TSGs have not been analyzed in great depth. Herein we show enhanced peripheral axon regeneration after PTEN deletion from facial motoneurons. This invokes a signaling cascade with novel PTEN partners, including CREB and PRAS40. In adult mice, PTEN loss induces hyperplasia of the skin epidermis, suggesting detrimental consequences when reaching adulthood in contrast to a beneficial TSG role for regeneration in young adult mice. Thus, our data highlight the double-edged sword nature of interfering with TSG function.

STAT6 mediates the effect of ethanol on neuroinflammatory response in TBI.

Traumatic brain injury (TBI) and ethanol intoxication (EI) frequently coincide, particularly in young subjects. However, the mechanisms of their interaction remain poorly understood. Among other pathogenic pathways, TBI induces glial activation and neuroinflammation in the hippocampus, resulting in acute and chronic hippocampal dysfunction. In this regard, we investigated the role of EI affecting these responses unfolding after TBI. We used a blunt, weight-drop approach to model TBI in mice. Male mice were pre-administered with ethanol or vehicle to simulate EI. The neuroinflammatory response in the hippocampus was assessed by monitoring the expression levels of >20 cytokines, the phosphorylation status of transcription factors and the phenotype of microglia and astrocytes. We used AS1517499, a brain-permeable STAT6 inhibitor, to elucidate the role of this pathway in the EI/TBI interaction. We showed that TBI causes the elevation of IL-33, IL-1ß, IL-38, TNF-α, IFN-α, IL-19 in the hippocampus at 3 h time point and concomitant EI results in the dose-dependent downregulation of IL-33, IL-1ß, IL-38, TNF-α and IL-19 (but not of IFN-α) and in the selective upregulation of IL-13 and IL-12. EI is associated with the phosphorylation of STAT6 and the transcription of STAT6-controlled genes. Moreover, ethanol-induced STAT6 phosphorylation and transcriptional activation can be recapitulated in vitro by concomitant exposure of neurons to ethanol, depolarization and inflammatory stimuli (simulating the acute trauma). Acute STAT6 inhibition prevents the effects of EI on IL-33 and TNF-α, but not on IL-13 and negates acute EI beneficial effects on TBI-associated neurological impairment. Additionally, EI is associated with reduced microglial activation and astrogliosis as well as preserved synaptic density and baseline neuronal activity 7 days after TBI and all these effects are prevented by acute administration of the STAT6 inhibitor concomitant to EI. EI concomitant to TBI exerts significant immunomodulatory effects on cytokine induction and microglial activation, largely through the activation of STAT6 pathway, ultimately with beneficial outcomes.

Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury.

Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce 'effector' RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types.

The transcription factor serum response factor stimulates axon regeneration through cytoplasmic localization and cofilin interaction.

Axonal injury generates growth inert retraction bulbs with dynamic cytoskeletal properties that are severely compromised. Conversion of "frozen" retraction bulbs into actively progressing growth cones is a major aim in axon regeneration. Here we report that murine serum response factor (SRF), a gene regulator linked to the actin cytoskeleton, modulates growth cone actin dynamics during axon regeneration. In regeneration-competent facial motoneurons, Srf deletion inhibited axonal regeneration. In wild-type mice after nerve injury, SRF translocated from the nucleus to the cytoplasm, suggesting a cytoplasmic SRF function in axonal regeneration. Indeed, adenoviral overexpression of cytoplasmic SRF (SRF-ΔNLS-GFP) stimulated axonal sprouting and facial nerve regeneration in vivo. In primary central and peripheral neurons, SRF-ΔNLS-GFP stimulated neurite outgrowth, branch formation, and growth cone morphology. Furthermore, we uncovered a link between SRF and the actin-severing factor cofilin during axonal regeneration in vivo. Facial nerve axotomy increased the total cofilin abundance and also nuclear localization of phosphorylated cofilin in a subpopulation of lesioned motoneurons. This cytoplasmic-to-nucleus translocation of P-cofilin upon axotomy was reduced in motoneurons expressing SRF-ΔNLS-GFP. Finally, we demonstrate that cytoplasmic SRF and cofilin formed a reciprocal regulatory unit. Overexpression of cytoplasmic SRF reduced cofilin phosphorylation and vice versa: overexpression of cofilin inhibited SRF phosphorylation. Therefore, a regulatory loop consisting of SRF and cofilin might take part in reactivating actin dynamics in growth-inert retraction bulbs and facilitating axon regeneration.

Paracrine control of oligodendrocyte differentiation by SRF-directed neuronal gene expression.

In neurons, serum response factor (SRF)-directed transcription regulates migration, axon pathfinding and synapse function. We found that forebrain-specific, neuron-restricted SRF ablation in mice elevated oligodendrocyte precursors while inhibiting terminal oligodendrocyte differentiation. Myelin gene and protein expression were downregulated and we observed a lack of oligodendrocytes in mixed neuron/glia and oligodendrocyte-enriched cultures derived from Srf(-/-) mutants. Ultrastructural inspection revealed myelination defects and axonal degeneration in Srf(-/-) mutants. Consistent with our finding that neuronal SRF depletion impaired oligodendrocyte fate in a non-cell autonomous manner, neuron-restricted expression of constitutively active SRF-VP16 affected neighboring oligodendrocyte maturation. Genome-wide transcriptomics identified candidate genes for paracrine regulation of oligodendrocyte development, including connective tissue growth factor (CTGF), whose expression is repressed by SRF. Adenovirus-mediated CTGF expression in vivo revealed that CTGF blocks excessive oligodendrocyte differentiation. In vitro, CTGF-mediated inhibition of oligodendrocyte maturation involved sequestration and thereby counteraction of insulin growth factor 1-stimulated oligodendrocyte differentiation.