Dynamic subcellular localization and transcription activity of the SRF cofactor MKL2 in the striatum are regulated by MAPK.

Dopamine type 1 receptor (D1R) signaling activates protein kinase A (PKA), which then activates mitogen-activated protein kinase (MAPK) through Rap1, in striatal medium spiny neurons (MSNs). MAPK plays a pivotal role in reward-related behavior through the activation of certain transcription factors. How D1R signaling regulates behavior through transcription factors remains largely unknown. CREB-binding protein (CBP) promotes transcription through hundreds of different transcription factors and is also important for reward-related behavior. To identify transcription factors regulated by dopamine signaling in MSNs, we performed a phosphoproteomic analysis using affinity beads coated with CBP. We obtained approximately 40 novel candidate proteins in the striatum of the C57BL/6 mouse brain after cocaine administration. Among them, the megakaryoblastic leukemia-2 (MKL2) protein, a transcriptional coactivator of serum response factor (SRF), was our focus. We found that the interaction between CBP and MKL2 was increased by cocaine administration. Additionally, MKL2, CBP and SRF formed a ternary complex in vivo. The C-terminal domain of MKL2 interacted with CBP-KIX and was phosphorylated by MAPK in COS7 cells. The activation of PKA-MAPK signaling induced the nuclear localization of MKL2 and increased SRF-dependent transcriptional activity in neurons. These results demonstrate that dopamine signaling regulates the interaction of MKL2 with CBP in a phosphorylation-dependent manner and thereby controls SRF-dependent gene expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15067.

Phosphorylation of Npas4 by MAPK Regulates Reward-Related Gene Expression and Behaviors.

Dopamine (DA) activates mitogen-activated protein kinase (MAPK) via protein kinase A (PKA)/Rap1 in medium spiny neurons (MSNs) expressing the dopamine D1 receptor (D1R) in the nucleus accumbens (NAc), thereby regulating reward-related behavior. However, how MAPK regulates reward-related learning and memory through gene expression is poorly understood. Here, to identify the relevant transcriptional factors, we perform proteomic analysis using affinity beads coated with cyclic AMP response element binding protein (CREB)-binding protein (CBP), a transcriptional coactivator involved in reward-related behavior. We identify more than 400 CBP-interacting proteins, including Neuronal Per Arnt Sim domain protein 4 (Npas4). We find that MAPK phosphorylates Npas4 downstream of PKA, increasing the Npas4-CBP interaction and the transcriptional activity of Npas4 at the brain-derived neurotrophic factor (BDNF) promoter. The deletion of Npas4 in D1R-expressing MSNs impairs cocaine-induced place preference, which is rescued by Npas4-wild-type (WT), but not by a phospho-deficient Npas4 mutant. These observations suggest that MAPK phosphorylates Npas4 in D1R-MSNs and increases transcriptional activity to enhance reward-related learning and memory.

Pioneering axons regulate neuronal polarization in the developing cerebral cortex.

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

Induction of peroxisomal lipid metabolism in mice fed a high-fat diet.

Peroxisomes catalyze a range of essential metabolic functions, mainly related to lipid metabolism. However, their roles in obesity have yet to be clarified. The aim of this study was to investigate the correlation between obesity and peroxisomal lipid metabolism, particularly very long-chain fatty acid (VLCFA) metabolism, gene expression of peroxisomal ß-oxidation enzymes, peroxisomal ATP-binding cassette (ABC) transporter adrenoleukodystrophy (ABCD1) gene and its related gene, ABCD2, the elongation of the VLCFA (ELOVL) gene family and the transcriptional factors involved in the regulation of these genes, including peroxisome proliferator-activated receptor α (PPARα) and sterol regulatory element-binding protein. These factors were analyzed in livers from mice fed a high-fat diet (HFD) or a regular diet (RD) for 20 weeks. Furthermore, the amounts of plasma saturated and unsaturated fatty acids, including VLCFAs, were measured. A HFD induced hepatic gene expression of not only hydroxysteroid 17-ß dehydrogenase 4 (HSD17b4) and sterol carrier protein 2 (SCP2) in peroxisomal ß-oxidation enzymes but also of ELOVL1, 2, 5 and 6, which are involved in the elongation of saturated and unsaturated VLCFAs. Furthermore, ABCD2 mRNA prominently increased in the HFD mice. The transcriptional regulator of these genes, PPARα, was also up-regulated in the HFD mice. VLCFA ratios including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0 are the most significant diagnostic markers of inherited peroxisomal diseases. These ratios were found to be low in the plasma of the HFD mice compared with the RD mice. The results suggest that HFD activates hepatic peroxisomal VLCFA metabolism, and may provide useful fundamental information to explain the role of peroxisomal function in obesity and lifestyle-related diseases.

X-linked adrenoleukodystrophy: diagnostic and follow-up system in Japan.

X-linked adrenoleukodystrophy (ALD) is an intractable neurodegenerative disease associated with the accumulation of very long-chain saturated fatty acids (VLCFA) in tissues and body fluids. We have established a Japanese referral center for the diagnosis of ALD, using VLCFA measurements and mutation analysis of the ABCD1 gene, and have identified 60 kinds of mutations in 69 Japanese ALD families, which included 38 missense mutations, 6 nonsense mutations, 8 frame-shift mutations, 3 amino acid deletions, 2 exon-skip mutations and 3 large deletions. A total of 24 kinds of mutations (40%) were identified only in Japanese patients by referring to the current worldwide ALD mutation database. There was no clear correlation between these mutations and phenotypes of 81 male patients in these 69 families. About 12% of the individuals with ALD had de novo mutations by mutation analysis in the male probands and their mothers, which should be helpful data for genetic counseling. The only effective therapy for the cerebral form of ALD should be hematopoietic stem cell transplantation at the early stages of the cerebral symptoms, therefore, we performed presymptomatic diagnosis of ALD by extended familial screening of the probands with careful genetic counseling, and established a long follow-up system for these patients to prevent the progression of brain involvement and to monitor the adrenocortical insufficiency. Further elucidation of pathology in ALD, especially concerning the mechanisms of the onset of brain involvement, is expected.