Influence of FOX genes on aging and aging-associated diseases. Elena Tschumak

It is known that vitamin D interacts with neurotrophic factors (NGF, NT3, NT4, GDNF) and its deficiency triggers neurodegenerative diseases in old age. (Jia et al, 2015) It would be of great interest to investigate if this effect is based on the interaction with FOXP2. Since FOXP2 seems to play an important role in many oncological diseases it would be also interesting to investigate if and to what extent this influence is controlled by vitamin D.

       FOXP2 regulation by internal factors Regulation by HuR According to Popovitchenko et al. (2016) „Depending on its degree of phosphorylation, the antigen R (HuR) dictates the amount of FOXP2 mRNA and the development of the neocorticals controlled by it, depending on its degree of phosphorylation“ HuR is the main factor in differential translation of autism-associated FoxP subfamily members in the developing neocortex subpopulation of the projection neurons. Regulation by Risperidone and NAP The study of a big Scottish family with severe mental disorders and schizophrenia (Liu et al. 2009) showed a balanced chromosomal translocation [(1:11) (q42.1; q14.3)] and an abnormally-truncated DisC1 protein (a microtubule-regulating and the FOXP2-influenced protein encoded on chromosome 1). The DISC1 is expressed in the cerebral cortex, in the hypothalamus, in the amygdala, in the hippocampal dentate gyrus, in the olfactory bulb and in the cerebellum. The truncated human DISC1 (hDISC1) alters its localization and can no longer interact with microtubules and microtubule-associated proteins. It leads to decreased dendritic growth and branching. Such anomalies have also been found in brain samples from patients with schizophrenia. (Harrison, 2004) This mutation has also been linked to depression and bipolar disorder. (Burdick et al., 2005) A connection between DISC1 and FOXP2 effects human verbal fluency as well as the ability to acquire spoken language. Several studies indicate that FOXP2 polymorphisms are in some cases associated with schizophrenia. (Nicodemus et al., 2014), (Walker et al., 2012), (Lai et al., 2001), (MacDermo et al., 2005), (Sanjuan et al., 2005), (Sanjuan et al. , 2006), (Tolosa et al., 2010) Vaisburd et al. (2015) showed in „Risperidone and NAP protect cognition and normalize gene expression in a schizophrenia mouse model“ that risperidone reduces effects of the DISC1 mutation. Risperidone is an analogue of the microtubule-stabilizing activity-dependent neuroprotective protein (ADNP) with a NAP (NAPVSIPQ) sequence and a SxIP motif (a microtubule junction for microtubule-end binding, responsible for microtubule dynamics and this is also important for synaptic plasticity and neuroprotection). (Oz et al., 2014), (Gozes et al., 2011), (Holtser-Cochav et al., 2006), (Jouroukhin et al., 2013), (Kumar & Wittmann, 2012).

      Both risperidone and NAP improved object recognition in the Morris water labyrinth. In contrast to Risperidone, NAP additionally reduced the anxiety in transgenic mice. Doxycycline blocked the expression of the mutated DISC1 gene. The candidate drugs were selected using bioinformatics programs and then affinity chromatographed. Mutations of the DISC1 gene were associated with increased FOXP2 level in hippocampus. FOXP2 levels could be significantly reduced by treatment with NAP, risperidone or their combination. This effect may be due to the blocking of the dopamine and 5HC2A serotonin receptors in the mesolimbic system, which leads to the reduction of negative schizophrenia symptoms. (Meltzer and McGurk, 1999), (Farde et al., 1995)

       An increased dopamine level leads to psychotic symptoms. The study by Mendoza et al. (2015), showed that FoxP-expressing neurons in Area-X also contain dopamine receptors 1A, 1B, and 2. Further studies may clarify whether the schizophrenic positive symptoms are due to the dopamine-FOXP2 interaction and how much FOXP 2 can influence dopamine dependent neurodegeneration in aging.

       Regulation of FOXP2 and other FOXP genes by SUMOilization

      

      Effect of sumoilization on the FOXP2 gene In „The Key Regulator for Language and Speech Development, FOXP2, is a Novel Substrate for SUMOylation“ Meredith et al. (2016) reported that the FOXP2 is covalently modified on its amino acid residues by both SUMO1 and SUMO3 at phylogenetically conserved lysine 674. The acid residues downstream of the FOXP2 SUMOylation motif are required for full SUMOylation capacity and modulation of its downstream target genes (DISC1, SRPX2 and MiR200c). Sumoylation is a form of posttranslational modification. The SUMOs (small ubiquitin-like modifiers) is a class of regulatory proteins that control the activity of the target protein and its interaction with other proteins. But the modification does not alter the localization and stability of FOXP2. The modulation effect can be reduced by SENP2 (a specific SUMOylation protease). It has also been observed that the human R553H mutation of FOXP2 reduces its SUMOylating ability compared to wild-type FOXP2. Isui et al. (2017) showed in „Sumoylation of FOXP2 Regulated Motor Function and Vocal Communication Through Purkinje Cell“ with the help of in utero electroporation that the SUMOylation of FOXP2 has, among others, an effect on the development of Purkinje cells and plays an important role in motor skills and vocal communication. They identified FOXP2 SUMOylation in the cerebellum of new-born K673 mice with the help of in vivo and in vitro coimmunoprecipitation and concluded that PIAS3 (an E3 ligase of the small ubiquitin-like modifier) catalyses FOXP2 SUMOylation. This SUMOylation modifies the transcription regulation of FOXP2. Estruch et al. (2016) showed in „The language-related transcription factor FOXP2 is post-translationally modified with small ubiquitin-li modifiers“ that post-translational FOXP2 modification is accomplished by members of the PIAS family. These members of the PIAS family are E3 ligases that promote SUMO transfer from the SUMO-conjugating enzyme UBC9 to an acceptor lysine residue of the target protein. (Rytinki et al., 2009) The SUMO2 and the SUMO3 are 95% identical. However, SUMO1 is only to 50% identical to SUMO2 and SUMO3 (Meulmeester and Melchior, 2008). Estruch research group studied various DNA constructs. They used myc-labelled PIAS1 and the His, mCherry and the YFP-labelled SUMOs and the bioluminescence resonance energy transfer assays. For FOXP2 detection they used Western blotting with the anti-V5 antibody and the anti-beta actin as reference. They took UBC9 enzyme to measure the extent of FOXP2 SUMOylation. During the study of FOXP2-PIAS interaction the working group used the BRET assay and the luciferase assays with the Renilla luciferase (Luc). Further methods were the gel-shift assay, the pull-down assay, the yeast two-hybrid assay and the fluorescence microscopy of HEK293 cells. The study revealed that FOXP-2 could be modified by all three human SUMO proteins and that PIAS1 promotes this process. The docking site of SUMO proteins in the FOXP1 and FOXP4 appears to be the most conserved N-terminal region of FOXP2 away from the polyglutamine tract. This region also interacts with the autism-relevant transcription factor TBR1. (Parikshak et al., 2013)

      Biochemical aspects of FOXP2 SUMOylation C-terminal carboxyl groups of SUMOS appear to interact with the amino group of FOXP2 via an isopeptide linkage. This amino group of the FOXP2 protein has a lysine side chain with the ΨKX (where Ψ is a hydrophobic amino acid and X is any other amino acid) consensus sequence . The SP-RING domain of the SUMO helps its FOXP2 recognition. N-terminal FOXP2 region contain some of the most important determinants of PIAS binding. These are K674 with PE motif and other residues, e.g. K285, K417 and K560 of the VE sequence. All of these residues are conserved in FOXP2. In this experiment a for SUMO conjugation required Arginine was removed and the SUMOLlyation did not happened. This showed that the K674 is the most important determinant for the SUMO1, SUMO2 and SUMO3 modifications. The mutants assay showed normal dimerization ability in the BRET, so the researchers concluded that the suppressed SUMOylation does not affect the FOXP2 ability to form homodimers or to interact with the CtBP129 co-repressor. This is because FOXP2 dimerization occurs with the help of the leucine zipper domain. However, the leucine zipper domain is not located near the SUMOylation site. This region includes domains that are highly conserved in FOXP1 and FOXP4. These domains serve as recognition sites for the autism-relevant transcription factor TBR1. This area is probably responsible for several protein-protein interactions. (Deriziotis et al., 2014) SUMOilization in FOXP1 / 4 genes Three SUMO proteomic studies of human cell lines identified FOXP1 and FOXP4 as substrates for SUMOilination. (Golebiowski et al., 2009; Tatham et al., 2011; Wen et al., 2014) FOXP1 and FOXP4 contain the SUMOylation relevant DNA motif that is identical to the corresponding FOXP2 motif of the surrounding PE sequence. In BRET study, FOXP1 also showed interactions with PIAS1, PIAS2, and PIAS4, but in contrast to FOXP2, FOXP1 showed little or no interaction with PIAS3. The FOXP1 also showed interactions with all three SUMOs. Probably FOXP1 is also Sumoylated by SUMO1, SUMO2 and SUMO3 at a location, like the K636 site of FOXP2. So, members of the PIAS