Masahito Shimojo

A Homozygous Mutation in Human PRICKLE1 Causes an Autosomal-Recessive Progressive Myoclonus Epilepsy-Ataxia Syndrome

Progressive myoclonus epilepsy (PME) is a syndrome characterized by myoclonic seizures (lightning-like jerks), generalized convulsive seizures, and varying degrees of neurological decline, especially ataxia and dementia. Previously, we characterized three pedigrees of individuals with PME and ataxia, where either clinical features or linkage mapping excluded known PME loci. This report identifies a mutation in PRICKLE1 (also known as RILP for REST/NRSF interacting LIM domain protein) in all three of these pedigrees. The identified PRICKLE1 mutation blocks the PRICKLE1 and REST interaction in vitro and disrupts the normal function of PRICKLE1 in an in vivo zebrafish overexpression system. PRICKLE1 is expressed in brain regions implicated in epilepsy and ataxia in mice and humans, and, to our knowledge, is the first molecule in the noncanonical WNT signaling pathway to be directly implicated in human epilepsy.

Protein Kinase A Regulates Cholinergic Gene Expression in PC12 Cells: REST4 Silences the Silencing Activity of Neuron-Restrictive Silencer Factor/REST

ABSTRACT The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A-deficient PC12 mutant, A126.1B2, in which transcription of the gene is reduced. The site of action of protein kinase A was localized to a neuron-restrictive silencer element/repressor element 1 (NRSE/RE-1) sequence within the cholinergic gene. Neuron-restrictive silencer factor (NRSF)/RE-1-silencing transcription factor (REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2 cells. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform REST4 was expressed in PC12 cells but not in A126.1B2. REST4 inhibited binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Coimmunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in A126.1B2 was sufficient to transcriptionally activate the cholinergic gene locus. Thus, in PC12 cells, protein kinase A promotes the production of REST4, which inhibits repression of the cholinergic gene locus by NRSF/REST.

Huntingtin Regulates RE1-silencing Transcription Factor/Neuron-restrictive Silencer Factor (REST/NRSF) Nuclear Trafficking Indirectly through a Complex with REST/NRSF-interacting LIM Domain Protein (RILP) and Dynactin p150Glued

Huntingtin has been reported to regulate the nuclear translocation of the transcriptional repressor RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). The REST/NRSF-interacting LIM domain protein (RILP) has also been shown to regulate REST/NRSF nuclear translocation. Therefore, we were prompted to address the question of how two distinct proteins could have the same function. We initially used a yeast two-hybrid screen to look for an interaction between huntingtin and RILP. This screen identified dynactin p150Glued as an interacting protein. Coimmunoprecipitation of proteins in vitro expressed in a reticulocyte lysate system showed an interaction between REST/NRSF and RILP as well as between RILP and dynactin p150Glued. Coimmunoprecipitation analysis further showed a complex containing RILP, dynactin p150Glued, and huntingtin. Huntingtin did not interact directly with either REST/NRSF or RILP, but did interact with dynactin p150Glued. The N-terminal fragment of wild-type huntingtin did not affect the interaction between dynactin p150Glued and RILP; however, mutant huntingtin weakened this interaction. We further show that HAP1 (huntingtin-associated protein-1) prevents this complex from translocating REST/NRSF to the nucleus. Thus, this study suggests that REST/NRSF, dynactin p150Glued, huntingtin, HAP1, and RILP form a complex involved in the translocation of REST/NRSF into the nucleus and that HAP1 controls REST/NRSF cellular localization in neurons.

REST/NRSF-Interacting LIM Domain Protein, a Putative Nuclear Translocation Receptor

ABSTRACT The transcriptional repressor REST/NRSF (RE-1 silencing transcription factor/neuron-restrictive silencer factor) and the transcriptional regulator REST4 share an N-terminal zinc finger domain structure involved in nuclear targeting. Using this domain as bait in a yeast two-hybrid screen, a novel protein that contains three LIM domains, putative nuclear localization sequences, protein kinase A phosphorylation sites, and a CAAX prenylation motif was isolated. This protein, which is localized around the nucleus, is involved in determining the nuclear localization of REST4 and REST/NRSF. We propose the name RILP, for REST/NRSF-interacting LIM domain protein, to label this novel protein. RILP appears to serve as a nuclear receptor for REST/NRSF, REST4, and possibly other transcription factors.

The Cholinergic Gene Locus Is Coordinately Regulated by Protein Kinase A II in PC12 Cells

Abstract: The vesicular acetylcholine transporter (VAChT) gene and the choline acetyltransferase (ChAT) gene comprise the cholinergic gene locus. We have studied the coordinate regulation of these genes by cyclic AMP‐dependent protein kinase (PKA) in the rat pheochromocytoma cell line PC12 and PC12 PKA‐deficient mutants. Both ChAT and VAChT mRNA increased approximately fourfold after treatment of PC12 cells with dibutyryl cyclic AMP (dbcAMP). ChAT and PKA activity were also increased by dbcAMP. The basal levels of ChAT and VAChT mRNAs in the PKA‐deficient cell lines were both about six times lower than in wild‐type PC12 cells, and were induced less than twofold by addition of dbcAMP. H‐89 and H‐9, specific inhibitors for PKA, reduced ChAT and VAChT mRNA levels to approximately one‐third that of untreated cells and ChAT activity to approximately one‐fourth that of untreated PC12 cells. Activation of PKA type II, but not PKA type I, increased ChAT activity approximately threefold. Analysis of reporter gene constructs indicates that PKA affects gene transcription at an upstream site in the cholinergic gene locus. These results demonstrate that the expression of the ChAT and VAChT genes is regulated coordinately at the transcriptional level, and a signaling pathway specifically involving PKA II plays an important role in this process.

Characterization of the REST/NRSF-interacting LIM domain protein (RILP): localization and interaction with REST/NRSF

We previously identified a nuclear envelope protein repressor element‐1 silencing transcription factor (REST)/neuron‐restrictive silencer factor (NRSF)‐interacting Lin‐11, Isl‐1 and Mec‐3 (LIM) domain protein (RILP) that we proposed functions in the nuclear translocation of the transcriptional repressor REST/NRSF. In this study we assessed the functionality of the prenylation motif, protein kinase A (PKA) phosphorylation sites and nuclear localization sequences (NLSs) of RILP. [3H]‐mevalonolactone labeled endogenous RILP, showing that RILP is indeed prenylated, while phosphorylation analysis showed that the two PKA sites are phosphorylated. Blocking RILP prenylation, mutating the NLSs or mutating the PKA phosphorylation sites caused RILP to mislocalize to the cytosol. Concurrent with this mislocalization of RILP, REST/NRSF and REST4, which are normally found in the nucleus, co‐localized in the cytosol with the RILP mutants. This provides additional evidence that RILP interacts with REST/NRSF and REST4 in vivo, and is involved in the nuclear localization of REST/NRSF and REST4. Reporter gene analysis using the promoter region of the human cholinergic gene locus revealed that these RILP mutants prevented repression of the reporter gene. By trapping REST/NRSF in the cytosol, the RILP mutants prevented translocation to the nucleus where REST/NRSF binds to an RE‐1/NRSE element to repress gene transcription. These results show that RILP is required for REST/NRSF nuclear targeting and function.

Characterization of the nuclear targeting signal of REST/NRSF.

RE-1 silencer transcription factor (REST), also known as neuron-restrictive silencer factor (NRSF), contains nine Cys2-His2 type zinc finger domains (ZFDs). REST/NRSF is localized to the nucleus, where it represses the transcriptional activity of a large number of neuronal genes in non-neuronal cells. It has been suggested that REST/NRSF contains a nuclear localization signal (NLS) corresponding to amino acids (512-522). However, our studies showed that REST4, a REST/NRSF splicing isoform, which contains the N-terminal 5 of 9 ZFDs, efficiently localized to the nucleus. On the other hand REST1, another REST/NRSF splicing isoform, which contains 4 of the 9 ZFDs, localized to the cytosol. In this study REST-DeltaC, which contains 8 ZFDs with the NLS (512-522) deleted, was found to localize to the nucleus in HeLa, COS and PC12 cells. Complete deletion or mutation of NLS (512-522) still permitted REST/NRSF to be localized to the nucleus in HeLa, COS and PC12 cells. In contrast REST/NRSF constructs which contain a deletion of ZFD-5 mislocalized to the cytosol. A point mutation in the zinc finger structure that disrupts its conformation remains nuclear. These data suggest that REST/NRSF contains a NLS around ZFD-5, while the putative NLS at residues 512-522 is non-functional.

Regulation of cholinergic gene expression by the neuron restrictive silencer factor/repressor element-1 silencing transcription factor.

The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A deficient PC12 mutant, A126.1B2 in which transcription of the locus is reduced. The site of action of protein kinase A was localized to a neuron restrictive silencer element/repressor element-1 (NRSE/RE-1) within the upstream region of the cholinergic gene locus. The neuron restrictive silencer factor/repressor element-1 silencing transcription factor (NRSF/REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform repressor element-1 silencing transcription factor-4 (REST4) was found to be expressed in PC12 cells, but not in the protein kinase A deficient PC12 cell line. REST4 inhibited the binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Co-immunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in the protein kinase A deficient PC12 cell line was sufficient to transcriptionally activate the cholinergic gene locus. Thus in PC12 cells protein kinase A promotes the production of REST4, which in turn de-represses of the cholinergic gene locus by inactivating the transcription repressor NRSF/REST.

Regulation of the cholinergic gene locus

DNase I hypersensitive site mapping of the human cholinergic gene locus has been used to detect cholinergic specific potential regulatory sites. Analysis of mutant PC12 cell lines provides evidence that protein kinase A II is required and coordinately regulates basal expression of both the ChAT and VAChT genes.