The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs PR50 and GR50 are highly toxic when expressed in Caenorhabditis elegans, and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNA interference (RNAi) screen for suppressors of PR50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by PR50 expression. All of these genes have vertebrate homologs, and 7 of 12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for GR50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that knockdown of bromodomain proteins in both C. elegans and mammalian neurons, which are known SPOP ubiquitination targets, suppresses the protective effect of SPOP inhibition. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent an entry point for therapeutic intervention to treat C9orf72 FTD/ALS.

The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting.

A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via 'tethering proteins' that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We previously identified a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify an evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation and dephosphorylation of the LBD. Furthermore, we identify a PP2A phosphatase recognition motif within the evolutionarily conserved C-terminal Tantalus domain of PRR14. Disruption of this motif affects PRR14 localization to the nuclear lamina. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two separable modular domains. Our findings also describe an optimal PRR14 LBD fragment that could be used for efficient targeting of fusion proteins to the nuclear lamina.

Neurons undergo pathogenic metabolic reprogramming in models of familial ALS.

OBJECTIVES: Normal cellular function requires a rate of ATP production sufficient to meet demand. In most neurodegenerative diseases (including Amyotrophic Lateral Sclerosis [ALS]), mitochondrial dysfunction is postulated raising the possibility of impaired ATP production and a need for compensatory maneuvers to sustain the ATP production/demand balance. We investigated intermediary metabolism of neurons expressing familial ALS (fALS) genes and interrogated the functional consequences of glycolysis genes in fitness assays and neuronal survival. METHODS: We created a pure neuronal model system for isotopologue investigations of fuel utilization. In a yeast platform we studied the functional contributions of glycolysis genes in a growth fitness assay iafter expressing of a fALS gene. RESULTS: We find in our rodent models of fALS, a reduction in neuronal lactate production with maintained or enhanced activity of the neuronal citric acid cycle. This rewiring of metabolism is associated with normal ATP levels, bioenergetics, and redox status, thus supporting the notion that gross mitochondrial function is not compromised in neurons soon after expressing fALS genes. Genetic loss-of-function manipulation of individual steps in the glycolysis and the pentose phosphate pathway blunt the negative phenotypes seen in various fALS models. CONCLUSIONS: We propose that neurons adjust fuel utilization in the setting of neurodegenerative disease-associated alteration in mitochondrial function in a baleful manner and targeting this process can be healthful.

The Protein Encoded by the CCDC170 Breast Cancer Gene Functions to Organize the Golgi-Microtubule Network.

Genome-Wide Association Studies (GWAS) and subsequent fine-mapping studies (>50) have implicated single nucleotide polymorphisms (SNPs) located at the CCDC170/C6ORF97-ESR1 locus (6q25.1) as being associated with the risk of breast cancer. Surprisingly, our analysis using genome-wide differential allele-specific expression (DASE), an indicator for breast cancer susceptibility, suggested that the genetic alterations of CCDC170, but not ESR1, account for GWAS-associated breast cancer risk at this locus. Breast cancer-associated CCDC170 nonsense mutations and rearrangements have also been detected, with the latter being specifically implicated in driving breast cancer. Here we report that the wild type CCDC170 protein localizes to the region of the Golgi apparatus and binds Golgi-associated microtubules (MTs), and that breast cancer-linked truncations of CCDC170 result in loss of Golgi localization. Overexpression of wild type CCDC170 triggers Golgi reorganization, and enhances Golgi-associated MT stabilization and acetyltransferase ATAT1-dependent α-tubulin acetylation. Golgi-derived MTs regulate cellular polarity and motility, and we provide evidence that dysregulation of CCDC170 affects polarized cell migration. Taken together, our findings demonstrate that CCDC170 plays an essential role in Golgi-associated MT organization and stabilization, and implicate a mechanism for how perturbations in the CCDC170 gene may contribute to the hallmark changes in cell polarity and motility seen in breast cancer.