Artificial microRNA suppresses C9ORF72 variants and decreases toxic dipeptide repeat proteins in vivo.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons, causing progressive muscle weakness and respiratory failure. The presence of an expanded hexanucleotide repeat in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent mutation causing familial ALS and frontotemporal dementia (FTD). To determine if suppressing expression of C9ORF72 gene products can reduce toxicity, we designed a set of artificial microRNAs (amiRNA) targeting the human C9ORF72 gene. Here we report that an AAV9-mediated amiRNA significantly suppresses expression of the C9ORF72 mRNA, protein, and toxic dipeptide repeat proteins generated by the expanded repeat in the brain and spinal cord of C9ORF72 transgenic mice.

Contrast-enhanced mammography versus breast MRI in the assessment of multifocal and multicentric breast cancer: a retrospective study.

BACKGROUND: Breast cancer multifocality and multicentricity diagnosis influences the surgeon's choice between applying breast conservative therapy or performing mastectomy. PURPOSE: To assess the role of contrast enhanced mammography (CEM) and breast magnetic resonance imaging (MRI) in the assessment of preoperative breast cancer multifocality and multicentricity and to assess their accuracy, agreement and impact on the surgical management. MATERIAL AND METHODS: The study retrospectively included cases over a 5-year period. After analysis and interpretation of suspicious breast lesions, a comparative evaluation of CEM and MRI was conducted with the assessment of diagnostic indices, including sensitivity, specificity and diagnostic accuracy. The kappa (κ) measure of agreement between both modalities was measured. The postoperative specimen pathology was the reference standard. RESULTS: One hundred and twenty-two female cases with 126 breast lesions were evaluated. Specimen pathology, MRI and CEM showed a single neoplastic lesion in 67.5%, 35% and 48.5% of cases, respectively, and multiple neoplastic lesions in 32.5%, 65% and 51.6% of cases, respectively. The sensitivity, specificity and accuracy of MRI were 95.12%, 49.41%,and 64.29%, and the CEM values were 85.37%, 64.71% and 71.43%, respectively. The κ value was 0.592 with an intermediate agreement between both modalities. When comparing between both modalities, enhancing foci showed a statistically significant difference, although there were no statistically significant difference in terms of high breast density or molecular subtype. CONCLUSION: In terms of breast cancer multifocality and multicentricity evaluation, MRI showed a higher sensitivity, while CEM showed a higher specificity, and there was moderate agreement between the two modalities.

FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions.

C9ORF72 repeat expansion is the most common genetic mutation in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Abnormal dipeptide repeat proteins (DPRs) generated from repeat-associated non-AUG (RAN) translation of repeat-containing RNAs are thought to be pathogenic; however, the mechanisms are unknown. Here we report that (GR)80 and (PR)80 are toxic in neuronal and non-neuronal cells in Drosophila. In contrast to reported shorter poly(GR) forms, (GR)80 is mostly localized throughout the cytosol without detectable accumulation in the nucleolus, accompanied by suppression of Notch signaling and cell loss in the wing. Some Notch target genes are also downregulated in brains and iPSC-derived cortical neurons of C9ORF72 patients. Increased Notch expression largely suppressed (GR)80-induced cell loss in the wing. When co-expressed in Drosophila, HeLa cells, or human neurons, (GA)80 recruited (GR)80 into cytoplasmic inclusions, partially decreasing the toxicity of (GR)80 and restoring Notch signaling in Drosophila. Thus, different DPRs have opposing roles in cell loss and we identify the Notch pathway as one of the receptor signaling pathways that might be compromised in C9ORF72 FTD/ALS.

Activated Cdc42 kinase regulates Dock localization in male germ cells during Drosophila spermatogenesis.

Deregulation of the non-receptor tyrosine kinase ACK1 (Activated Cdc42-associated kinase) correlates with poor prognosis in cancers and has been implicated in promoting metastasis. To further understand its in vivo function, we have characterized the developmental defects of a null mutation in Drosophila Ack, which bears a high degree of sequence similarity to mammalian ACK1 but lacks a CRIB domain. We show that Ack, while not essential for viability, is critical for sperm formation. This function depends on Ack tyrosine kinase activity and is required cell autonomously in differentiating male germ cells at or after the spermatocyte stage. Ack associates predominantly with endocytic clathrin sites in spermatocytes, but disruption of Ack function has no apparent effect on clathrin localization and receptor-mediated internalization of Boss (Bride of sevenless) protein in eye discs. Instead, Ack is required for the subcellular distribution of Dock (dreadlocks), the Drosophila homolog of the SH2- and SH3-containing adaptor protein Nck. Moreover, Dock forms a complex with Ack, and the localization of Dock in male germ cells depends on its SH2 domain. Together, our results suggest that Ack-dependent tyrosine phosphorylation recruits Dock to promote sperm differentiation.

CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro.

A GGGGCC24+ hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), fatal neurodegenerative diseases with no cure or approved treatments that substantially slow disease progression or extend survival. Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. Here, we used an adeno-associated viral vector system to deliver CRISPR/Cas9 gene-editing machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus. We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion (500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, major hallmarks of C9-ALS/FTD, making this a promising therapeutic approach to these diseases.

Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD.

BACKGROUND: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. METHODS AND RESULTS: Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. CONCLUSIONS: Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.