Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Enhancing the Natural Biological Control in the Thyroid Hormone Homeostasis As a First-Order Control System.

This study explores the natural control system that exists within the pituitary gland. More specifically, this study investigates the regulation of the thyroid stimulating hormone (TSH), released by the anterior pituitary, with regards to the thyroid releasing hormone (TRH), which is released by the hypothalamus. Using appropriate assumptions on the behavior of the hormones, along with relevant boundary conditions, we modeled an output of TSH using constant TRH input over the course of a six-hour period. Other relevant hormones such as thyroxine (T4), triiodothyronine (T3), and their relevant intermediaries were also modeled as a means to complete the natural feedback found physiologically. Due to our boundary conditions, we do not consider the consumption or final function of these hormones since they leave the pituitary gland, our control system; instead, we consider a constant TRH since it is produced by the hypothalamus. Finally, we explore the results of reducing the TRH input while observing the TSH response. We append a short loop controller feedback that uses the TSH output to regulate a TRH input to remedy the reduction of TRH. The open-loop transfer function derived presented three poles at the clearance exponents for T4, TSH, and central T3, with a phase margin of 74.1°, characterizing a stable but slow system that can be improved with a simple proportional control.

RAS-mediated suppression of PAR3 and its effects on SCC initiation and tissue architecture occur independently of hyperplasia.

Proper epithelial development and homeostasis depends on strict control of oriented cell division. Current evidence shows that this process is regulated by intrinsic polarity factors and external spatial cues. Owing to the lack of an appropriate model system that can recapitulate the architecture of the skin, deregulation of spindle orientation in human epithelial carcinoma has never been investigated. Here, using an inducible model of human squamous cell carcinoma (SCC), we demonstrate that RAS-dependent suppression of PAR3 (encoded by PARD3) accelerates epithelial disorganization during early tumorigenesis. Diminished PAR3 led to loss of E-cadherin-mediated cell adhesion, which in turn contributed to misoriented cell division. Pharmacological inhibition of the MAPK pathway downstream of RAS activation reversed the defects in PAR3 expression, E-cadherin-mediated cell adhesion and mitotic spindle orientation. Thus, temporal analysis of human neoplasia provides a powerful approach to study cellular and molecular transformations during early oncogenesis, which allowed identification of PAR3 as a critical regulator of tissue architecture during initial human SCC development.