Generation of three isogenic gene-edited Huntington's disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus.

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.

Nicotinamide Adenine Dinucleotide Phosphate Oxidases Are Everywhere in Brain Disease, but Not in Huntington's Disease?

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by neuronal loss and tissue atrophy mainly in the striatum and cortex. In the early stages of the disease, impairment of neuronal function, synaptic dysfunction and white matter loss precedes neuronal death itself. Relative to other neurodegenerative diseases such as Alzheimer's and Parkinson's disease and Amyotrophic Lateral Sclerosis, where the effects of either microglia or NADPH oxidases (NOXs) are recognized as important contributors to disease pathogenesis and progression, there is a pronounced lack of information in HD. This information void contrasts with evidence from human HD patients where blood monocytes and microglia are activated well before HD clinical symptoms (PET scans), and the clear signs of oxidative stress and inflammation in post mortem HD brain. Habitually, NOX activity and oxidative stress in the central nervous system (CNS) are equated with microglia, but research of the last two decades has carved out important roles for NOX enzyme function in neurons. Here, we will convey recent information about the function of NOX enzymes in neurons, and contemplate on putative roles of neuronal NOX in HD. We will focus on NOX-produced reactive oxygen species (ROS) as redox signaling molecules in/among neurons, and the specific roles of NOXs in important processes such as neurogenesis and lineage specification, neurite outgrowth and growth cone dynamics, and synaptic plasticity where NMDAR-dependent signaling, and long-term depression/potentiation are redox-regulated phenomena. HD animal models and induced pluripotent stem cell (iPSC) studies have made it clear that the very same physiological processes are also affected in HD, and we will speculate on possible roles for NOX in the pathogenesis and development of disease. Finally, we also take into account the limited information on microglia in HD and relate this to any contribution of NOX enzymes.