ResearchALS.org
is available for sale
About ResearchALS.org
Former two words, brandable domain representing Research ALS - the Alzheimer Research Forum, also known as the Alzforum, is an online research community dedicated to furthering research into Alzheimer's disease and related neurodegenerative disorders.
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Induced Motor Neurons May Provide New Insights on the Role of Cellular Aging in ALS
February 2, 2016
Motor neurons created by direct lineage reprogramming of FUS-ALS fibroblasts model several important aspects of ALS, according to a report in the January 5 online issue of Cell Reports. Unlike induced pluripotent stem cells (iPSCs), the directly reprogrammed cells bypass the embryonic-like state, and thus retain some of the molecular characteristics of the aging cells from which they are derived (see Jan 2010 news). This may offer a cellular model of ALS in which the effects of aging, as well as disease-linked mutations, can be explored.
Recapitulation of the cell donor phenotype is the sine qua non of cell-based models of disease, and questions have arisen over how faithfully iPSC-derived cells can reproduce that phenotype, since the fibroblasts they arise from are wiped clean of the epigenetic markers that define the differentiated cell. “The neurons created from iPSCs are actually embryonic neurons, because all the aging markers have been removed,” noted senior author of the new study Chun-Li Zhang, of UT Southwestern Medical Center in Dallas, Texas.
Chun-Li Zhang. Image courtesy of CL Zhang.
Recently, for instance, Mertens et al. showed that induced neurons (iNs) generated by direct conversion from fibroblasts had an age-related reduction in the level of a nuclear transport receptor, which decreased the degree of nucleocytoplasmic compartmentalization. This characteristic that was lost in iPSC-derived neurons.
To explore the use of human induced motor neurons (hiMNs) for modeling ALS, Zhang developed a protocol for direct lineage reprogramming of human skin fibroblasts from both healthy and FUS-ALS donors. Zhang had previously shown that cholinergic neurons could be created by direct lineage reprogramming (Liu et. al., 2013). Here, he went further, adding the transcription factors ISL1 and LHX3, delivered via lentivirus, along with other factors delivered in the growth media, in order to induce a motor neuron phenotype.
Working with co-first authors Meng-Lu Liu and Tong Zang, Zhang found that about 90% of healthy adult fibroblasts were converted to motor neuron-like cells, characterized by development of axons and dendrites and expression of multiple motor neuron markers, including HB9 and ChAT. Discrete puncta of the presynaptic marker synaptotagmin suggested the development of synaptic terminals. Further analysis indicated the neurons were a mix of cervical and thoracic motor neurons. When cocultured with astrocytes, the cells lived for 49 days, and continued to strongly express ChAT, indicating continued synthesis of acetylcholine neurotransmitter. The neurons fired repetitive action potentials when current was applied, and formed functional neuromuscular junctions when allowed to contact skeletal myotubes.
The team then performed direct conversion on fibroblast samples from three different FUS-ALS patients, and observed similar differentiation into HB9- and ChAT-expressing hiMNs by 21 days. While FUS in patient-derived fibroblasts localized in a similar pattern to that in healthy controls, in the hiMNs derived from ALS patients, an abnormal fraction of the protein was cytoplasmic. The neurons displayed a shrunken soma and an increased death rate compared to non-ALS hiMNs neurons. FUS neurons fired infrequently and abnormally, and while they did form synapses, they rarely formed neuromuscular junctions.