Differential Influence of Sample Sex and Neuronal Maturation on mRNA and Protein Transport in Induced Human Neurons

Abstract

Nucleocytoplasmic transport (NCT) across the nuclear envelope (NE) is tightly regulated in eukaryotic cells and is critical for maintaining cellular homeostasis. Its dysregulation leads to aging and neurodegeneration. Because they maintain aging-associated hallmarks, directly reprogrammed neurons from human fibroblasts are invaluable in understanding NCT. However, it is not clear whether NCT activity is influenced by neuronal maturation and sample sex [a key biological variable emphasized by the National Institutes of Health (NIH) policy]. We examined here NCT activity at the single-cell level by measuring mRNA subcellular distribution and protein transport in directly induced motor neurons (diMNs) from adult human fibroblasts. The results show that mRNA subcellular distribution but not protein transport is affected by neuronal maturation stages, whereas both transport processes are not influenced by the sample sex. This study also provides quantitative methods and optimized conditions for measuring NCTs of mRNAs or protein cargoes, establishing a robust way for future functional examinations of NCT activity in directly induced neurons from diseased human patients.

Disease modeling with human neurons reveals LMNB1 dysregulation underlying DYT1 dystonia

“…The fibroblasts of four DYT1 patients and four age-matched healthy controls were used for this study (Table S1). Although sex does not affect the reprogramming efficiency 25 , all healthy controls were selected to be sex-matched to patient cell lines. Transduction with reprogramming factor-expressing lentivirus enabled adult human fibroblasts to gradually become neuron-like and grow complex neurites with time ( Fig.…”

“…In torsin mutants, disruption of mRNA nuclear export led to slowed growth and impaired development and plasticity of neuromuscular junctions 14 . To examine whether mRNA export was similarly impaired in DYT1 diMNs, we performed fluorescence in situ hybridization (FISH), as previously described 25,33,34 . We used digoxigenin-labeled oligo-dT as probes to detect the subcellular distribution of Poly (A) mRNAs.…”

“…2C), suggesting that protein NCT might also be compromised. We used a dual reporter system (2Gi2R) to further examine NCT 25 . This reporter system consists of a fused double-GFP with a nuclear export signal (2GFP-NES) and a double RFP containing a nuclear localization signal (2RFP-NLS) 35 .…”

“…In situ hybridization was performed as described previously 25 . Briefly, cultured cells were washed once with PBS, and fixed with 4% PFA for 30 min at room temperature.…”

Aging-Relevant Human Basal Forebrain Cholinergic Neurons as a Cell Model for Alzheimer’s Disease

“…TAU hyperphosphoration leads to disrupted nucleocytoplasmic transport (NCT) in AD neurons [57]. We examined NCT activity in hiBFCNs by using a well-established reporter assay [15,57,58]. This reporter (2Gi2R) consists of 2xGFP containing an NES sequence (GFP-NES), an internal ribosome entry site (IRES), followed by 2xRFP fused to an NLS sequence (RFP-NLS).…”