Photobiomodulation Inhibits Ischemia-Induced Brain Endothelial Senescence via Endothelial Nitric Oxide Synthase.

Recent research suggests that photobiomodulation therapy (PBMT) positively impacts the vascular function associated with various cerebrovascular diseases. Nevertheless, the specific mechanisms by which PBMT improves vascular function remain ambiguous. Since endothelial nitric oxide synthase (eNOS) is crucial in regulating vascular function following cerebral ischemia, we investigated whether eNOS is a key element controlling cerebrovascular function and the senescence of vascular endothelial cells following PBMT treatment. Both rat photothrombotic (PT) stroke and in vitro oxygen-glucose deprivation (OGD)-induced vascular endothelial injury models were utilized. We demonstrated that treatment with PBMT (808 nm, 350 mW/cm2, 2 min/day) for 7 days significantly reduced PT-stroke-induced vascular permeability. Additionally, PBMT inhibited the levels of endothelial senescence markers (senescence green and p21) and antiangiogenic factor (endostatin), while increasing the phospho-eNOS (Ser1177) in the peri-infarct region following PT stroke. In vitro study further indicated that OGD increased p21, endostatin, and DNA damage (γH2AX) levels in the brain endothelial cell line, but they were reversed by PBMT. Intriguingly, the beneficial effects of PBMT were attenuated by a NOS inhibitor. In summary, these findings provide novel insights into the role of eNOS in PBMT-mediated protection against cerebrovascular senescence and endothelial dysfunction following ischemia. The use of PBMT as a therapeutic is a promising strategy to improve endothelial function in cerebrovascular disease.

RANBP17 Overexpression Restores Nucleocytoplasmic Transport and Ameliorates Neurodevelopment in Induced DYT1 Dystonia Motor Neurons.

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patient-specific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including a deformed nucleus, disrupted neurodevelopment, and compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes in gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RAN-binding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating the nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies.

Generation of two induced pluripotent stem cell lines with heterozygous and homozygous amyotrophic lateral sclerosis-causing mutation P525L (c.1574C > T) in FUS gene.

Mutations in the FUS (fused in sarcoma) gene are implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, the pathophysiology underlying these mutations remains elusive. In this study, we created two induced pluripotent stem cell (iPSC) lines through genetic modification of a healthy hiPSC line (WTC11, UCSFi001-A). These iPSC lines carry the heterozygous and homozygous P525L (c.1574C > T) mutation in the FUS gene. We confirmed that both cell lines possess typical stem cell morphology, normal karyotype, and pluripotency. Our iPSC lines offer a valuable resource for investigating the pathological mechanisms underlying the FUS mutation P525L in ALS.

Generation of two induced pluripotent stem cell lines with heterozygous and homozygous amyotrophic lateral sclerosis-causing mutation R521G (c.1561C > G) in FUS gene.

Mutations in the RNA-binding protein FUS (fused in sarcoma) are linked to amyotrophic lateral sclerosis (ALS), but the pathogenesis is not fully understood. For modeling ALS, here we generated two induced pluripotent stem cell (iPSC) lines carrying the heterozygous and homozygous R521G (c.1561C > G) mutation in the FUS gene via genetic modification of a healthy hiPSC line (WTC11, UCSFi001-A). Both lines show normal stem cell morphology and karyotype, express pluripotent markers, and can differentiate into three germ layers, providing a valuable resource in determining the pathological mechanisms underlying the FUS mutation of R521G in ALS.

Modeling Movement Disorders via Generation of hiPSC-Derived Motor Neurons.

Generation of motor neurons (MNs) from human-induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain tissues and provides an unprecedent approach for modeling MN-related diseases. In this review, we discuss the recent progression in understanding the regulatory mechanisms of MN differentiation and their applications in the generation of MNs from hiPSCs, with a particular focus on two approaches: induction by small molecules and induction by lentiviral delivery of transcription factors. At each induction stage, different culture media and supplements, typical growth conditions and cellular morphology, and specific markers for validation of cell identity and quality control are specifically discussed. Both approaches can generate functional MNs. Currently, the major challenges in modeling neurological diseases using iPSC-derived neurons are: obtaining neurons with high purity and yield; long-term neuron culture to reach full maturation; and how to culture neurons more physiologically to maximize relevance to in vivo conditions.

Protocol to image and quantify nucleocytoplasmic transport in cultured cells using fluorescent in situ hybridization and a dual reporter system.

Nucleocytoplasmic transport (NCT) plays critical roles in maintaining cellular homeostasis. Here, we present a protocol to measure NCT for both transcript and protein cargos in cultured cells. We first describe the fluorescent in situ hybridization (FISH) assay to measure the nuclear mRNA export. We then detail a dual reporter system to measure the protein NCT. This protocol also includes image analysis and data output using CellProfiler™. The combined approach can be used to unbiasedly analyze NCT activities in cultured cells. For complete details on the use and execution of this protocol, please refer to Ding et al. (2020, 2021).

Generation of gene-corrected isogenic control cell lines from a DYT1 dystonia patient iPSC line carrying a heterozygous GAG mutation in TOR1A gene.

Childhood-onset torsin dystonia (DYT1) is a rare hereditary movement disorder and usually caused by a heterozygous GAG deletion (c.907-909) in the TOR1A gene (ΔE, p.Glu303del). The neuronal functions of torsin proteins and the pathogenesis of ΔE mutation are not clear. Previously, we have generated a hiPSC line from DYT1 patient fibroblast cells. In this study, we genetically corrected GAG deletion and obtained two isogenic control lines. These hiPSC lines contain the wild-type TOR1A sequence, showed the normal stem cell morphology and karyotype, expressed pluripotency markers, and differentiated into three germ layers, providing a valuable resource in DYT1 research.

Generation of highly pure motor neurons from human induced pluripotent stem cells.

Generation of human motor neurons (MNs) overcomes the inaccessibility to patient brain tissues and greatly facilitates the research in MN-related diseases. Here, we describe a protocol for generation of neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs), followed by preparation of functional MNs. The optimized induction condition with the expression of three transcription factors in a single lentiviral vector significantly improved the yield and purity, making it possible to biochemically identify dysregulated factors in diseased neurons. For complete details on the use and execution of this protocol, please refer to Ding (2021), Ding et al. (2021), and Sepehrimanesh and Ding (2020).

Direct conversion of adult fibroblasts into motor neurons.

Generation of patient-derived neurons provides an unprecedented approach in modeling neurological diseases. Here, we describe the direct conversion of adult fibroblasts into motor neurons via lentiviral delivery of transcription factors. Compared with iPSC-based approach, directly converted neurons from donors retain features associated with age, making them ideal systems for modeling age-related neurological diseases. Low yield is the major challenge of this protocol. High quality lentiviruses and optimized cell culture conditions are critical to improve the final yield. For complete details on the use and execution of this protocol, please refer to Ding et al. (2020), Ding et al. (2021), and Liu et al. (2016).

Generation of two induced pluripotent stem cell lines with heterozygous and homozygous GAG deletion in TOR1A gene from a healthy hiPSC line.

A typical DYT1 dystonia is caused by a heterozygous GAG deletion (c.907-909) in the TOR1A gene (ΔE, p.Glu303del) and the pathogenesis is not clear. In this study, human induced pluripotent stem cell (hiPSC) lines carrying the heterozygous or homozygous GAG deletion in TOR1A gene were generated by genetic modification of a healthy hiPSC line (WTC11, UCSFi001-A). These hiPSC lines showed the normal stem cell morphology and karyotype, expressed the same pluripotency markers as their parental line, and had the capacity to differentiate into three germ layers, providing a valuable resource in determining the pathogenesis of human DYT1 dystonia.

Nucleocytoplasmic Transport: Regulatory Mechanisms and the Implications in Neurodegeneration.

Nucleocytoplasmic transport (NCT) across the nuclear envelope is precisely regulated in eukaryotic cells, and it plays critical roles in maintenance of cellular homeostasis. Accumulating evidence has demonstrated that dysregulations of NCT are implicated in aging and age-related neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and Huntington disease (HD). This is an emerging research field. The molecular mechanisms underlying impaired NCT and the pathogenesis leading to neurodegeneration are not clear. In this review, we comprehensively described the components of NCT machinery, including nuclear envelope (NE), nuclear pore complex (NPC), importins and exportins, RanGTPase and its regulators, and the regulatory mechanisms of nuclear transport of both protein and transcript cargos. Additionally, we discussed the possible molecular mechanisms of impaired NCT underlying aging and neurodegenerative diseases, such as ALS/FTD, HD, and AD.

Disease Modeling with Human Neurons Reveals LMNB1 Dysregulation Underlying DYT1 Dystonia.

DYT1 dystonia is a hereditary neurologic movement disorder characterized by uncontrollable muscle contractions. It is caused by a heterozygous mutation in Torsin A (TOR1A), a gene encoding a membrane-embedded ATPase. While animal models provide insights into disease mechanisms, significant species-dependent differences exist since animals with the identical heterozygous mutation fail to show pathology. Here, we model DYT1 by using human patient-specific cholinergic motor neurons (MNs) that are generated through either direct conversion of patients' skin fibroblasts or differentiation of induced pluripotent stem cells (iPSCs). These human MNs with the heterozygous TOR1A mutation show reduced neurite length and branches, markedly thickened nuclear lamina, disrupted nuclear morphology, and impaired nucleocytoplasmic transport (NCT) of mRNAs and proteins, whereas they lack the perinuclear "blebs" that are often observed in animal models. Furthermore, we uncover that the nuclear lamina protein LMNB1 is upregulated in DYT1 cells and exhibits abnormal subcellular distribution in a cholinergic MNs-specific manner. Such dysregulation of LMNB1 can be recapitulated by either ectopic expression of the mutant TOR1A gene or shRNA-mediated downregulation of endogenous TOR1A in healthy control MNs. Interestingly, downregulation of LMNB1 can largely ameliorate all the cellular defects in DYT1 MNs. These results reveal the value of disease modeling with human patient-specific neurons and indicate that dysregulation of LMNB1, a crucial component of the nuclear lamina, may constitute a major molecular mechanism underlying DYT1 pathology.SIGNIFICANCE STATEMENT Inaccessibility to patient neurons greatly impedes our understanding of the pathologic mechanisms for dystonia. In this study, we employ reprogrammed human patient-specific motor neurons (MNs) to model DYT1, the most severe hereditary form of dystonia. Our results reveal disease-dependent deficits in nuclear morphology and nucleocytoplasmic transport (NCT). Most importantly, we further identify LMNB1 dysregulation as a major contributor to these deficits, uncovering a new pathologic mechanism for DYT1 dystonia.

Generation and optimization of highly pure motor neurons from human induced pluripotent stem cells via lentiviral delivery of transcription factors.

Generation of neurons from human induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain samples and greatly facilitates the progress of research in neurological diseases. However, it is still a challenge to generate a particular neuronal subtype with high purity and yield for determining the pathogenesis of diseased neurons using biochemical approaches. Motor neurons (MNs) are a specialized neuronal subtype responsible for governing both autonomic and volitional movement. Dysfunctions in MNs are implicated in a variety of movement diseases, such as amyotrophic lateral sclerosis (ALS). In this study, we generated functional MNs from human iPSCs via lentiviral delivery of transcription factors. Moreover, we optimized induction conditions by using different combinations of transcription factors and found that a single lentiviral vector expressing three factors [neurogenin-2 (NGN2), insulin gene enhancer 1 (ISL1), and LIM/homeobox 3 (LHX3)] is necessary and sufficient to induce iPSC-derived MNs (iPSC-MNs). These MNs robustly expressed general neuron markers [microtubule-associated protein 2 (MAP2), neurofilament protein (SMI-32), and tubulin ß-3 class III (TUBB3)] and MN-specific markers [HB9 and choline acetyltransferase (ChAT)] and showed electrical maturation and firing of action potentials within 3 wk. This approach significantly improved the neuronal survival, yield, and purity, making it feasible to obtain abundant materials for biochemical studies in modeling movement diseases.

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

Nucleocytoplasmic transport (NCT) across thenuclear envelope (NE) is tightly regulated in eukaryotic cells and iscritical for maintaining cellular homeostasis. Its dysregulationleads to aging and neurodegeneration. Because they maintainaging-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.

Evidence that Moderate Eviction of Spt5 and Promotion of Error-Free Transcriptional Bypass by Rad26 Facilitates Transcription Coupled Nucleotide Excision Repair.

Transcription coupled repair (TC-NER) is a subpathway of nucleotide excision repair triggered by stalling of RNA polymerase at DNA lesions. It has been suspected that transcriptional misincorporations of certain nucleotides opposite lesions that result in irreversible transcription stalling might be important for TC-NER. However, the spectra of nucleotide misincorporations opposite UV photoproducts and how they are implicated in transcriptional stalling and TC-NER in the cell remain unknown. Rad26, a low abundant yeast protein, and its human homolog CSB have been proposed to facilitate TC-NER in part by positioning and stabilizing stalling of RNA polymerase II (RNAPII) at DNA lesions. Here, we found that substantial AMPs but no other nucleotides are transcriptionally misincoporated and extended opposite UV photoproducts and adjacent bases in Saccharomyces cerevisiae. Rad26 does not significantly affect either the misincorporation or extension of AMPs. At normally low or moderately increased levels, Rad26 promotes error-free transcriptional bypass and TC-NER of UV photoproducts. However, Rad26 completely loses these functions when it is overexpressed to ~1/3 the level of RNAPII molecules. Also, Rad26 does not directly displace RNAPII but constitutively evicts Spt5, a key transcription elongation factor and TC-NER repressor, from the chromatin. Our results indicate that transcriptional nucleotide misincorporation is not implicated in TC-NER, and moderate eviction of Spt5 and promotion of error-free transcriptional bypass of DNA lesions by Rad26 facilitates TC-NER.

BDNF activates an NFI-dependent neurodevelopmental timing program by sequestering NFATc4.

We show that BDNF regulates the timing of neurodevelopment via a novel mechanism of extranuclear sequestration of NFATc4 in Golgi. This leads to accelerated derepression of an NFI temporal occupancy gene program in cerebellar granule cells that includes Bdnf itself, revealing an autoregulatory loop within the program driven by BDNF and NFATc4.

Lamin Mutations Accelerate Aging via Defective Export of Mitochondrial mRNAs through Nuclear Envelope Budding.

Defective RNA metabolism and transport are implicated in aging and degeneration [1, 2], but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration [3]. Here, we link a novel mechanism for RNA export through nuclear envelope (NE) budding [4, 5] that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. We identified mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin), as well as other transcripts required for mitochondrial integrity and function, in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates [6, 7] and myofibril disorganization [8, 9]. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marf was insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis of NE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging.

Reciprocal autoregulation by NFI occupancy and ETV1 promotes the developmental expression of dendrite-synapse genes in cerebellar granule neurons.

Nuclear Factor One (NFI) transcription factors regulate temporal gene expression required for dendritogenesis and synaptogenesis via delayed occupancy of target promoters in developing cerebellar granule neurons (CGNs). Mechanisms that promote NFI temporal occupancy have not been previously defined. We show here that the transcription factor ETV1 directly binds to and is required for expression and NFI occupancy of a cohort of NFI-dependent genes in CGNs maturing in vivo. Expression of ETV1 is low in early postnatal cerebellum and increases with maturation, mirroring NFI temporal occupancy of coregulated target genes. Precocious expression of ETV1 in mouse CGNs accelerated onset of expression and NFI temporal occupancy of late target genes and enhanced Map2(+) neurite outgrowth. ETV1 also activated expression and NFI occupancy of the Etv1 gene itself, and this autoregulatory loop preceded ETV1 binding and activation of other coregulated target genes in vivo. These findings suggest a potential model in which ETV1 activates NFI temporal binding to a subset of late-expressed genes in a stepwise manner by initial positive feedback regulation of the Etv1 gene itself followed by activation of downstream coregulated targets as ETV1 expression increases. Sequential transcription factor autoregulation and subsequent binding to downstream promoters may provide an intrinsic developmental timer for dendrite/synapse gene expression.